JPH1081685A - Erythro-hydroxynonyladenine analog having high lipophilicity and antiischemic characteristics - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject compound capable of inhibiting adenosine deaminase (hereinafter ADA) and reducing the enzymatic degradation of chemotherapeutics and antiviral medicines to enhance the therapeutic usefulness. SOLUTION: This chemically modified analog of erythro-hydroxynonyladenine of the formula at least one of R1 and R2 is H or a chemical part except hydroxyl and contains a part enabling that the compound of the formula is pharmacologically permissible and nontoxic, that the compound of the formula can more effectively and reversibly inhibit the intracellular enzymatic activity of ADA than erythro-hydroxynonyladenine or 9-[2(S),9-dihydroxy-3-(R)- nonyl]adenine in a cell culture test, and that the compound of the formula can effectively reduce hypoxia and ischemic injuries in cardiac muscles and cerebral tissues}. The compound of the formula preferably has a Ki value of approximately 10<-7> to approximately 10<-10> mole with respect to the inhibition of the ADA. The compound of the formula can thereby protect cardiac muscles or cerebral tissues, and protect lesions due to ischemia and hypoxia developed in paroxysmal syndromes such as apoplexy and cardiac standstill.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention belongs to the field of chemistry and pharmacology and relates to an enzyme (A) called adenosine deaminase.
DA, also known as adenosine aminohydrolase). ADA-
The use of inhibitory agents may enhance the therapeutic utility of chemotherapeutic agents and antiviral agents by reducing enzymatic degradation of the agent. As disclosed herein, the use of ADA-inhibiting agents may result in ischemia (insufficient blood flow) occurring during stroke, cardiac arrest, heart attack, suffocation, and various other seizure symptoms. It can also protect myocardial and brain tissue from damage induced by hypoxia (insufficient oxygen supply).

[0002]

BACKGROUND OF THE INVENTION A mammalian enzyme called adenosine deaminase (ADA) has been designated EC 3.5.4.4. Under the International Enzyme Classification System,
Adenosine is converted to inosine by removing the amino group from carbon 6 in the ring adenyl structure. ADA
Can also degrade some other molecules, including some nucleoside analogs used in cancer chemotherapy or antiviral therapy. Since ADA is known to reduce the therapeutic utility of various drugs used to treat cancer and viral infections, a great deal of research has been done on the development of drugs that function as ADA inhibitors. I have been. The use of ADA inhibitors as adjuvants (ie, secondary agents that increase the efficacy of a primary agent) can increase the metabolic half-life of a therapeutic agent during cancer or antiviral chemotherapy. Also, the use of ADA inhibitors can artificially create ADA deletions, so some researchers are interested in this.

[0003]

DETAILED DESCRIPTION OF THE INVENTION A compound called erythro-hydroxynonyl adenine (abbreviated as EHNA and usually pronounced "enah") is a relatively mild ADA.
Inhibitors, which are of particular interest here. EHNA is a stereoisomer having the following chemical structure and indicates how to number carbon atoms in nonyl "side chains" (ie, in erythro-hydroxy-nonyl straight chains attached to bicyclic adenyl groups). ing:

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[0004] The prefix "erythro-" indicates certain stereoisomeric configurations of the atoms attached to the second and third carbons in the nonyl side chain. Since both the second and third carbon atoms are chiral atoms (ie, carbon atoms with four different groups attached thereto),
The spatial configuration of the four groups will be in the (+) or (-) configuration depending on how they rotate the polarized light passing through the aqueous solution of the "optically purified" stereoisomer. Other optically pure stereoisomers that have the same atoms as the erythro compounds but have different stereoisomeric configurations are referred to as "threo-" compounds.

A "racemic" mixture of EHNA (ie,
50-5 of the + and-isomers in erythro configuration
0) was identified as an ADA inhibitor by Schaeffer and Schbender (1974). Subsequent reports, including Bastian et al. (1981) and Baker and Hawkins (1982), have identified the (+)-2S, 3R isomer (erythro isomer) as the most potent ADA inhibitor among the various hydroxynonyl adenine isomers. ) Was identified.

[0006] Various analogs and derivatives of EHNA are
For example, it is described in the report of Harriman et al. (1992). Those other analogs are not related to the EHNA analogs described in this specification.

Apparently, EHNA is metabolized and purified from the bloodstream of mammals quite rapidly (McConnell et al., 1).
980, Rambe and Nelson, 1982). further,
The activity of EHNA as an ADA inhibitory agent is not as strong as various other ADA inhibitory agents including deoxycoformycin (dCF, also known as pentostatin). The so-called "Ki" value of dCF (i.e., the negative logarithm of the molar concentration of dCF required for inactivation of standard ADA) is very low, about 2.5 x ^10-12 mol.
(M; all Ki values here are molar). About 1
0 ^-12 is the molar range this very low value indicates that dCF is very firmly bound and ADA. dC
F, sometimes referred to as a "suicide inhibitor", indicates that the binding between dCF and ADA is virtually irreversible, and neither molecule can be regenerated. This irreversible binding process is also referred to as "disabling" the enzyme.

[0008] Due to its potency as an ADA inhibitor, several research teams have tested dCF and determined whether it can be used therapeutically. Reportedly, dCF exhibited some beneficial activity in cardiovascular models (eg, Dorheim et al., 1991), neuroprotection (eg, Phyllis and Olegan, 1989) and cancer treatment, but induced severe and adverse side effects. Have been found (eg, Odoyer et al., 1986). Therefore, a more gradual AD
Attention has been returned to EHNA and various other mild or "light" ADA inhibitors, with the hope that A inhibitors would have fewer side effects and lower toxicity. (±) -EH
The Ki value of NA is about 6 × 10 ^-9 and EHNA is dCF
Binding to ADA with approximately 1000 times less force.

The present invention provides a method for treating EHNA wherein the hydrogen atom bonded to one side chain of the "distal terminal" carbon atom (ie, the 8th or 9th carbon atom) is replaced by a moiety, other than hydrogen or a hydroxyl group. Form 8 or 9 analogs of one or more internal organs (such as the heart or brain) that are vulnerable to ischemic injury, with (+) EHNA against ischemic injury. + Stereoisomer of EHNA to write,
Alternatively, it has better therapeutic utility compared to the 9-hydroxy analog of (+) EHNA described in U.S. Patent No. 5,491,146, issued February 1996 to Abushanab.

[0010] The control analogs of interest herein have a combination of two desired properties, as follows: (1) the Ki value is between about 10 ^-7 and about 10 ^-10 moles;
Has a desired range of binding affinity to ADA appeal.
A Ki value in this range means that the control analog of interest has a relatively high level of efficacy without becoming a "suicide inhibitor" that adversely inactivates ADA enzyme molecules.

(2) They may also cause ischemia (insufficient blood flow) or hypoxia (insufficient blood flow) during stroke, heart attack, cardiac arrest, suffocation and various other types of seizure symptoms or conditions. Unprotected (+) EHN in protecting heart and / or brain tissue from damage induced by oxygenation
It is substantially more effective than the 9-hydroxy analog of A or (+) EHNA.

Prior to the present invention, the usefulness of ADA-inhibiting agents in protecting myocardial or brain tissue from ischemia / hypoxia damage was scarcely stated; for example, Zhu et al 1990 and See Marangos et al 1990. However, despite these publications, almost all studies on ADA inhibitors (prior to the present invention) have
The focus has been on their potential to enhance the efficacy of anti-cancer or anti-viral drugs by delaying the degradation of the anti-cancer or anti-viral drug by enzymes.

It should be noted that the ADA enzyme is active in almost all whole cells. This enzymatic activity can affect the amount of adenosine released by cells, and extracellular adenosine plays various roles initiated by adenosine receptors on the cell surface; It is said that it is detected by. However, the fact remains that ADA enzymes function almost exclusively in cells. Thus, an ADA inhibitor must enter a mammalian cell in order to function properly, and its potency depends largely on how easily it can be taken into the cell.

It is an object of the present invention to substantially improve the rate of incorporation into mammalian cells so that intracellular ADA enzyme molecules can be reached and inhibited at higher concentrations than unmodified or hydroxylated EHNA. In a method, a group of analogs of EHNA is disclosed in which the 8th or 9th carbon atom in the side chain has been modified.

Another object of the present invention is to substantially improve the therapeutic efficacy of these analogs against ischemic or hypoxic damage to myocardial or brain tissue as compared to either unmodified or hydroxylated EHNA. The disclosure is to disclose a group of analogs of EHNA in which the 8th or 9th carbon atom in the side chain has been modified in such a way.

Another object of the present invention is to provide a binding affinity (preferably about 10%) for ADA enzymes within a desired range.
EHNAs with modified 8 or 9 carbon atoms in the side chains in a manner that retains these analogs with various therapeutic benefits while retaining a Ki value between ^{-7 and} about 10 ^-10 ).
Are to disclose a family of analogs. With this condition as a relatively mild reversible ADA inhibitor, the analog can inhibit ADA activity at therapeutically effective levels and
It does not irreversibly inactivate the DA enzyme or increase the risk of harmful side effects.

Another object of the invention is to include certain types of halide atoms, carboxylic acids or salts, ester or ether linkages or nitrogen-containing moieties at various controllable positions in the nonyl side chain, especially EHNA molecules. Containing a novel moiety attached to the 8th or 9th carbon atom in the side chain of
It is to disclose synthetic reagents and methods that can be used to generate pharmacologically valuable EHNA analogs of HNA.

Another object of the present invention is to disclose a novel set of EHNA analogs that can be used to slow down the ADA enzyme degradation of certain types of anticancer, antiviral or other therapeutic agents. is there.

These and other objects of the invention will become more apparent and apparent from the following summary, detailed description and examples.

[0020]

SUMMARY OF THE INVENTION Various types of moieties are numbered 8 or 9 in the "side chain" portion of the molecule (ie, the 9-carbon erythro-hydroxynonyl linear portion in EHNA, attached to the adenosine ring structure). Erythro-hydroxynonyl adenine (E
HNA) are disclosed. EHNA analogs in which a relatively lipophilic moiety is attached at position 8 or 9 in the side chain may be responsible for cell death and ischemia (insufficient blood flow) and / or hypoxia (insufficient oxygenation). It has been found to have an improved effect on reducing tissue damage. In an initial series of tests, one of the following hydroxylated EHNA analogs (referred to herein as 9-OH-EHNA, where the hydroxyl moiety is attached to carbon atom 9 of the EHNA side chain) was compared to unmodified EHNA. Have been found to have significant benefits in protecting the myocardium from blood damage. This assay is described in Example 4.

Subsequent work on EHNA analogs has shown that some analogs not only surpass unmodified EHNA,
It has been shown to have significant therapeutic advantages even when compared to the 9th or 8th hydroxylated analog. This subsequent study uses the somewhat different cardiac perfusion technique described in Example 6, where the 9-OH-EHNA analog is more potent than unmodified EHNA in most of the parameters used to measure cardioprotection. It did not show that it had a substantially superior effect. However, by the time it is confirmed, several other EHNA analogs have
Was found to show a significant improvement over either the unmodified or the hydroxylated form of, and subsequent work was directed to their other analogs, ignoring 9-OH-EHNA and further testing Did not.

Analogs that have shown the best effect on ischemic tissue damage in studies performed to date are more lipophilic than hydroxylated analogs (ie, soluble in lipids and other fatty nonpolar fluids). High solubility, poor water solubility)
it is conceivable that. These lipophilic analogs are disclosed below, along with their chemical synthetic methods and other desirable analogs of EHNA, where the 8 or 9 carbon atom of the side chain has been modified. All of the above analogs synthesized in the manner described herein can be screened using the assays described below or other methods known to those skilled in the art, and specific analogs described herein. It is determined whether it has the desired combination of properties. These features include (1) reversible ADA inhibitors,
Non-toxic efficacy, (2) the desired level of lipophilicity to increase cellular uptake, and (3) reduced damage from ischemia or hypoxia, or the degradation of ADA by anti-cancer, anti-viral or other agents. Therapeutic utility in reduction is included.

[0023]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows the formation of 9′-hydroxy (+)-EHNA, designated as compound [10], an intermediate compound used to generate other more analogous EHNAs that are more lipophilic. FIG. 3 is a diagram showing a series of chemical reactions used for the present invention. FIG. 2 shows 8′-defined as compound [23].
FIG. 3 shows the reaction used to generate hydroxy (+)-EHNA. FIG. 3 shows the reaction used to generate 8 ′, 9′-dihydroxy (+)-EHNA, designated as compound [14]. FIG. 4 illustrates the reaction used to generate analogs of EHNA containing various non-hydroxy moieties attached to carbon number 9 (see Example 5). FIGS. 5, 6 and 7 show that 9-chloro-EHNA (compound [29]) and 9-phthalimido-EHNA (compound [27]) were tested in the test described in Example 6 with unmodified EHNA or 9-hydroxy-EHNA. 4 is a bar graph showing that myocardium was more effective in protecting myocardium from ischemic damage.

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, the side chain (ie, the linear erythro-hydroxynonyl moiety, attached to the adenyl ring structure) is
Certain types of chemical groups (parts) can be added to the "long end"
Chemically modified EHNA analogs have been reported by attaching to the number 9 or other 9 carbon atoms. As described herein, preferred moieties for anti-ischemic analogs are those in which the resulting analogs are provided with certain pharmacological and therapeutic activities, such as unmodified EHNA or US Pat. No. 5,491,146.
No. 9-hydroxy analog.

In one embodiment of the present invention, a hydroxyl group is attached to the 8th or 9th carbon atom of the EHNA side chain, whereby 8-OH-EHNA or 9-OH-
A hydroxylated analog referred to as EHNA (or a dihydroxylated analog having a hydroxyl moiety attached to both carbon atoms) can be produced. These hydroxylated analogs were synthesized as described in Examples 1 and 2 (and more particularly, the example of US Pat. No. 5,491,146) and tested with intact perfused hearts isolated from rats. When tested with a model, it was discovered that the analogs had minor but significant advantages over unmodified EHNA in protecting myocardium from ischemic damage. Limited tests performed with these hydroxylated analogs indicate that
It has been shown that 9-OH-EHNA analogs are preferred over 8-OH-EHNA analogs.

Based on their initial findings, other analogs of EHNA were synthesized and tested in various ways. Studies of these analogs have shown that unmodified EHNA or 9-
Shows that some have significant therapeutic benefits compared to hydroxy analogs.

The 9-OH analog was synthesized as an optically pure (+) stereoisomer, and all other analogs subsequently synthesized were produced as pure (+) stereoisomers.

The analogs which have demonstrated the best combination properties in studies completed to date are hydroxylated 9-OH-
It is believed to be more lipophilic (ie, more soluble in lipids and other fatty non-polar fluids) and less water soluble than EHNA analogs. These analogs, including 9-chloro-EHNA and 9-phthalimido-EHNA, are disclosed below. Alternatively, the chemical synthesis methods disclosed herein (and others well known to those skilled in the chemical synthesis art) can be used to add other functional groups to carbon atoms 8 or 9 of the side chain. By doing so, other analogs of the modified EHNA can be generated. After synthesis, the analogs can be screened and tested using the assays described herein or other assays well known to those skilled in the biomedical testing industry to obtain the desired combination of the specified analogs. Was determined.

In the present invention, three characteristics are of primary interest. As a property of the foregoing, an EHNA analog intended for human therapy should have adequate potency as a reversible ADA inhibitor, and
The binding affinity for A gives a Ki value in the range of about ^10-7 to about ^10-10 . EH with Ki values in this desired range
NA analogs can reversibly inhibit the ADA enzyme, without permanently disabling the enzyme molecule, and in some patients or test animals, a potent "suicide inhibitor"
Neither does it elicit the type of toxic side effects that could be induced, for example, by deoxycoformycin.
For any EHNA analog, its Ki value can be measured as a test, for example, by the spectrophotometric method described in Harriman et al. (1992) (further detailed in Example 3).

A second desirable property for EHNA analogs intended for human therapy is an appropriate level of lipid solubility (also called lipophilicity). In general, lipophilic drugs tend to be easier and more massively incorporated into cells than hydrophilic drugs because of two chemical factors. First
In addition, in the case of lipophilic drugs such as oil droplets in water, surface tension is generated between the drug itself and water molecules. They are not "comfortably" suspended in the aqueous liquid surrounding the cells, but are seeking configurations that minimize the area of the surface in contact with water. This surface tension causes lipophilic molecules to bind and adhere to the lipophilic surfaces they encounter, including the cell surface, and minimize the surface area where they are in contact with water.

Second, since the membrane of a mammalian cell is itself made of a lipid bilayer membrane, molecules soluble in lipids are easily dissolved in the cell membrane and easily transferred into the cell membrane. This is a major step in cellular uptake and does not require an active transport mechanism to facilitate these drugs across cell membranes and into cells.

Both of these factors are hydrophilic and / or
Or they tend to promote and increase cellular uptake of lipophilic drugs as compared to highly charged drugs. This is hypothesized and believed to be a potentially important factor for ADA-inhibiting EHNA analogs, since EHNA and its analogs act via a molecular mechanism that resides inside the cell.

However, the increased hydrophobicity does not, without limitation, enhance the desirable property of high uptake into cells. For highly hydrophobic drugs, administration to patients by conventional routes, such as injection or ingestion, can be difficult, and once inside the body, these drugs are often in lipid vesicles or spherules. Or they tend to adhere to various membranes, plaque deposits or granules in the small intestine or blood vessels.

For these reasons, therapeutic agents that must be taken up intracellularly to produce sufficient efficacy often have a moderately high but not extreme level of lipophilicity (hydrophobicity). preferable. Thus, the analog attached to the other 8 or 9 carbon atom is more likely to have a hydroxylated 9
Other EHNA analogs with other lipophilic moieties have been discovered to be more effective at protecting against ischemic damage than -OH-EHNA analogs, and the methods and assays disclosed herein. Various molecules that are synthesized and tested using the methods, thus providing good candidates for producing this therapeutic analog, are evaluated.

The lipophilicity level of an EHNA analog with any candidate moiety can be assessed using a dual solvent assay, such as the widely used octanol-water partition assay. The partition coefficient is usually indicated as P _{o / w} (where “o / w” indicates oil and water). Usually this value is indicated by the logarithm of the base 10, comparable to the pH value. logP
_A high _{o / w} value indicates a high solubility for the oil, while a low value (or negative)
Indicates high water solubility.

The octanol-water partition coefficient can be calculated using commercially available computer software (for example, ACD /
LogP software program, Advanced Chemistry Development, Toronto, Canada, Incorporated Sales). Using this software, the octanol-water partition coefficients listed in Table 1 were calculated. The details of the methods used to calculate and evaluate the partition coefficients are based on their chemical structure and are described in Border et al. (1989).

Without imposing or limiting the present invention to a particular theory, it is generally believed that the lipophilic solubility levels are close to or somewhat greater than the chloro- or phthalimide-analogs described below. It is to be appreciated that EHNA analogs are preferred over hydroxylated analogs and other more hydrophilic (water soluble) analogs for use in the present invention.

A third major desirable property of EHNA analogs intended for the uses described herein includes laboratory tests or a laboratory test that provides a suitable model of therapeutic utility against certain types of cell damage or drug degradation. There is therapeutic utility in mammalian patients. At present, the two major and most urgent uses for the EHNA analogs described herein are: (a) the amount of damage induced by ischemia or hypoxia in vulnerable tissues, especially myocardial or brain tissue Prolonging the half-life of the drug by reducing the rate of ADA enzyme degradation of the drug that is being used to treat a patient suffering from a cancer, viral infection or other medical condition; It is to increase the therapeutic effect. Also, various applications may be discovered in the future, although they may still be used for various other applications, or after these compounds have been published and made available to scientists and medical researchers.

The present invention also relates to No. 8 or 9 in the side chain.
EHNA to which another functional group attached to carbon atom No.
Methods for synthesizing analogs are disclosed. The method comprises the following steps.

A. An epoxide reagent having a desired chiral orientation and an alkyl halide reagent having an unsaturated bond between two selected carbon atoms are combined with a first portion having a desired chiral orientation by the reagent, and EHNA. Reacting under conditions that produce an unsaturated aliphatic compound comprising a second moiety having an unsaturated bond located between the two carbon atoms that result in carbon atoms 8 and 9 of the analog; and b. The unsaturated side chain is attached to the adenyl ring using a simple step. One such route, as shown in FIG. 1, is a synthetic route leading to compounds [6], [7] and [8], in which 5-amino-4,6-dichloropyrimidine (A
DCP). Another route, if desired, is "Mits
Using the unobu "state, the bicyclic adenyl derivative is attached to the side chain and then the adenyl derivative is modified.

C. Combining the unsaturated aliphatic compound and at least one third reagent with at least one hydroxyl group (or other desired group or atom) by the third reagent at carbon atoms 8 and 9; The double bond between the 8th and 9th carbon atom is modified by adding an unsaturated bond between the hydroxyl group (the atom such as another desired group or halide atom, or an ester or ether). The reaction is carried out under the conditions for producing a group) saturated aliphatic compound.

This makes an EHNA analog having a saturated side chain with a hydroxyl group or other desired functional group attached to the 8th or 9th carbon atom and having all atoms in its desired configuration.

When a hydroxyl group is attached to the EHNA side chain, the hydroxyl group can be prepared using conventional chemical synthesis methods described herein to produce a hydroxylated intermediate that can be used in the next step. Thus, the final compound may be replaced or converted by different moieties which make it more lipophilic.

When all of the initial steps are completed, the analog of interest is synthesized, if any, with additional processing steps, for example, by removal of benzyl or other protecting groups. Such groups are commonly used during synthesis and prevent undesired reactions involving the protected components. The final deprotected protected analog is then purified by any suitable means, for example, chromatography, gel electrophoresis or isoelectric focusing.

The particular processing and purification steps used to make a particular analog will depend on the exact molecular structure of the analog of interest. The above steps are within the ordinary skill in the art and various examples of suitable reagents and reactions that can be used for said purpose are described below. In the examples and figures, each major starting reagent or intermediate is indicated by a number in parentheses. For convenience, the number in parentheses is then used to indicate the compound in a subsequent processing step.

Example 1 (below) details the reagents and reactions used in the synthesis of EHNA analogs having a hydroxyl group attached to carbon number 9 in the side chain. Here, 9-hydroxy-EHNA or 9-
This compound, termed OH-EHNA, was converted to compound [10]
Is determined. Its complete chemical name is 9- [2 (S), 9-
Dihydroxy-3 (R) -nonyl] adenine, the synthesis of which is depicted in FIG. The full chemical name includes the word "dihydroxy" because this analog has two hydroxy groups in the side chain. One hydroxy group is attached to the second chiral carbon atom in the same [S] orientation as found in unmodified EHNA. Addition of the other hydroxy group to carbon number 9 produces a 9-OH-EHNA analog.

Example 2 describes the reagents and reactions used in the synthesis of EHNA analogs having a hydroxyl group attached to carbon number 8 of the side chain. Here, 8-
This compound, designated hydroxy-EHNA or 8-OH-EHNA, is designated as compound [23]. Its full chemical name is 9- [2 (S), 8-dihydroxy-3.
(R) -Nonyl] adenine, the synthesis of which is depicted in FIG.

Example 2 also shows that dihydroxylated EHN in which a hydroxy group has been added to both 8 'and 9' carbon atoms (in addition to the standard hydroxy group at carbon number 2).
It also describes the synthesis of compound [14], an A analog. The synthesis is depicted in FIG.

All three of these hydroxylated analogs were prepared in a reversible manner as described in Example 3.
It was shown to inhibit DA activity and have a Ki value in the desired range. Then, as described in Example 4,
They were tested in certain types of in vitro tissue tests, including perfused hearts. In both the ADA inhibition test and the myocardial test, the 9-OH analog showed slightly better potency than the 8-OH analog or the 8,9-dihydroxy analog. The 9-OH analog is the strongest binder of the three and has a Ki value of 3.8 x ^10-9 . The 8-OH analog is the weakest, with a Ki value of 15.8 × 10 ^-9 and 8,9
-The dihydroxy analogue had an intermediate strength with a Ki value of 6.4 x ^10-9 . The 9-OH analog can also be
The OH analog showed a useful protective effect in a myocardial reperfusion assay where the OH analog did not show significant levels, including an undesirable decrease in muscle stiffness.

Thus, the 9-OH analog was identified as a preferred candidate and used as a starting reagent in the synthesis of other analogs (more precisely, the 9-OH compound defined in Example 1 as compound [9]). The benzyl protected precursor of the OH analog was used as starting material, the benzyl group protecting the hydroxy group attached to carbon number 2). If desired, other selected moieties may be substituted for the 9th carbon atom using the same general methods and reagents described herein, and substituting an 8-hydroxy or 8,9-dihydroxy analog instead. Or, in addition, an equivalent lipophilic analog attached to carbon number 8 may be produced.

Alternatively, the synthetic chemist may need to go through a hydroxylated intermediate and use another reagent suitable for one or more of the reactions used to make the hydroxylated derivative (eg, In the reaction of converting compound [8] of Example 1 and FIG. 1 to [9], during the step used to convert and saturate the unsaturated bond between carbons 8 and 9), the side chain It will be appreciated that other non-hydroxy moieties pointing to the 8th or 9th carbon atom can be added. The more straightforward synthetic methods described above will generally yield better yields and require fewer purification steps, so they are generally less than the indirect methods via hydroxyl used during the initial work described herein. Is also preferred.

Nevertheless, relatives of intermediates having a hydroxyl group attached to carbon number 8 or 9 of the side chain (and having a benzyl protecting group attached to the oxygen atom on carbon number 2 by depletion) Because of stability and ease of manipulation, the hydroxyl intermediate pathway has been recognized as a useful route for the synthesis of various analogs that can be produced by substitution or derivatization of hydroxyl groups.

For example, a hydroxyl group at the 8th or 9th carbon atom may be an oxygen-containing group (an aldehyde or ketone group,
(Including carboxylic acid groups, esters or ethers) can be prepared relatively easily using, for example, the techniques disclosed in the Examples or other techniques known to those skilled in the chemical synthesis arts. As another example, a hydroxyl group can be converted to a halide group (eg, a chlorine, fluorine, bromine or iodine atom) by, for example, the method in Example 5 for compounds [28] and [29]. Furthermore, hydroxyl groups can be converted into numerous other groups by known methods.

As an example, the hydroxyl group can be prepared by reacting the hydroxyl with p-toluenesulfonyl chloride (TsCl) to form an O-tosyl group (abbreviated as OTs in the drawings, where tosyl stands for toluenesulfonyl), and then The compound can be converted to an azido group by reacting the compound with sodium azide (NaN ₃ ) to displace the O-tosyl group, leaving an N ₃ group attached to the carbon chain.

Analogs that can be synthesized as described herein include chemical moieties attached to carbon atoms 8 or 9 of the nonyl side chain, including halides, nitrogen-containing moieties such as amines, amides, and the like. There are, but are not limited to, azides, imides or lactams, carboxylic acids or salts thereof, or analogs composed of moieties linked to the 8th or 9th carbon atom by ester or ether linkages. In order to be encompassed by the claims herein, all of the analogs must exhibit properties that render them therapeutically useful as disclosed herein (i.e., generated The analog is about 1
It should have a Ki in the desired range from ^0-7 to about ^10-10 . It must be pharmacologically acceptable. Furthermore, it must be therapeutically useful in protecting tissues from ischemia or hypoxia damage or as an adjuvant with one or more anti-cancer, anti-viral or other agents).

Epoxide [1] can be obtained from Abshanab or the like (1).
984 and 1988). It controls the orientation of substituents at the two chiral carbon atoms in the final EHNA analog, provided by carbons 3 and 4 in the epoxide. E being considered here
To synthesize any of the different stereoisomers of the HNA analog, different epoxide stereoisomers having the desired chiral configuration can be used as starting reagents.

Benzyl group (—CH ₂ C ₆ H ₅ ) bonded to the third carbon atom via an oxygen atom in the starting epoxide
Acted as a protecting group for the oxygen atom. In each final synthesis step of the hydroxylated EHNA analog,
Displacing the benzyl group with hydrogen created a hydroxyl group on carbon number 2 of the side chain. The second hydroxyl group is part of the normal EHNA molecule. If desired, the hydroxyl group can be removed by using a starting epoxide without a protected oxygen atom, or according to the procedures described above, which can be modified during synthesis to form halides, carboxylic acids, esters, ethers, Azide or other groups may be provided. If a moiety is desired at carbon number 1 in the final EHNA analog, it can be provided by using a starting epoxide or a precursor at carbon number 4 of the epoxide having the desired moiety.

The synthetic reactions described herein also involve derivatization of the 4, 5, 6, or 7 carbon atoms in the nonyl side chain (ie, attaching a moiety thereto).
Provide a way. In the synthesis method used here,
Those carbon atoms are provided by the reagent 1-pentenylmagnesium bromide, which has the structure shown in FIG. 1 in a reaction that converts epoxide [1] to compound [2]. The indication 1-pentenyl means
2 shows that the unsaturated double bond is located between the 1st and 2nd carbon atoms in 1-pentenyl magnesium bromide. Those carbon atoms will eventually be the 8th and 9th carbon atoms in the EHNA analogs of this invention. The unsaturated carbon atom in the double bond pentenyl compound became the point of attachment of the hydroxyl group during the reaction for converting compound [8] to compound [9]. Hydroxyl groups were added to both unsaturated carbon atoms, followed by purification of the compound with the hydroxyl moiety at the desired position. In another method, the double bond provided by the pentenyl compound is a compound [1
5] was converted to an epoxide intermediate as shown in FIG.

Using either of these methods, EH
The position of the hydroxyl (or other desired) group in the side chain of the NA analog can be controlled by using a pentenyl magnesium bromide (or similar) compound having a double bond at the desired position. The 2-pentenyl compound has a double bond between its second and third carbon atoms,
They will be the 8th and 7th carbon atoms in the final EHNA analog. The 3-pentenyl reagent (having a double bond between its third and fourth carbon atoms) generates a hydroxyl group attached to the seventh or sixth carbon in the EHNA analog.

FIG. 2 also shows the halogenated analog, compound [21]. In compound [21], a halogen (chlorine) atom was substituted in the adenine ring structure. The chlorine atom was replaced by an amine group during the synthesis of compound [22], but the specific reaction (i.e., replacement of the amine group by a halide atom) can be omitted if desired, so that the halogen moiety has an adenyl ring structure. Will remain. In this attempt, the second benzyl protecting group on the side chain was removed by a non-reducing method.
(Using a reagent such as TMSI)
Removal of halide groups from the adenyl ring structure must be avoided.

The methods used to generate the adenyl structure of the EHNA analogs described herein provide a general method of making various changes to the adenine group.
The adenyl structure may be used to form the heterocyclic compound, 5-amino-4,6, shown in FIG.
-Dichloropyrimidine (ADCP) was obtained and then worked up. By using this same reagent, the compound [20] shown in FIG. 2 was produced. ADCP was attached to the side chain by replacing one of the chlorine atoms of ADCP with an amine group attached to the side chain. Next, a five-membered ring in the adenine structure was closed by forming a carbon bond between two adjacent nitrogen atoms.

If desired, the alternative heterocyclic reagent may be replaced with ADCP
Can be used to produce an EHNA analog with a modified adenine structure, either as a moiety attached to one of the rings, or as a different atom incorporated into either of the rings. Crystally et al. (1988 and 1991)
Report that certain EHNA analogs with modified adenine structures (eg, 3-deaza-EHNA derivatives) are active as ADA inhibitors. The above modifications made to the adenyl structure can be incorporated into analogs of the invention having modified side chains.

As stated above, all three hydroxylated EHNA analogs tested for ADA inhibition (as described in Example 4) were shown to exhibit activity. 9-hydroxy analog (compound [1
0]) was the strongest binder of the three and had a Ki value of 3.8 × 10 ^-9 mol. The 8,9-dihydroxy analog (compound [14]) is the weakest with a Ki value of 15.8 ×.
10 ^-9 , the 8-OH analog (compound [23]) had intermediate strength and a Ki value of 6.4 × 10 ^-9 .

All three of these Ki values fall within the desired range, ranging from about 10 ^-7 to about 10 ^-10 . At one end of the desired range, very effective ADA inhibitors with a Ki value of less than about 10 ^-10 bind very tightly to the enzyme, effectively making the reaction irreversible, There is a risk of "neutralizing" the enzyme. At the other end of the desired range, weak ADA inhibitors with Ki values greater than about 10 ^-7 tend to be unable to achieve the desired level of ADA inhibition due to insufficient potency. They need to be administered in relatively large amounts, and even in large amounts the potency may not be sufficient.

Since the desired range of Ki values is relatively wide, the candidate compound can be administered to a patient in a desired amount by various routes. Analogs having a Ki value in the range of about 10 ^-9 should be administered in relatively low doses, for example, less than about 10 milligrams per kilogram of body weight per day for intravenous injection, In this case, it is about 50 mg / kg / day or less. Low potency analogs having Ki values in the range of about 10 ^-7 can be administered at high doses, for example, when administered orally or injected in response to significant seizure symptoms, about 2
5 mg / kg / day or less, or 20m for intravenous injection
g / kg / day or less. Short-term doses may be somewhat larger as metabolic problems induced by ADA deficiency tend to accumulate slowly.

After testing for ADA inhibition, the hydroxylated EHNA analogs were tested for protection against ischemic injury to the heart using the method described in Example 4. Briefly, in these studies, a heart isolated from a laboratory rat was mounted on a perfusion device, subjected to electrical stimulation to sustain the heartbeat, treated with a candidate drug, allowed to undergo ischemia for a period of time,
It was then evaluated how well the heart could restore its pump function by reperfusion. In these initial assays, 9-OH-EHNA exhibited a higher level of protection than unmodified EHNA in certain parameters, including reduction of unwanted muscle stiffness after ischemia. In a subsequent assay using a somewhat different heart preparation as described in Example 6, the advantage of 9-OH-EHNA was not significant compared to unmodified EHNA. However, other analogs have been generated by the time their subsequent tests were performed, and the results of those other tests are
The other preferred analogs clearly showed that they were substantially better than 9-OH-EHNA or unmodified EHNA in protecting the heart from ischemia or hypoxia damage.

The synthesis of some other analogs using the benzyl protected precursor of the 9-OH analog (compound [9]) as the starting reagent is described in Example 5 and shown in FIG. I have. These analogs include two relatively lipophilic analogs, here 9-chloro-EHNA
(Compound [29]) and 9-phthalimide-EHNA
(Compound [26]). These two analogs showed the best therapeutic results observed to date in protecting both myocardial and brain tissue from ischemic damage.

Since significant quantities were still available after the completion of the first biological test, some additional analogs were also deprotected by Cypros Pharmaceutical Corporation as starting reagents with 9-OH-EHNA
Produced using analogs. One such analog is the silicon-containing analog described in Example 5 (compound [33]). The silicon-containing part was chosen for two reasons. That is, (1) the calculated results indicate that it has very high lipophilicity and could provide a potentially useful test compound to help evaluate that factor, and (2) that it could be deprotected It can be added to the ninth atom in the 9-OH-EHNA molecule, so as not to interfere with the hydroxyl group at the second carbon atom in the side chain.

For convenience, the Ki and oil / water solubility values collected or calculated for the final (deprotected) analog listed in Examples 1, 2 or 5 are compiled in Table 1.
In this table, these analogs are given simple names indicating which type of modifying group was added to the side chain and to which carbon atom it was attached. Full chemical names are given in Examples 1, 2 and 5 and are correlated with compound numbers in parentheses. It should be noted that the Ki values in Table 1 used the extracellular ADA enzyme and did not represent the apparent ability of the lipophilic analog to enter cells more easily and in large quantities.

[0070]

Table 1 Chemical data for various EHNA analogs Compound Modifier Ki value (mol) Log Po / w number x ^10-9 (calculated) --- Unmodified racemic EHNA 6 2.60 ± 0.41 --- Non Modified (+) EHNA 3 2.60 ± 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 [25] 9-benzoyloxy-EHNA 0.2 3.50 ± 0.41 [27] 9-phthalimido-EHNA 2.3 2.86 ± 0.44 [29] 9-chloro-EHNA 3.7 2.28 ± 0.41 [31] 9-carboxymethyl-EHNA 5.0 0.95 ± 0 .41 [32] 8,9-unsaturated EHNA 2.5 2.06 ± 0.41 [33] 9-t-BDPSi-EHNA Not measured 9.46 ± 0.6

Due to the availability of the compounds, a racemic mixture of unmodified EHNA was tested rather than the pure (+) stereoisomer. The (-) isomer is (+) for inhibition of the ADA enzyme.
Because of its much lower potency than the isomer, the Ki value of the pure (+) isomer is almost half that of the racemic mixture (ie, about 3 × 10 ⁻⁹ , rather 6 × 10 ⁻⁹ ). .

The test for measuring the Ki value is carried out by using ADA in a solution.
Requires cell release preparation of the enzyme. By contrast, cell culture studies comparing the potency of the pure (+) isomer to the racemic mixture in cells challenged with ischemic or hypoxic conditions showed that the two preparations (racemic mixture versus pure
The difference in the protective ability of the (+) isomer) is due to the fact that the pure isomer, which has only about 10% efficacy in many tests, rather than having twice the efficacy of the racemic mixture, Cases were shown to be relatively insignificant.
These tests are described in Example 10 and the data is given in Tables 9 and 10.

Example 6 describes a test evaluating the ability of various analogs to protect myocardium from ischemia. The results show that the relatively lipophilic analog (9-chloro-
EHNA and 9-phthalimide-EHNA) are better than unmodified or hydroxylated EHNA
The results showed that the ability to protect the myocardium from ischemic damage was substantially stronger.

Example 7 describes the results of a cell culture test assessing the ability of both EHNA and some analogs to (1) enter human cells and (2) inhibit ADA activity in cells. doing. In these tests, the cells were not stressed or the analogs were not tested for ischemic damage. Instead, in the exam,
Various analogs were evaluated for their ability to reach intracellular enzyme molecules and inhibit their activity. In these studies, erythrocytes (easy to study) and human astrocytoma cells (brain cells that can be regenerated in cell culture. By using them, EHNA analogs reduce ischemic damage in brain tissue) An indicator of whether or not it could help was provided). These results, according to where the lower an IC ₅₀ value is shown, unmodified EHNA or 9-OH @ -
Some EHNA analogs, which are more lipophilic than EHNA, are more resistant to unmodified E in inhibiting ADA activity in cells.
It was shown to be substantially more potent than HNA or 9-OH-EHNA.

Example 8 describes the results of cell culture tests using several different methods to produce ischemic damage in brain cells or blood cells. In some of these tests, toxins such as 2-deoxyglucose or sodium azide were used to prevent respiration and glycolysis. In other tests, a culture medium containing no free oxygen, obtained by blowing nitrogen gas instead of oxygen gas into the cell culture medium, was used. In all of these tests, the cells were subjected to a period of oxygen deprivation (usually lasting several minutes) and then the oxygen supply was restored. After allowing the cells to return to equilibrium in a short period of time, the selected metabolic indicators were evaluated to determine how carefully the cells had regained their proper metabolic rate. The result is EHNA
It has been shown that some analogs, especially lipophilic analogs, can actually protect brain cells from ischemic damage.

Example 9 describes several assays for EHNA analogs that can be used to quantitatively test their ability to protect intact mammalian brain tissue against ischemia. Rather than using excised cultured brain cells as in Example 7, these tests use intact brain tissue sections obtained from the hippocampal region of sacrificed rats. The hippocampal region is used because it is very susceptible to ischemic injury, and the use of intact hippocampal pieces, which can still generate electroencephalograms in response to electrical stimulation, can improve the metabolic rate of isolated cells. The overall organizational function is secured in a better condition. These trials are ongoing. Although final results are not yet available, at least some of the EHNA analogs described herein can significantly and therapeutically reduce ischemia or hypoxic damage in brain tissue (perfused hearts). (Based on the level of protection provided by other tests, including tests and cultured brain cell tests).

Analogs showing promising results in the hippocampal slice test described in Example 9 are further tested in in vivo tests on intact animals. For example, according to the literature described by Nelgard and Wieloch (1992), Buchan and Prusinelli (1990), Michelfelder et al. (1989) and Laniel et al. Alternatively, other methods of inducing local or global ischemia in the test animal brain may be used.

In summary, the Examples, Tables and Figures show that certain EHNA analogs described herein are useful and previously known in protecting myocardial and brain cells from ischemia damage. It shows that there are no therapeutic benefits. The advantages provided by the relatively lipophilic analogs outweigh and exceed those provided by unmodified or hydroxylated EHNA analogs.

Isomers, Salts, and Analogs A group of agents useful for the purposes described herein include isomers (including the “threo” isomer), analogs or salts of the compounds described herein. Is functionally effective as an ADA inhibitor, is pharmacologically acceptable and is therapeutically effective in reducing ischemia or hypoxic damage or delaying the degradation of anticancer, antiviral or other agents. If valid, they are all included. The efficacy of a candidate isomer, analog or salt in inhibiting ADA activity can be tested, for example, using the methods described in Example 3. The therapeutic efficacy of candidate isomers, analogs or salts against ischemia or hypoxic injury can be tested initially using the in vitro methods described in each Example, and candidate compounds that show good efficacy in in vitro tests Can then be further evaluated in an in vivo test.

Initial in vivo tests that can demonstrate ADA-inhibitory efficacy without the need for more difficult tests to assess ischemic damage are usually performed as “probe drugs”
This can be done with nucleoside analogs that are rapidly degraded by ADA. Candidate EHNA analogs can be administered to experimental animals (or humans) that have already or will soon receive a nucleoside probe drug. Appropriate period (depending on drug, usually in the range of 2 to 24 hours)
Thereafter, the amount of the probe agent present in the blood or tissue of the test animal or human can be measured and compared to the amount of the probe agent in an animal or human that has not been treated with the EHNA analog. If the EHNA analog were able to withstand blood flow degradation, enter cells and successfully inhibit ADA, it would increase the level of probe agent that could be detected in the treated animal or human.

The term "pharmacologically acceptable" encompasses properties that make a drug suitable and practical for administration to humans. The compounds must be chemically stable enough to have adequate shelf life under reasonable storage conditions, and they may not be physiologically compatible when introduced into the body by appropriate routes of administration. Must be economically acceptable.

The term “salts” is used herein in its conventional, medical sense. Acceptable salts can include alkali metal salts and addition salts of free acids or free bases. Examples of acids that are widely used to form pharmacologically acceptable acid addition salts include inorganic acids such as hydrochloric, sulfuric and phosphoric acids, and organic acids such as maleic, succinic and citric acids. Alkali metal or alkaline earth metal salts can include, for example, sodium, potassium, calcium or magnesium salts. All of these salts can be prepared by conventional means. Non-toxic and does not substantially interfere with the desired activity,
The nature of the salt chosen is not critical.

The analogs encompassed in the claims are E
Limited to analogs modified at the 8th and / or 9th carbon atom of the HNA (referent molecule) side chain. As used herein, the term "analog" is used in its conventional pharmaceutical sense and refers to the referent molecule (in this case, EH
NA, 9-OH-EHNA or 8-OH-EHNA)
Structurally similar to that described above, but in a targeted and controlled manner, certain substituents on the referent molecule have been replaced by other substituents than hydrogen (9
Replacing the 9th hydroxyl group of -OH-EHNA with a hydrogen atom would result in an unmodified EHNA rather than a true analog of 9-OH-EHNA). Chemical analogs require a "progeny" type of relationship, where analogs are generated by chemical modification of a known compound (often referred to as a parent or referent compound). Therefore, hydroxylated compounds [10], [14]
And [23] are analogs of EHNA,
Are not considered analogs of those hydroxylated compounds.

Similarly, “analogs” as used herein
Does not include a bifunctional conjugate containing an EHNA molecule associated with a second component molecule having distinct and distinct pharmacological activity. For example, if EHNA is bound to a protein, the resulting conjugate will be:
It is not considered an "analog" as used herein. As used herein, the EHNA analogs are relatively small, low molecular weight substances that can be used to improve the known and desired activity of EHNA as potential therapeutic ADA inhibitors. Limited to molecules that are attached to the EHNA structure at the atomic position.

Also, analogs of EHNA modified at the 8th or 9th carbon atom can meet the requirements of pharmacological tolerance, ADA inhibitory potency and therapeutic utility disclosed herein. If present, the analog is AD
A, as long as they retain their ability to inhibit A and have Ki values within the desired range of about 10 ^-7 to about 10 ^-10 (i.e., they are not sufficiently potent to be therapeutically effective). Should not be irreversible "suicide" inhibitors of the ADA enzyme.) It should be noted that they are also encompassed by the claims of the present invention.

Analogs encompassed by this specification include, but are not limited to, those wherein the moiety attached to the 8th or 9th carbon atom of the side chain is a halogen atom, for example:
Analogs consisting of chlorine or fluorine; a carboxylic acid or a salt thereof; a moiety attached to the 8th or 9th carbon atom through an ester or ether bond; or a suitable nitrogen-containing moiety.

Ether-bonded moiety (EH
Alkyl, aryl, alkene and alkyne groups attached to the NA side chain) are expected to be promising groups for further evaluation. Ester-linked moieties are generally more susceptible to hydrolysis than various other chemical structures,
And because it seems to be easily cleaved from the EHNA molecule,
It is not considered the best candidate for evaluation.

Various nitrogen-containing moieties may also be used, provided that EHNA analogs having such moieties are of a suitable standard (eg, the analog must be pharmacologically acceptable and can be stored for weeks or months without the need for freezing). It should have suitable chemical stability and is unstable in human blood, must not degrade rapidly, etc.) and provides suitable candidates for evaluation to determine whether it satisfies. Nitrogen-containing moieties that may be suitable include amine, amide, azide, nitrile, lactam, imine, and imide moieties.

To be encompassed by the teachings and claims herein, any EHNA analog must exhibit the property of producing a therapeutically useful analog as described herein. That is, such an analog must be an ADA inhibitor that is effective, preferably having a Ki value in the desired range of about 10 ^-7 to 10 ^-10 moles; Must be able to do so, including criteria such as non-toxicity and non-carcinogenicity;
For at least one of the two uses described herein (reducing ischemia or hypoxic damage in internal organs, or improving the efficacy of drugs that are degraded by ADA in the absence of ADA inhibitors) Must be therapeutically effective.

If desired, EHNA analogs having other carbon atoms directly attached to carbon number 8 or 9 of the side chain (to make a branched or long side chain) are also provided. The uses described herein can be evaluated. Such analogs are currently not available since Schäfer and Schwender reported the synthesis of some such compounds in 1974, indicating that the nonyl side chain provided the highest level of ADA inhibition. , Is not considered the best candidate for a quick assessment. Nevertheless, EHNA
Further development of analogs may indicate that it is worthwhile to carefully re-evaluate early studies of EHNA variants with long or branched side chains.

The Log P _{O / W} (oil / water partition) coefficient discussed herein can be used alone as a potentially useful indicator for the compounds described in various assays described herein (cell culture studies, perfusion). It is used and intended to suggest that it provides suitable candidates for testing and analysis using cardiac tests (including brain tissue tests). Log P _{O / W} values are not used or intended as strict requirements;
The findings and inventions disclosed herein focus on the following facts: (1) The various EHNA analogs that are modified at position 8 and / or 9 of the side chain carbon atoms include heart and heart. And / or the unmodified form of EHNA already known in preventing and / or reducing ischemic / hypoxic damage in brain tissue or the 8/9 hydrolysis of EHNA already disclosed in US Pat. No. 5,491,146 It has greater potency than either of the analogs; moreover, (2) Applicants have noted that the EHNA analogs with the best efficacy for reducing ischemia and hypoxic damage are the previously disclosed hydrolysis EH
It is more lipophilic than the NA analog.

It should also be noted that the tissue protective efficacy of an analog depends on a combination of factors, rather than on any single factor in the isolated state. Other parameters are
The combination of a low Ki value and a high Po / w value determines either characteristic alone, until it is shown to represent better accuracy in predicting protective benefit against ischemia or hypoxic injury It should be regarded as a more reliable indicator than it is. At this point, based on the tests completed to date, the analogs have (a) a Ki value for adenosine deaminase inhibition of less than about 5 × 10 ^-9 and (b) an octanol / water partition coefficient of at least about 2 It is considered that both should have.

This combination of characteristics is not always a reliable performance index. For example, the unmodified EHNA pure
The (+) isomer is in both desired ranges (Ki = 3 × 10 ⁻⁹ ; L
ogP _{O / W} = 2.6) and still 9-chloro-EHN on protection against ischemia and hypoxic injury
It showed substantially less benefit than either A or 9-phthalimide-EHNA. Thus, the combination of the desired Ki and Log P _{O / W} values should be considered merely as an indicator that the candidate analog is a candidate for further evaluation. Any such candidate compounds must be evaluated in tests involving actual ischemia and / or hypoxia in order to reach a reliable assessment of their ability to reduce ischemia / hypoxia damage .

It was also noted that the 9-benzoyloxy-EHNA analog had the best combination of low Ki and high Po / w values among all the analogs listed in Table 1. In the future, it will be tested in both cell culture and intact tissue tests. It has been tested in assays performed to date because the benzoyloxy group is easily cleaved from EHNA molecules by various mammalian enzymes, thereby converting to 9-OH-EHNA and having relatively low tissue protective efficacy. Did not.

It has also been recognized that different analogs appear to have different activities in different tissues and organs. For example, from the tests completed so far,
9-Chloro-E in protecting myocardium against ischemic injury
The difference between HNA and 9-phthalimide-EHNA is shown to be relatively minor. However,
Phthalimide analogs tended to show significantly better potency in cell culture studies on brain cells. Thus, it is anticipated that one analog may be best for protecting myocardium and another may be best for protecting brain tissue.

Administration of the compounds of the present invention to humans or animals can be by any technique which allows the compounds to be introduced into the bloodstream, including oral administration or intravenous or intramuscular injection routes. The active compounds are usually administered in pharmaceutical preparations, such as aqueous carriers for injection, or capsules, tablets or liquid form for oral ingestion. The above formulation,
It can include a mixture of one or more active compounds in admixture with one or more pharmaceutically acceptable carriers or diluents. When lipophilic drugs are formulated for injection, they are usually mixed with water, a buffer compound (eg, a mixture of carboxylic acids and salts thereof), and an organic compound having multiple hydroxyl groups. Propylene glycol, dextran compounds and cyclodextrin compounds are also frequently used for the above purpose.

If desired, other therapeutic agents, such as anticancer or antiviral nucleoside analogs, may also be present in injectable or ingestible preparations containing the EHNA analogs described above. EHNA analogs can prolong the half-life of anti-cancer or anti-viral drugs (and increase their therapeutic efficacy) by inhibiting the degradation of nucleoside analogs by ADA enzymes, and in particular ADA -Mixtures of EHNA analogs with anticancer or antiviral nucleoside analogs, usually degraded by ADA, mixed with inhibitory EHNA analogs can be very useful.

[0098]

【Example】

Example 1 Synthesis of 9-OH-EHNA analogs This example lists the full chemical names and compound numbers of the starting reagents and intermediates used in the synthesis of 9-OH-EHNA. Its molecular structure is shown in FIG. Synthetic methods are described in detail in the examples of US Pat. No. 5,491,146 (February 13, 1993), the contents of which are incorporated herein by reference. These synthetic methods are also described by Valgies et al.
1994, the teachings of which are also incorporated herein by reference.

Compound 1: (2S, 3S) -3- (benzyloxy) -1,2-epoxybutane Compound 2: (2S, 3S) -2-O-benzyl-2,3-
Nona-8-ene-diol Compound 3: (2S, 3S) -2-O-benzyl-3-O
-Tosyl-2,3-non-8-en-diol Compound 4: (2S, 3R) -3-azido-2-O-benzyl-2-non-8-en-ol Compound 5: (2S, 3R) -3-Amino-2-O-benzyl-2-non-8-en-ol Compound 6: 5-amino-6-chloro-4 [2 (S) -O
-Benzyl-3 (R) -non-8-enyl] aminopyrimidine compound 7: 6-chloro-9- [2 (S) -O-benzyl-3 (R) -non-8-enyl] purine compound 8: 9- [2 (S) -O-benzyl-3 (R)-
Nona-8-enyl] adenine Compound 9: 9- [2 (S) -O-benzyl-9-hydroxy-3 (R) -nonyl] adenine Compound 10: 9- [2 (S), 9-dihydroxy-3
(R) -nonyl] adenine. This is the 9-OH-EHNA analog tested in Example 3.

Example 2 Synthesis of 8-OH-EHNA and 8,9-dihydroxy analogs This example was used to synthesize the full chemical name and 8-OH-EHNA and 8,9-dihydroxy-EHNA. The compound numbers of starting reagents and intermediates are listed. Its molecular structure is shown in FIGS. Synthetic methods are described in detail in the examples of US Pat. No. 5,491,146 (February 13, 1993), the contents of which are incorporated herein by reference. Compound 11: 6-chloro-9 [2 (S) -O-benzyl-8,9-epoxy-3 (R) -nonyl] purine Compound 12: 6-chloro-9 [2 (S) -O-benzyl- 8,9-Dihydroxy-3 (R) -nonyl] purine Compound 13: 9- [2 (S) -O-benzyl-8,9-
Dihydroxy-3 (R) -nonyl] adenine compound [12] was obtained from [11] according to the method described for the preparation of [8], with a yield of 90%. Compound 14: 9- [2 (S), 8,9-trihydroxy-
3 (R) -Nonyl] adenine. This compound is an 8,9-dihydroxy analog of EHNA and was tested as described in Example 3. It has been shown to be a modestly effective inhibitor of ADA activity, but its potency is 9-OH-EH
Since it was lower than the NA analog, it was not tested further. Compound 15: (2S, 3S) -2-O-benzyl-3-
O-Tosyl-8,9-epoxy-2,3-nonanediol Compound 16: (2S, 3S) -2-O-benzyl-3-
O-Tosyl-2,3,8-nonenetriol Compound 17: (2S, 3S) -2-O-benzyl-3-
O-tosyl-8-O-tetrahydropyranyl-2,3,8
-Nonantriol Compound 18: (2S, 3R) -3-azido-2-O-benzyl-8-O-tetrahydropyranyl-2,8-nonanediol Compound 19: (2S, 3R) -3-amino-2 -O-benzyl-8-O-tetrahydropyranyl-2,8-nonanediol Compound 20: 5-amino-6-chloro-4 [2 (S)-
O-benzyl-8-O-tetrahydropyranyl-3
(R) -2,8-dihydroxynonyl] aminopyrimidine compound 21: 6-chloro-9- [2 (S) -O-benzyl-2,8-dihydroxy-3 (R) -nonyl] purine compound 22: 9 -[2 (S) -O-benzyl-2,8-
Dihydroxy-3 (R) -nonyl] adenine Compound 23: 9- [2 (S), 8-dihydroxy-3
(R) -nonyl] adenine. This compound is EHNA 8
-Hydroxy analogue, tested as described in Example 3. It was shown to inhibit ADA activity within the desired range, but was not tested further because its potency was lower than the 9-OH-EHNA analog.

Example 3 Assay for ADA Inhibition Bovine intestine A measured at 30 ° C. by a direct spectrophotometric assay at 265 nm as described by Harriman et al. (1992).
By using DA (Type III, Sigma Chemical Company), compound [10] (9-hydroxy analog), compound [23] (8-hydroxy analog) and compound [14] (8,9-dihydroxy analog) were obtained. Analog)
ADA activity was tested. Because these tests used an extracellular enzyme product, it was not necessary for any of the analogs to enter the cell to reach the enzyme. Table 1
Where the Ki values listed in Table 1 indicate
The 9-OH analog was more potent than any of the other hydroxylated analogs because less was needed to achieve 50% inactivation of the DA enzyme. Because it is more powerful
The 9-hydroxy analog was used as a starting compound for the synthesis of other analogs.

Table 1 summarizes the ADA inhibition data and the octanol-water partition coefficient (lipophilicity index) for all of the final (deprotected) analogs listed in Examples 1, 2 or 5.

Example 4 9-OH-EHN for protection against ischemic damage to tissue
Testing of A After synthesis of the 9-hydroxy and 8-hydroxy analogs of EHNA, Applicants filed samples with the University of
Provided to Dr. Robert Rogers of the Department of Pharmacology and Toxicology in Rhode Island. The amount of 9-OH-EHNA was sufficient and the amount of 8-OH-EHNA was less. Therefore, most studies used 9-OH-EHNA and compared it with unmodified EHNA and disulfiram, an unrelated compound known to have some protective anti-ischemic effect in cardiovascular tissue. .
In a study conducted by Dr. Rogers, "working heart"
A commonly used protocol known as specimen was used. In these tests, laboratory animals (using male Sprague-Dawley rats) are killed and intact hearts are removed and the heart is perfused with a liquid containing a modest amount (or lack of) of oxygen and glucose for a period of time. did. The methods used in these experiments were Davidoff and Rogers,
"Hyper tension" 15: 633-642 (199
0) is described in detail, but with certain minor modifications. Meets the left atrium at 15 cmH ₂ O pressure, the left ventricle was drained to pressure equal to 72mmHg to buffer the packed column in ischemia period was exceptional. The perfusate was HCO ₃ ⁻ (25 mmol), Ca
Krebs-Henseleate buffer containing ⁺⁺ (2.2 mmol) and glucose (10 mmol). 9
When treated with 5% O ₂ and 5% CO ₂ , the pH of the perfusate was 7.4 ± 0.2. 37 ° C perfusion and ambient temperature
The heart beat was left naturally.

After the start of perfusion, the isolated heart was allowed to stabilize for 10 minutes, and then the test agent (unmodified EHNA, 9-OH
-EHNA or disulfiram) or dilute ethyl alcohol (EHNA or 9-hydroxy-EHN)
A used to increase the solubility of A) or dilute dimethyl sulfoxide (used to increase the solubility of disulfiram)
They were treated with buffered saline containing for 10 minutes.

After stabilization and processing, the heart was placed in a simulated ischemic state for 20 minutes. During this period, no oxygen was added to the perfusate. At the end of the ischemic period, oxygen was added again to the perfusion buffer and the following parameters were measured over 10 minutes. LVPP-Left ventricular pulse pressure (calculated as time-dependent pressure, peak pressure-diastolic pressure, in mmHg, millimeters of mercury) LVEDP-left ventricular end diastolic pressure (i.e., for ventricular relaxation during diastolic filling). Corresponding time dependent pressure, mmH
g) CFR-coronary flow velocity (ml / min) HR-spontaneous beating rate (beats / min) ECG-electrocardiogram (surface potential, mV)

The results showed that both EHNA and 9-hydroxy-EHNA reduced the occurrence of fibrillation. The differences between them were not significant. Also, both EHNA and 9-OH-EHNA moderately increased both LVPP and coronary flow rates after ischemia. Also, their effects did not differ from each other at the effective level.

The most important differences observed between EHNA and 9-OH-EHNA in these initial studies were:
Appeared on measurement of LVEDP (left ventricular end diastolic pressure). This parameter indicates whether the muscles of the left ventricular wall can quickly relax after contraction. During diastolic relaxation during systole, said relaxation is essential because the pumping chamber of the heart is filled with blood by rapid relaxation. High levels of LVEDP indicate that the myocardium has stiffened due to ischemic injury and is no longer flexible and elastic enough to adequately fill the pumping chamber with blood during diastolic relaxation. Therefore, it is extremely undesirable.
In these studies, 9-OH-EHNA showed a higher level of protection from muscle stiffness than unmodified EHNA.

8-OH- in these perfused heart preparations
Testing of EHNA analogs was limited because only small quantities were available. However, in those limited studies, the 8-OH-EHNA analog showed 9-OH-EH in reducing left ventricular stiffness and LVEDP.
It was shown to be less potent than NA.

9-OH-EHNA is more potent than 8-OH-EHNA as an ADA inhibitor, and 9-OH-EHN
Since A appeared to have a more beneficial effect in reducing myocardial stiffness, subsequent studies showed that 9-OH-EHNA, compound [1
0] or its benzyl protected precursor, compound [9].

The method used by Prof. Rogers was slightly different from the myocardial test described in Example 6 in some respects, after which Colomed, Inc. (Troy,
(New York), a contract research company. In tests performed by Colomedo under contract with Applicant, Cypros Pharmaceutical Corporation, 9-OH-EHNA showed little or no improvement over unmodified EHNA for myocardial protection, May not show as potent as EHNA.
However, by then, other more lipophilic analogs, including 9-chloro-EHNA and 9-phthalimido-EHNA, had already been synthesized and tested. As described in Example 6, these other analogs showed significant advantages in protecting the myocardium against ischemia compared to EHNA or 9-OH-EHNA. Therefore, subsequent work has been directed to those other analogs, and 9-OH-EHNA and 8-OH-EHNA have not been further actively tested.

Example 5 Synthesis of Other Analogues of EHNA with Improved Anti-Ischemic Properties This example and FIG. 4 describe the synthesis of some additional analogs of EHNA. Except where otherwise noted, the following synthesis reactions used the benzyl protected compound [9] (described in Example 1) as a starting reagent, dried all organic solutions over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Let dry. The ratio of the chromatography solvent is v /
Indicated by v. Silica gel (Davison, grade H, 230-245mesh) suitable for flash column chromatography
purchased from her Scientific. Chromatotron (accelerated centrifugally, preparative thin layer, radial chromatograph) model 73
24T was used to simplify various separations.

The molecular structure was confirmed in a number of ways. Elemental analysis (for comparison of measured versus calculated values) was performed by MHW Labora
tories (Phoenix, AZ). Melting points were measured on a Buchi 535 melting point apparatus and ¹ H NMR spectra were obtained using Varian EM-39.
0, recorded on a Bruker AM-300 or Bruker AM-500 spectrometer. Optical rpm is Perkin Elmer Model 141
Obtained with a digital reading polarimeter. All of these analyzes provided a limited range of data, confirming that each compound was produced in analytically pure form,
However, compounds [26] and [28] were exceptions. These were intermediates, not finished analogs, and were not sufficiently purified.

By using the method described in Example 3,
For ADA enzyme inhibition, the final (deprotected) analogs described herein (compounds [25], [27], [2
9], [31] and [32]) were all found to have the desired range of potency and they
It has been shown that it can inhibit the DA enzyme and does not irreversibly neutralize it. These Ki values are shown in Table 1.

Compound 24: 9- [9-benzoyloxy-2 (S) -O-benzyl-3 (R) -nonyl] adenine A benzoyloxy group bonded to carbon number 2 and a benzyloxy group on carbon number 9 This analog having THF
(5ml) in the compound [9] (314mg, 0.82 mmol), benzoic acid (BzOH, 122mg, 1 mmol) and triphenylphosphine (PPh _3, 262
mg, 1 mmol) in a stirred solution of N, N-diisopropylazo-dicarboxylate (DIAD, 202
mg, 1 mmol). The mixture was stirred at room temperature for 24 hours and the precipitated triphenylphosphine oxide was filtered. The filtrate is concentrated and the residue is chromatographed on silica gel using ethyl acetate (EtOAc) to give [24], 9: 1
Further purification by preparative thin-layer chromatography (TLC) using EtOAc and methanol (MeOH). The yield was 250 mg (63%).

Compound 25: 9- [9-benzoyloxy-2 (S) -hydroxy-3 (R) -nonyl] adenine This alcohol having a benzoyloxy group bonded to the ninth carbon atom is ethanol (EtOH, 18 ml). )) And compound [24] (20) in cyclohexane (6 ml).
0 mg) in 20% palladium hydroxide-carbon (PdOH ₂
/ 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 under reduced pressure. The residue was chromatographed on silica (EtOAc and MeOH, 9:
1), pure [25], 130 mg (80
%)was gotten.

Compound 26: 9- [2 (S) -O-benzyl-9-phthalimido-3 (R) -nonyl] adenine Compound [26] was prepared in the same manner as in the case of compound [24]. Was produced in 87% yield from
Phthalimide (1 mmol, 147) instead of benzoic acid
mg) was used. Since the compound always contains trace amounts of triphenylphosphine oxide and dihydro-DIAD, no analytically pure compound was obtained. It was used as a reagent in the next step, producing compound [27].

Compound 27: 9- [2 (S) -hydroxy-9-phthalimido-3 (R) -nonyl] adenine This alcohol is the same palladium on carbon (PdOH ₂ / C) Prepared in 85% yield using catalytic method and using [26] as starting reagent (255 mg, 0.5 mmol). Compound 28: 9- [2 (S) -O-benzyl-9-chloro-3 (R) -nonyl] adenine

A benzyl ring at the 2nd carbon atom and 9
Analogs with chlorine atoms bound to carbon atom [28]
It is anhydrous CCl ₄ in (5ml) [9] (500mg , 1.
3 mmol) and NaHCO ₃ (50 mg) PPh ₃ To a stirred solution consisting of (400 mg, prepared by adding 1.5 mmol). The mixture was refluxed for 12 hours, then filtered and the filtrate was concentrated. The residue was chromatographed on silica gel (EtOAc) to give 38
0 mg (75%) was obtained. Since the compound contained trace amounts of triphenylphosphine oxide, no analytically pure compound was obtained. It was used as a reagent in the next step to produce compound [29].

Compound 29: 9- [9-chloro-2 (S)
-Hydroxy-3 (R) -nonyl] adenine With a halide moiety at carbon 9 and an alcohol group at carbon 2 this analog is the same palladium on carbon (PdOH ₂ / C) By using a catalytic method and using [28] as the starting reagent,
Produced in 82% yield.

Other halide analogs involving fluorine, bromine or iodine atoms can be produced, if desired, by appropriate choice of reagents containing said atoms. For example, the synthetic method described for compound [28] can be modified by using CBr ₄ or CI ₄ instead of CCl ₄ . As another example, a 9-fluoro-EHNA analog is
It can be prepared by reacting compound [9] (a benzyl protected 9-OH-EHNA analog) with the well-known fluorinating agent diethylaminosulfur trifluoride (DAST).

Compound 30: Methyl-7 (R) -adenin-9-yl) -8 (S) -O-benzyl-nonoate Analog having an ester group at the 9th carbon and a benzyl group at the 2nd carbon [30] Is dimethylformamide (DMF,
[9] (1.369 g, 3.4 mmol) in 2 ml) was added to pyridinium dichromate (PDC, 2.7).
55 g, 7.3 mmol). The mixture was stirred at room temperature for 24 hours, then diluted with ethyl acetate and passed through a mixture consisting of silica gel and Na ₂ SO ₄ (1: 1) to give the corresponding acid (220 m 2).
g, 15.7% yield). The acid was converted to the methyl ester by using methyl alcohol and sulfuric acid, followed by neutralization and drying.

Compound 31: Methyl-7 (R) -adenin-9-yl) -8 (S) -hydroxy-nonoate An analog [31] having an ester group at the 9th carbon and a hydroxy group at the 2nd carbon is using benzyl analogs [30] as a starting reagent, produced in 82% yield using the same palladium-carbon (PdOH ₂ / C) catalyst methods as those used for the preparation of [25]. Ester 31 is also 9- [2
(S) -hydroxy-8-carboxymethyl-3 (R)
-Nonyl] adenine.

Compound 32: 9- [2 (S) -Hydroxy-3 (R) -non-8-enyl] adenine The unsaturated analog [32] is shown in FIG. Prepared by using the described unsaturated benzyl protected analog [7]. Toluene (10
Compound [7] (200 mg, 0.55 mmol) in (ml) was cooled with dry ice / acetone and ammonia was bubbled through the solution until the volume of the mixture reached 40 ml. Sodium metal was added in portions with vigorous stirring until the mixture became a strong blue color. After stirring for 2 hours,
The mixture was neutralized with NH ₄ Cl and methanol and concentrated to dryness. Then the compound was extracted with CH ₂ Cl _2, the extracts were dried over Na ₂ SO _4, and evaporated. The product was purified by preparative thin layer chromatography using ethyl acetate to give [32] in 70% yield.

Compound 33: 9- [9-tert-butyldiphenylsilyloxy-2 (S) -hydroxy-3 (R)-
Nonyl] adenine Notable additional analogs were available after the completion of the first biological test of 9-OH-EHNA, so additional compounds having a silicon-containing group attached to the 9th carbon atom, Synthesized using deprotection of 9-OH-EHNA as starting material. The silicon-containing moiety has (1) calculated that it has a very high level of lipophilicity (compared to 2 or 3 for the chloro and phthalimide analogs,
9 (logPo / w values in the range of 9), which have been shown to be able to provide potentially useful compounds useful for assessing high levels of lipophilicity, and
The choice was made for two reasons: it could be added to the 9th atom in the deprotected 9-OH-EHNA molecule without interfering with the unprotected hydroxyl group at the carbon atom.

Therefore, compound [10] (30 mg, 0.1 mg) was obtained.
102 mmol) in 0.5 ml of DMF
It was added to a solution consisting of 50 μl diisopropylethylamine and 15 mg dimethylaminopyridine in 1 CH ₂ Cl ₂ . To this solution was added 40 mg (0.14 mmol) of t-butylchlorodiphenyl-silane and the mixture was stirred at room temperature for 16 hours. The solvent is evaporated and the product is chromatographed on silica gel (10% C
Purification by H ₃ OH / CHCl ₃ ) gave 26 mg (4
8%) 9-t-BDPSi-EHNA compound [33]
was gotten.

Example 6 Protection of Myocardium Against Ischemia Using the so-called "Langendorff Heart Specimen", testing some of the analogs described in Example 5 to evaluate their ability to protect myocardium from ischemia did. Briefly, male Sprague-Dawley rats weighing 250-350 grams were anesthetized with heparin sodium,
Killed by CO ₂ . The heart is quickly removed by thoracotomy,
Placed in saline (PSS) until contraction ceased. The heart is then attached to the cannula via the base of the aorta and at 80 mm Hg, 37 ° C., NaCl (118 in mmol, the same applies hereinafter), KCl (4.7), CaCl _2.
(2.2), KH ₂ PO ₄ (1.18), MgSO ₄ (1.1
7), NaHCO ₃ (25), dextrose (11)
Perfused retrograde with PSS containing. 95 perfusion solution
The pH was maintained at 7.4 by aeration with% O ₂ /5% CO ₂ . The heart was allowed to equilibrate for 15 minutes, during which time a balloon-tipped catheter was introduced into the left ventricular lumen through a small incision made in the left atrium. The catheter is connected to a pressure transducer and the left ventricular hemodynamic capabilities, ie, left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVED).
P), left ventricular pressure (LVDP), + dP / dtmax
(Maximum velocity when pressure is generated in the left ventricle during each contraction),-
It was used to measure dP / dtmax (the maximum rate at which left ventricular pressure falls after each contraction) and heart rate. After placing a catheter fitted with a balloon at the tip, a coronary artery effluent was collected by inserting a cannula into the pulmonary artery, and coronary artery flow was measured.

At the end of the stabilization period, measurements of left ventricular hemodynamic performance, heart rate and coronary flow were taken. The heart was then perfused with PSS containing vehicle, unmodified EHNA (racemic), 9-chloro-EHNA or 9-phthalimide-EHNA for 10 minutes and the measurement was repeated. Global ischemia was generated by clamping the aortic cannula, and measurements of these parameters were taken at 5 minute intervals. 35 minutes after global ischemia, the heart was reperfused with PSS at 80 mm Hg pressure for 20 minutes. Measurements were taken again at 5 minute intervals during the reperfusion period.

The results in Table 2-4 and FIGS. 5-7 show that the more lipophilic analogs have substantially better ability to protect myocardium from ischemic damage than unmodified EHNA or 9-OH-EHNA. I understood that.

Example 7 Cell culture studies, unstressed cells In a first series of cell culture studies, human erythroid cells (relatively easy to study) and human astrocytoma cells (which can be regenerated in cell culture) Brain cells, which have been used to provide an indication of whether an EHNA analog can promote a reduction in ischemic damage in brain tissue. Analogs were evaluated for their ability to inhibit ADA activity.

The first series of tests consisted of "normox (normox
ic) "conditions (ie, cells were not stressed by hypoxia or simulated ischemia with deoxyglucose or sodium azide) and were associated with EHNA and some analogs. IC ₅₀ values were measured. The IC ₅₀ value is defined as A
Shows the drug concentration required to inhibit half DA activity. This value indicates the ability of the drug to enter the cell and reach the ADA enzyme, and to bind to the ADA enzyme inside the cell,
It reflects both the efficacy of the drug in inhibiting it.
Low IC ₅₀ values indicate that the drug is a potent ADA inhibitor and can easily enter cells.

To perform these tests, cell populations were preincubated with EHNA or EHNA analogs at various concentrations for one hour. The cells are then treated with 10 micromolar of 5-micron, which inhibits the activity of a different enzyme called adenosine kinase, which adds a phosphate group to adenosine.
Incubated with iodotubercidin for 30 minutes. This step ensured that adenosine levels were not altered by phosphorylation pathways that could consume adenosine in intact cells. Next, cells are removed for 30 minutes.
Incubated with 00 micromolar radiolabeled adenosine. Thirty minutes later, the concentrations of radiolabeled inosine and hypoxanthine (a molecule produced when the ADA enzyme degrades adenosine) were measured in cell culture medium after separation using cellulose thin-layer chromatography. Several concentrations of EHNA or EHNA analog were used in each series of tests, and the IC ₅₀ for each compound was calculated based on the dose response curve for that compound.

The results in Table 5 show that 9-chloro-EHN
A was substantially more potent than unmodified EHNA or 9-OH-EHNA when inhibiting ADA activity inside red blood cells (indicated by lower inhibitory concentration values), and 9-phthalimide -Shows that EHNA was substantially more potent than unmodified EHNA or 9-OH-EHNA when inhibiting ADA activity inside brain (astrocytoma) cells. The IC ₅₀ values in this table show the standard deviation after the mean.

Table 5 Inhibition of ADA activity in cell culture tests Cell type / compound IC50 (μM) Red blood cell EHNA (racemic) 1.200 ± 0.70 9-OH-EHNA compound [10] 2.100 ± 0. 909 9-Chloro-EHNA compound [29] 0.220 ± 0.14 Astrocytoma cells EHNA (racemic) 3.0 ± 2.5 9-OH-EHNA [10] 3.5 ± 0.8 9- Chloro-EHNA compound [29] 4.8 ± 1.9 9-phthalimido-EHNA compound [27] 1.1 ± 0.9 8,9-unsaturated-EHNA compound [32] 2.5 ± 0.8

Example 8 Cell Culture Tests for Ischemic Protection In a second series of cell culture tests, cells were stressed by one of two methods: simulating hypoxia or ischemic damage. In these studies, one population of astrocytoma cells containing radiolabeled ATP was incubated with EHNA or analog for 60 minutes. Then, to interrupt glycolysis and respiration, 2-deoxyglucose or sodium azide (5.5 mM final concentration for any toxin) was added and the cells were incubated for 60 minutes. After incubation, the cultures were tested to determine how much of them released radiolabeled adenosine into the medium. Adenosine release is a normal and proper metabolic function of cells during ischemia or hypoxia, and was similarly stressed by the same toxin as the amount of adenosine released by EHNA-treated or analog-treated cells, The amount of adenosine released by control cells without any treatment with EHNA or analog was compared. The results in Tables 6 and 7 are expressed as percentage of adenosine released by treated cells compared to untreated control cells.

Table 6 Adenosine release by astrocytoma cells during hypoxia simulation (stressed by deoxyglucose) Protective agent Percent of control value Untreated = 100% (baseline) EHNA (racemic) 339 ± 25 9− OH-EHNA [10] 289 ± 12 9-Chloro-EHNA compound [29] 375 ± 259 9-phthalimido-EHNA compound [27] 559 ± 198,9-Unsaturated-EHNA compound [32] 300 ± 489- Butyldiphenylsilyloxy-EHNA [33] 244 ± 21

[0134]

Table 7 Adenosine release by astrocytoma cells under normoxic and hypoxic simulations Protective agent (μM, condition) Percent of control value Untreated = 100% (baseline) EHNA (racemic) 0.01 Normal Under oxygen 99 ± 10 0.1 Under normal oxygen 92 ± 8 0.01 Under stress 139 ± 7 0.1 Under stress 253 ± 21 9-phthalimide-EHNA compound [27] 0.01 Under normoxic 102 ± 11 0.1 1 Under normoxia 111 ± 13 0.01 Under stress 242 ± 9 0.1 Under stress 425 ± 408 8,9-unsaturated-EHNA compound [32] 0.01 Under normoxia 115 ± 22 0.1 Under normoxia 119 ± 12 0.01 Under stress 159 ± 11 0.1 Under stress 225 ± 31

These results indicate that 9-chloro and 9-phthalimide EHNA analogs have a greater ability to stimulate adenosine release during hypoxic injury in brain cell cultures than unmodified EHNA.

In addition, the lack of significant effect on cells cultured under normoxic conditions indicates that EHNA does not destroy the normal metabolic activity of unstressed cells. That's important. EHNA is active only in cells that are exclusively under stress.

In a third series of cell culture tests, astrocytoma cells were placed under actual hypoxic conditions for 2 hours in an anaerobic chamber and nitrogen gas (not oxygen) was blown into the cell culture medium. The release of radiolabeled adenosine into the culture medium after a period of hypoxia (Table 8) was measured.

Table 8 Adenosine release by brain cells after 2 hours of hypoxia Protective agent (μM) Percent of control value Untreated = 100% (baseline) EHNA (racemic) 0.05 257 ± 34 0.5 769 ± 67 9-phthalimide-EHNA 0.05 386 ± 51 0.5 866 ± 83

These results indicate that EHNA and its 9-
It has been shown that phthalimide analogs enhance adenosine release under both simulated and non-simulated hypoxic conditions, and that phthalimide analogs exhibit an even higher level of protection. This ability to promote the maintenance of normal metabolic function in brain cells in the face of an ischemic attack indicates that phthalimide analogs can help protect brain tissue against ischemic damage.

Example 9 Additional Assays for Protection of Brain Tissue Various assays are currently in progress, and some EHNA analogs have been shown to reduce hypoxic or ischemic damage in intact brain tissue, as distinct from cultured cells. Quantifies the ability to reduce In the case of these tests, the applicant, Cypros
Sponsored and funded by Pharmaceutical Corporation, it is being conducted by unrelated researchers at the UCLA Medical Center in Los Angeles.

In the currently performed in vitro assays,
Intact hippocampal explants from the brains of sacrificed rats are used for two reasons. First, tissue collected from the hippocampal region of the brain is known to be very vulnerable to ischemic damage. And secondly, from intact connective tissue (including other types of supporting cells such as neurons and glial cells), destructed cell debris or excised cells cultured under artificial conditions (especially those cells are cancerous Perfused intact brain tissue pieces, often resulting in better models of in vivo cell and tissue behavior than either Tests using are often preferred in neurological studies.

When properly perfused, the so-called "CA1" neurons in the hippocampal slices are capable of producing an electrophysiological response (in living animals) that can be measured quickly and easily using a device equivalent to an electroencephalograph for several hours. Equivalent to the brain wave of Thus, by testing a candidate neuroprotective agent, it prolongs, restores, or otherwise increases the ability of nerve cells in a perfused hippocampal slice to generate electrical spikes having normal and desirable amplitudes and frequencies. Can be found (as distinguished from seizure-inducing convulsants that induce spikes with abnormal amplitudes or frequencies). If hippocampal slices could restore or prolong their normal electrophysiological responses despite the ischemic attack by the candidate drug, this would in fact indicate that the candidate drug had substantial protective activity against ischemic damage to brain tissue Provide strong and direct evidence of having The above test is widely used and accepted, and various references such as Bitingham et al. (1984) and Shuul and Rigger (1)
992).

The following protocol is described by Wallis et al. (1992)
In more detail. Briefly, Sprague-Dawley rats are anesthetized with halothane and then decapitated. The brain is quickly removed and placed in cold artificial cerebrospinal fluid (CSF) for 1 minute. This cerebrospinal fluid contains NaCl, 12
6 (mmol, the same applies hereinafter), KCl, 4.0, KH ₂ PO
_{4, 1.4, MgSO 4, 1.3} , CaCl 2, 2.4, Na
Contains HCO ₃ , 26 and glucose 4.0, pH
Is 7.4 and is saturated with a gas mixture consisting of 95% O ₂ and 5% CO ₂ . Next, a hippocampal tissue slice obtained by slicing the cooled brain tissue is placed in a self-recording well, and the temperature of the surrounding bath is controlled to 34 ° C. by a thermostat.

One hour after placing the slices in the wells, the forward CA1 population spike (PS) is measured for each tissue slice. This output, indicative of cellular function, is induced in response to electrical stimulation using a kinked bipolar electrode placed on the CA3 shuffer collateral. The response is recorded using a tungsten electrode inserted into the pyramidal layer of CA1. By adjusting the current intensity (at the stimulating electrode) and the electrode depth (for the measuring electrode), the maximum amplitude of the CA1 spike is achieved. Only slices showing a forward CA1 PS of 3 mV or greater at the initial evaluation are used for further tests.

In control samples, the tissue pieces are soaked in an artificial CSF solution without any EHNA analogs or other neuroprotective agents. Test samples are processed similarly, but the CSF fluid contains known concentrations of EHNA or an EHNA analog described herein.

In these assays, hippocampal explants are exposed to hypoxic conditions for a limited period of time, and then the ability of CA1 cells to respond to electrical stimulation after the hypoxic period is measured.
To initiate hypoxia, paired hippocampal slices (ie, two tissue sections taken from the same animal) were placed in two autologous wells, and the perfusate in both wells was added to free oxygen-free artificial CSF. Change. The CSF solution is saturated with 95% N ₂ (instead of O ₂₎ and 5% CO _2.

One piece of each pair is exposed 30 seconds before the onset of oxygen tension reduction and further exposed to EHNA or an EHNA analog and continued until 15 minutes after the end of oxygen pressure reduction. The deficiency period due to hypoxia varies with different slices. During hypoxia, each control explant (ie, EHNA or EHN
Monitoring of each slice that has not been treated with the A-analog) provides confirmation that the "potential hypoxic injury" (HIP, Fairchild et al., 1988) is still present.

5 minutes after disappearance of HIP in untreated slices
Addition of oxygen to the perfusion medium terminates the depletion due to reduced oxygen pressure in the paired slices. Then 2
The paired slices are monitored for an additional 90 seconds and the EEG amplitude is recorded. The final CA1 forward PS amplitude is then compared to the original CA1 forward PS amplitude measured before processing. In addition, the backward PS amplitude is evaluated before oxygen pressure drop and 90 minutes after recovery.

In some tests, hippocampal tissue sections are electrically stimulated every 30 seconds throughout the perfusion period, including the hypoxic period. In these tests, continued periodic stimulation forces additional metabolic demands, which exacerbates toxin-stimulated injury and makes the test more precise.

Data collected from the stimulus slices, monitored for mean PS recovery throughout hypoxia, is analyzed using the Student's t-test. Data collected from unstimulated slices, evaluated for CA1 orthodrome and antidromic PS amplitude only at the beginning and end of the experiment, are analyzed using the Wilcoxon rank-to-count test.

Due to the design of these test protocols, the CA1 injury induced by hypoxia in this assay was strict and usually absolutely reliable for non-medicated unstimulated strips and was therefore used for comparison purposes. A useful baseline is provided.

The first assay completed to date shows that unmodified EHNA provides neuroprotection against ischemia at an EC ₅₀ level of 10 micromolar (ie, the molar concentration that results in an average 50% recovery level). . The 9-hydroxy analog does not provide significant protection at concentrations up to 50 μM. The benzoyloxy analog (compound [25]) was unprotected at concentrations up to 20 μM and the 8,9-didehydro analog was
Provides protection at a concentration between 10 and 20 μM. The 9-phthalimide analog provides the best protection to date, with an EC ₅₀ concentration of about 7.5 μM.

Certain EHNA analogs that substantially reduce hypoxia or ischemic injury in these relatively simple and inexpensive in vitro tests are then described, for example, in Nergard and Biloch (1992) (Surgical studies in rats). Induced ischemia), Buchan and Prusinelli (1
990) (surgical ischemia in gerbils), Michelen Felder et al. (1989) (surgical ischemia in dogs) and Laniel et al. (1988) (cervical tourniquets in primates). Can be used or tested in intact animals using other protocols known to those skilled in the art.

Example 10: Racemic (±) EHNA vs. (+) E
Comparison of HNA Due to changes in the status of compound availability, the test described here uses a racemic mixture of (±) EHNA as the target compound, while other tests are more effective than the (-) isomer as ADA inhibitors The (+) stereoisomer was used. To make a good comparison of the potential different activity levels of these different types of controls, some direct comparisons of the racemic mixture with the (+) isomer in protection against hypoxia or ischemic injury The test was performed. To date, the only tests performed have been cell culture tests using human astrocytoma (HAC) cells, as described in Examples 7 and 8. Studies performed to date have not shown a significant difference in ischemia / hypoxia protection between the racemic mixture and the pure (+) isomer.

The first test involves measuring the ADA activity of intact HAC cells. These tests are based on the measurement of radiolabeled inosine and hypoxanthine concentrations and are produced by cells using the method described in Example 7 after adding radiolabeled adenosine (100 μM final concentration) to the cells, Including the use of Indian tubers. These tests used various concentrations of EHNA (either racemic or (+) isomer) and were added shortly before the onset of hypoxia in which the cells were subjected to an anoxic chamber. After 2 hours of hypoxia, the authors measured inosine and hypoxanthine concentrations and determined percent inhibition as EHN.
Determined by comparison to control cells treated simultaneously with the level of A-treated cells but not exposed to EHNA.

Table 9 shows the results. The (+) isomer shows slightly better results than the racemic mixture, but the difference is not significant. Further testing was performed at a 0.005M concentration but the results were exceptional and were removed as unreliable.

TABLE 9 ADA inhibition by racemic or (+) EHNA in hypoxic astrocytes ADA inhibition (%) EHNA concentration (μM) Racemic (+) isomer Improvement 0.05 39.1 42.2 8% 0 .25 71.6 76.7 7% 0.5 83.9 82.7 -1% 1.0 81.6 83.1 2%

The second test involves the measurement of adenosine released from astrocytoma cells after 2 hours of hypoxia under nitrogen gas in an anoxic chamber, as described in Example 8.
Control cells were treated and measured immediately, but not with EHNA. The data is shown in Table 10. As mentioned above,
The test was also performed at 0.005M, but the results were exceptional and were removed as unreliable.

[0157]

Table 10 Adenosine release by hypoxic astrocytes Inhibition of Ado release (% of control concentration) EHNA concentration (μM) Racemic (+) isomer improvement 0.05 128 129 <1% 0.225 128 181 41% 0.5 156 181 16% 1.0 182 214 18%

As mentioned above, the (+) isomer works somewhat better; however, the level of effect of the (+) isomer is
On average, it was not 20% higher than the racemic mixture.

Thus, a new class of EHNA analogs useful for reducing ischemia or hypoxic injury and delaying the metabolic degradation of useful anticancer and antiviral agents is disclosed and described. While this invention has been illustrated and described with reference to certain embodiments for purposes of description and description, it will be apparent to those skilled in the art that various modifications and variations of the described embodiments are possible. It should be. It is intended that the present invention encompasses any modifications that come directly from the teachings of this specification and do not depart from the spirit and scope of the invention.

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[Brief description of the drawings]

FIG. 1: 9′-hydroxy (+)-EHNA defined as compound [10], ie EH with even higher lipophilicity
FIG. 3 shows a series of chemical reactions used to generate intermediate compounds used to generate other subsequent analogs of NA.

FIG. 2 shows the reaction used to generate 8′-hydroxy (+)-EHNA designated compound [23].

FIG. 3 shows the reaction used to generate 8 ′, 9′-dihydroxy (+)-EHNA, designated compound [14].

FIG. 4 illustrates the reaction used to generate analogs of EHNA containing various non-hydroxy moieties attached to carbon number 9 (see Example 5).

FIG. 5: 9-Chloro-EHNA (compound [29]) and 9-phthalimido-EHNA (compound [27])
Is a bar graph showing that the test described in Example 6 was more effective in protecting myocardium from ischemic damage than unmodified EHNA or 9-hydroxy-EHNA.

FIG. 6: 9-chloro-EHNA (compound [29]) and 9-phthalimide-EHNA (compound [27])
Is a bar graph showing that the test described in Example 6 was more effective in protecting myocardium from ischemic damage than unmodified EHNA or 9-hydroxy-EHNA.

FIG. 7: 9-chloro-EHNA (compound [29]) and 9-phthalimide-EHNA (compound [27])
Is a bar graph showing that the test described in Example 6 was more effective in protecting myocardium from ischemic damage than unmodified EHNA or 9-hydroxy-EHNA.

 ──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Paul Jay Marangos 22224 Running Spring Place, Entinitas, California, USA

Claims (28)

[Claims] 1. The following molecular structure: Wherein at least one of R 1 and R 2 is a chemical moiety other than a hydrogen atom or a hydroxyl group, wherein a chemically modified analog of erythro-hydroxynonyl adenine is administered to humans and experimental animals If pharmacologically acceptable and non-toxic, (b) in cell culture tests, from erythro-hydroxynonyl adenine or 9- [2 (S), 9-dihydroxy-3 (R) -nonyl] adenine Can even more effectively enter human cells and inhibit the intracellular enzymatic activity of adenosine deaminase in a reversible manner, (c) erythro-hydroxynonyl adenine or 9-
More effectively reduces hypoxia and ischemic injury in at least one type of mammalian tissue selected from the group consisting of myocardial and brain tissues, more effectively than [2 (S), 9-dihydroxy-3 (R) -nonyl] adenine Comprising a chemically modified analog of erythro-hydroxynonyl adenine. 2. The method of claim 1, wherein the chemically modified analog of erythro-hydroxynonyl adenine has a Ki value for adenosine deaminase inhibition in the range of about 10 ^-7 to about 10 ^-10 mole. 3. The method of claim 1, wherein the chemically modified analog of erythro-hydroxynonyl adenine comprises: (a) a Ki value for adenosine deaminase inhibition of less than about 5 × 10 ⁻⁹ mole; 2. The medicament of claim 1, having an octanol / water partition coefficient of about 2. 4. At least one of R1 and R2 comprises: a. A halide atom, b. An ester linking group capable of providing a chemically modified analog of erythro-hydroxynonyl adenine having a Ki value for adenosine deaminase inhibition that is in the range of about ^10-7 to about ^10-10 moles; and c. The chemically modified analog of erythro-hydroxynonyl adenine is selected from the group consisting of an ether linking group capable of having a Ki value for adenosine deaminase inhibition in the range of about ^10-7 to about ^10-10 mole. Item 10. The drug according to Item 1. 5. The method of claim 1, wherein at least one of R1 and R2 is a nitrogen-containing moiety selected from the group consisting of amine, amide, azide, nitrile, lactam, imine, and imide moieties. 6. An agent comprising a chemically modified analog of erythro-hydroxynonyl adenine containing an adenyl ring structure attached to a nonyl side chain, wherein the side chain has at least one chemical moiety other than a hydroxyl moiety. A chemically modified analog of erythro-hydroxynonyl adenine, which is modified by attaching to the side chain a carbon atom selected from the group consisting of carbon atoms 8 and 9 of the nonyl side chain; And pharmacologically acceptable and non-toxic when administered to experimental animals; (b) erythro-hydroxynonyl adenine or 9- [2 (S), 9-dihydroxy-3 (R ) -Nonyl] adenine can more effectively enter human cells and inhibit the intracellular enzymatic activity of adenosine deaminase in a reversible manner; Littrow - hydroxy nonyl adenine or 9-
More effectively reduces hypoxia and ischemic injury in at least one type of mammalian tissue selected from the group consisting of myocardial and brain tissues, more effectively than [2 (S), 9-dihydroxy-3 (R) -nonyl] adenine A drug that is therapeutically effective to 7. A chemically modified analog of erythro-hydroxynonyl adenine comprising: (a) a Ki value for adenosine deaminase binding that is less than about 5 × 10 ⁻⁹ mole; and (b) a logarithmic value of base 10 at least. 7. The medicament of claim 6, having an octanol / water partition coefficient of about 2. 8. At least one of R1 and R2 comprises: a. A halide atom, b. An ester linking group capable of providing a chemically modified analog of erythro-hydroxynonyl adenine having a Ki value for adenosine deaminase inhibition that is in the range of about ^10-7 to about ^10-10 moles; and c. The chemically modified analog of erythro-hydroxynonyl adenine is selected from the group consisting of an ether linking group capable of having a Ki value for adenosine deaminase inhibition in the range of about ^10-7 to about ^10-10 mole. Item 7. The drug according to Item 6. 9. The method of claim 6, wherein at least one of R1 and R2 is a nitrogen-containing moiety selected from the group consisting of an amine, amide, azide, nitrile, lactam, imine, and imide moiety. 10. A method of inhibiting adenosine deaminase activity in a mammalian patient in need of treatment, comprising:
The patient has the following molecular structure: Wherein at least one of R 1 and R 2 is a chemical moiety other than a hydrogen atom or a hydroxyl group, wherein a chemically modified analog of erythro-hydroxynonyl adenine is administered to humans and experimental animals If pharmacologically acceptable and non-toxic, (b) in cell culture tests, from erythro-hydroxynonyl adenine or 9- [2 (S), 9-dihydroxy-3 (R) -nonyl] adenine Can even more effectively enter human cells and inhibit the intracellular enzymatic activity of adenosine deaminase in a reversible manner, (c) erythro-hydroxynonyl adenine or 9-
More effectively reduces hypoxia and ischemic injury in at least one type of mammalian tissue selected from the group consisting of myocardial and brain tissues, more effectively than [2 (S), 9-dihydroxy-3 (R) -nonyl] adenine Including a moiety that renders it therapeutically effective to treat a erythro-hydroxynonyl adenine. 11. The method of claim 10, wherein the patient is receiving a useful therapeutic agent 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 from ischemia or hypoxic brain injury. 13. The method of claim 10, wherein the patient is receiving a useful therapeutic agent that is degraded by adenosine deaminase in the absence of an adenosine deaminase inhibitor. 14. A chemically modified analog of erythro-hydroxynonyl adenine comprising: (a) a Ki value for adenosine deaminase inhibition that is less than about 5 × 10 ^-9 moles; The method of claim 10 having an octanol / water partition coefficient of about 2. 15. At least one of R1 and R2
One is: a. A halide atom, b. A chemically modified analog of erythro-hydroxynonyl adenine, which can have a Ki value for adenosine deaminase inhibition that is in the range of about 10 ^-7 to about 10 ^-10 mole, and d. The chemically modified analog of erythro-hydroxynonyl adenine is selected from the group consisting of an ether linking group capable of having a Ki value for adenosine deaminase inhibition in the range of about ^10-7 to about ^10-10 mole. Item 11. The method according to Item 10. 16. At least one of R1 and R2 is
11. The method of claim 10, wherein the nitrogen-containing moiety is selected from the group consisting of an amine, amide, azide, nitrile, lactam, imine, and imide moiety. 17. A method of inhibiting adenosine deaminase activity in a mammalian patient in need of treatment, comprising:
The patient is provided with a chemically modified analog of erythro-hydroxynonyl adenine containing an adenyl ring structure attached to the nonyl side chain, wherein the side chain has at least one chemical moiety other than the hydroxyl moiety at position 8 of the nonyl side chain. And a chemically modified analog of erythro-hydroxynonyl adenine, which is modified by attachment to a side chain at a carbon atom selected from the group consisting of: and 9 carbon atoms, wherein (a) administering to humans and experimental animals If pharmacologically acceptable and non-toxic, (b) in cell culture tests, from erythro-hydroxynonyl adenine or 9- [2 (S), 9-dihydroxy-3 (R) -nonyl] adenine Can also more effectively enter human cells and inhibit the intracellular enzymatic activity of adenosine deaminase in a reversible manner, (c) erythro-hydroxy Synonyladenine or 9-
More effectively reduces hypoxia and ischemic injury in at least one type of mammalian tissue selected from the group consisting of myocardial and brain tissues, more effectively than [2 (S), 9-dihydroxy-3 (R) -nonyl] adenine Including a moiety that renders it therapeutically effective to treat a erythro-hydroxynonyl adenine. 18. A chemically modified analog of erythro-hydroxynonyl adenine comprising: (a) a Ki value for adenosine deaminase inhibition of less than about 5 × 10 ⁻⁹ mole; and (b) a logarithmic value of base 10 at least. 18. The method of claim 17, having an octanol / water partition coefficient of about 2. 19. At least one of R1 and R2
One is: a. A halide atom, b. A chemically modified analog of erythro-hydroxynonyl adenine, which can have a Ki value for adenosine deaminase inhibition that is in the range of about 10 ^-7 to about 10 ^-10 mole, and d. The chemically modified analog of erythro-hydroxynonyl adenine is selected from the group consisting of an ether linking group capable of having a Ki value for adenosine deaminase inhibition in the range of about ^10-7 to about ^10-10 mole. Item 18. The method according to Item 17. 20. at least one of R1 and R2 is
20. The method of claim 17, wherein the nitrogen-containing moiety is selected from the group consisting of an amine, amide, azide, nitrile, lactam, imine, and imide moiety. 21. The use of a chemically modified analog of erythro-hydroxynonyl adenine in the manufacture of a medicament for reducing adenosine deaminase activity in a mammalian patient, wherein the chemically modified erythro-hydroxynonyl adenine analog comprises: The following molecular structure: Wherein at least one of R 1 and R 2 is a chemical moiety other than a hydrogen atom or a hydroxyl group, wherein a chemically modified analog of erythro-hydroxynonyl adenine is administered to humans and experimental animals If pharmacologically acceptable and non-toxic, (b) in cell culture tests, from erythro-hydroxynonyl adenine or 9- [2 (S), 9-dihydroxy-3 (R) -nonyl] adenine Can even more effectively enter human cells and inhibit the intracellular enzymatic activity of adenosine deaminase in a reversible manner, (c) erythro-hydroxynonyl adenine or 9-
More effectively reduces hypoxia and ischemic injury in at least one type of mammalian tissue selected from the group consisting of myocardial and brain tissues, more effectively than [2 (S), 9-dihydroxy-3 (R) -nonyl] adenine Including moieties that make them therapeutically effective to do so]. 22. A chemically modified analog of erythro-hydroxynonyl adenine comprising: (a) a Ki value for adenosine deaminase inhibition that is less than about 5 × 10 ⁻⁹ mole; and (b) a logarithmic value of base 10 at least. 22. The method of claim 21 having an octanol / water partition coefficient of about 2. 23. At least one of R1 and R2
One is: a. A halide atom, b. An ester linking group capable of providing a chemically modified analog of erythro-hydroxynonyl adenine having a Ki value for adenosine deaminase inhibition that is in the range of about ^10-7 to about ^10-10 moles; and c. The chemically modified analog of erythro-hydroxynonyl adenine is selected from the group consisting of an ether linking group capable of having a Ki value for adenosine deaminase inhibition in the range of about ^10-7 to about ^10-10 mole. Item 22. The method according to Item 21. 24. at least one of R1 and R2 is
22. The method of claim 21, wherein the nitrogen-containing moiety is selected from the group consisting of an amine, amide, azide, nitrile, lactam, imine, and imide moiety. 25. Use of a chemically modified analog of erythro-hydroxynonyl adenine in the manufacture of a medicament for reducing adenosine deaminase activity in a mammalian patient, wherein the chemically modified analog of erythro-hydroxy nonyl adenine is used. Comprises an adenyl ring structure attached to a nonyl side chain, wherein the side chain has at least one chemical moiety other than a hydroxyl moiety, a carbon atom selected from the group consisting of carbon atoms 8 and 9 of the nonyl side chain. And a chemically modified analog of erythro-hydroxynonyl adenine, which (a) is pharmacologically acceptable and non-toxic when administered to humans and experimental animals. (B) In cell culture tests, erythro-hydroxynonyl adenine or 9- [2 (S), 9-dihydroxy- 3 (R) -nonyl] adenine can more effectively enter human cells and inhibit the intracellular enzymatic activity of adenosine deaminase in a reversible manner; and (c) erythro-hydroxynonyl adenine or 9-
More effectively reduces hypoxia and ischemic injury in at least one type of mammalian tissue selected from the group consisting of myocardial and brain tissues, more effectively than [2 (S), 9-dihydroxy-3 (R) -nonyl] adenine Uses that are therapeutically effective to do. 26. A chemically modified analog of erythro-hydroxynonyl adenine comprising: (a) a Ki value for adenosine deaminase binding that is less than about 5 × 10 ⁻⁹ mol; and (b) a logarithmic value of base 10 at least. 26. The method of claim 25, having an octanol / water partition coefficient of about 2. 27. At least one of R1 and R2 comprises: a. A halide atom, b. An ester linking group capable of providing a chemically modified analog of erythro-hydroxynonyl adenine having a Ki value for adenosine deaminase inhibition that is in the range of about ^10-7 to about ^10-10 moles; and c. The chemically modified analog of erythro-hydroxynonyl adenine is selected from the group consisting of an ether linking group capable of having a Ki value for adenosine deaminase inhibition in the range of about ^10-7 to about ^10-10 mole. Item 29. The method according to Item 25. 28. at least one of R1 and R2 is
26. The method of claim 25, wherein the nitrogen-containing moiety is selected from the group consisting of an amine, amide, azide, nitrile, lactam, imine, and imide moiety.