EP0871449A1 - Hydroxynonyladenine analogs with enhanced lipophilic and anti-ischemic traits - Google Patents

Hydroxynonyladenine analogs with enhanced lipophilic and anti-ischemic traits

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Publication number
EP0871449A1
EP0871449A1 EP96912400A EP96912400A EP0871449A1 EP 0871449 A1 EP0871449 A1 EP 0871449A1 EP 96912400 A EP96912400 A EP 96912400A EP 96912400 A EP96912400 A EP 96912400A EP 0871449 A1 EP0871449 A1 EP 0871449A1
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EP
European Patent Office
Prior art keywords
erythro
hydroxynonyladenine
chemically modified
modified form
adenosine deaminase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96912400A
Other languages
German (de)
English (en)
French (fr)
Other versions
EP0871449A4 (enrdf_load_stackoverflow
Inventor
Elie Abushanab
Paul J. Marangos
Lewis R. Makings
Anne M. Danks
Jerzy A. Barankiewicz
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Mallinckrodt ARD Inc
Original Assignee
Cypros Pharmaceuticals Corp
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Publication date
Application filed by Cypros Pharmaceuticals Corp filed Critical Cypros Pharmaceuticals Corp
Publication of EP0871449A4 publication Critical patent/EP0871449A4/xx
Publication of EP0871449A1 publication Critical patent/EP0871449A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine

Definitions

  • This invention is in the fields of chemistry and pharmacology, and relates to drugs that can inhibit an enzyme called adenosine deaminase (ADA, also known as adenosine aminohydrolase) .
  • ADA-inhibiting drugs can be used to reduce the enzymatic degradation of chemotherapeutic and anti-viral drugs, thereby increasing the therapeutic utility of such drugs.
  • ADA-inhibiting drugs can also be used to protect heart muscle and brain tissue against damage caused by ischemia (inadequate blood flow) or hypoxia (inadequate oxygen supply) , as occurs during stroke, cardiac arrest, heart attack, asphyxiation, and various other crises.
  • adenosine deaminase The mammalian enzyme called adenosine deaminase (ADA) , which is designated E.C.3.5.4.4 under the international enzyme classification system, converts adenosine into inosine by removing an amine group from the #6 carbon in the two-ring adenyl structure of adenosine.
  • ADA can also degrade a number of other molecules, including several nucleoside analogs that are used in cancer chemotherapy or for anti-viral therapy. Since ADA is known to reduce the therapeutic utility of various drugs used to treat cancer and viral infections, a substantial amount of work has been done to develop drugs which function as ADA inhibitors.
  • ADA inhibitor drugs can be used as adjuncts (i.e., as secondary agents to increase the effectiveness of a primary drug) to prolong the metabolic half-lives of therapeutic drugs during cancer or anti-viral chemotherapy.
  • ADA inhibitors can also be used to artificially create ADA deficiencies, whic are of interest to some researchers.
  • EHNA erythro-hydroxynonyladenine
  • eenah a relatively mild ADA inhibitor
  • EHNA erythro-hydroxynonyladenine
  • eenah a relatively mild ADA inhibitor
  • EHNA is a stereoisomer with the following chemical structure, which shows the numbering of the carbon atoms in the nonyl "side chain" (i.e., in the erythro-hydroxy-nonyl straight chain which is attached to the double-ringed adenyl group) :
  • the "erythro-" pref x nd cates a certain stereoisomeric arrangement of the atoms attached to the #2 and #3 carbon atoms in the nonyl side chain.
  • Both the #2 and #3 carbon atoms are chiral atoms (i.e., carbon atoms with four different groups attached to them, so that the spatial arrangement of the four groups will have either a dextrorotatory (or right, or +) or levorotatory (or sinister, or -) configuration, depending on how they rotate polarized light passing through an aqueous solution of a purified stereoisomer. These rotations are abbreviated as D/L, R/S, or +/-.
  • Other purified steroeisomers having the same atoms as erythro compounds, but in a different stereoisomeric arrangement, are referred to as "threo-" compounds.
  • EHNA apparently is metabolized and cleared from the mammalian bloodstream fairly rapidly (McConnell et al 1980; Lambe and Nelson 1982).
  • EHNA's activity as an ADA inhibitor drug is not as strong as various other ADA inhibitor drugs, including deoxycoformycin (dCF, also known as Pentostatin) .
  • dCF deoxycoformycin
  • the so-called "Ki" value of dCF i.e., the negative log value of a molar concentration of dCF required to inactivate a standardized quantity of ADA is very low, about
  • dCF was tested by several research teams to determine whether it can be used therapeutically. Although dCF reportedly provided some beneficial activity in cardiovascular models (e.g., Dorheim et al 1991), neuroprotection (e.g., Phillis and O'Regan 1989), and cancer therapy, it was found to cause serious toxic side effects (e.g., O'Dwyer et al 1986). Therefore, attention subsequently returned to EHNA and various other milder or "softer" ADA inhibitors, in the hope that the milder ADA inhibitors would have fewer side effects and would be less toxic.
  • cardiovascular models e.g., Dorheim et al 1991
  • neuroprotection e.g., Phillis and O'Regan 1989
  • cancer therapy e.g., O'Dwyer et al 1986. Therefore, attention subsequently returned to EHNA and various other milder or "softer" ADA inhibitors, in the hope that the milder ADA inhibitors would have fewer side effects and would be less toxic.
  • the Ki value of ( ⁇ )-EHNA is about 6 x 10 "9 , which indicates that EHNA binds to ADA about a thousand times less tightly than dCF.
  • This invention discloses a class of compounds in which a hydrogen atom coupled to one of the "far end" carbon atoms (i.e., the #8 or #9 carbon atoms) is replaced by a hydroxyl group, to create a #8 or #9 hydroxylated EHNA, or by various other types of moieties to create other #8 or #9 analogs (including analogs which are more soluble in lipids than the hydroxylated analogs, and which have shown better therapeutic utility against ischemia) .
  • analogs that are of interest herein have both (1) a binding affinity for the ADA enzyme which is in the desired range, with a Ki value between about 10 "7 and about 10 '10 , and (2) additional properties which render them substantially more useful and beneficial than unmodified EHNA in protecting heart tissue and/or brain tissue against damage caused by ischemia (inadequate blood flow) or hypoxia (inadequate oxygen supply), as occurs during stroke, heart attack, cardiac arrest, asphyxiation, and various other types of crises or conditions.
  • ischemia inadequate blood flow
  • hypoxia inadequate oxygen supply
  • ADA-inhibiting drugs in protecting heart muscle or brain tissue against ischemic or hypoxic damage has not been widely recognized prior to this invention. Instead, nearly all research on ADA inhibitors has focused on their potential ability, as adjuncts, to slow the degradation of anti ⁇ cancer or anti-viral drugs by the ADA enzyme, in order to increase the efficacy of such anti-cancer or anti-viral drugs.
  • EHNA analogs have also been shown to provide substantial protection for the heart against ischemic or hypoxic damage, as would occur during a heart attack, cardiac arrest, or surgery requiring cardiopulmonary bypass. It is also believed that at least some of these analogs may also provide substantial protection for brain tissue against ischemic or hypoxic damage due to stroke, cardiac arrest, asphyxiation, etc.
  • ADA enzyme acts inside cells. Despite the fact that this enzyme activity can affect the quantity of adenosine released by a cell, which will react with adenosine receptors on other cells, the fact remains that the ADA enzyme, itself, functions almost exclusively inside cells. Therefore, an ADA inhibitor drug must enter mammalian cells in order to function properly, and its efficacy will depend to a large extent on how readily it can be taken into cells.
  • One object of this invention is to disclose a class of analogs of EHNA which have been modified at the #8 or #9 carbon atoms on the side chain, in a manner which substantially improves the therapeutic efficacy of these analogs against ischemic or hypoxic damage to heart muscle or brain tissue, compared to either unmodified or hydroxylated EHNA.
  • Another object of this invention is to disclose a class of analogs of EHNA which have been modified at the #8 or #9 carbon atoms on the side chain, in a manner which provides various therapeutic advantages for these analogs while retaining a binding affinity for the ADA enzyme which is in the desired range (preferably with a Ki value between about 10 '7 and about 10 '10 ) .
  • This status as a relatively mild and reversible ADA inhibitor allows such analogs to inhibit ADA activity at therapeutically effective levels, without irreversibly inactivating (poisoning) the ADA enzyme and increasing the risk of toxic side effects.
  • Another object of this invention is to disclose synthetic reagents and methods that can be used to create pharmacologically valuable analogs of EHNA which contain hydroxyl, halide, acid, ester, ether, amine, amide, imide, azide, nitrile, or other moieties at various controllable locations in the nonyl side chain, and particularly containing novel moieties coupled to the #8 or #9 carbon atoms in the side chain .
  • Another object of this invention is to disclose a new set of EHNA analogs which can be used to slow down the degradation by the ADA enzyme of certain types of anti-cancer, anti-viral, or other therapeutic drugs.
  • EHNA erythro-hydroxynonyladenine
  • the analogs which have showed the best combinations of traits in the research done to date are believed to be more lipophilic (i.e., more soluble in lipids and other fatty, non- polar fluids, and less soluble in water) than the hydroxylated analogs.
  • lipophilic analogs are disclosed below, along with chemical methods for synthesizing them and any other desired analog of EHNA which has been modified at the #8 or #9 carbon atom of the side chain.
  • Any such analog which is synthesized as described herein can be screened, using assays as described below or otherwise known to those skilled in the art, to determine whether a particular analog has a desired combination of traits as described herein.
  • These traits primarily involve (1) non-toxic potency as a mild, reversible ADA inhibitor; (2) a desirable level of lipophilicity, to increase cellular uptake; and (3) therapeutic utility in reducing ischemic or hypoxic tissue damage, or in reducing ADA degradation of anti-cancer, anti ⁇ viral, or other drugs.
  • FIGURE 1 depicts a series of chemical reactions used to create 9 '-hydroxy(+)-EHNA, designated as Compound [10], an intermediate compound that was used to create other subsequent analogs of EHNA that are more lipophilic.
  • FIGURE 2 depicts the reactions that were used to create 8 ⁇ - hydroxy(+) -EHNA, designated as Compound [23].
  • FIGURE 3 depicts the reactions that were used to create 8 • ,9 '-dihydroxy(+)-EHNA, designated as Compound [14] .
  • FIGURE 4 depicts the reactions that were used (see Example 5) to create analogs of EHNA that contained various non-hydroxy moieties bonded to the #9 carbon atom.
  • FIGURES 5, 6, AND 7 are bar graphs showing that 9-chloro- EHNA (compound [29]) and 9-phthalimido-EHNA (compound [27] provided better protection for heart muscle against ischemic damage than either unmodified EHNA or 9-hydroxy-EHNA, in the tests described in Example 6.
  • This invention describe ⁇ analogs of EHNA in which the side chain (i.e., the straight chain erythro-hydroxynonyl portion, which is attached to an adenyl ring structure) has been chemically modified by bonding certain types of chemical groups (moieties) to it.
  • the preferred moieties provide the resulting analogs with certain pharmacological and therapeutic activities, which are substantially improved compared to unmodified EHNA.
  • a hydroxyl group can be bonded to either the #8 or #9 carbon atom on the EHNA side chain, to generate hydroxylated analogs referred to herein as 8- OH-EHNA or 9-OH-EHNA (or a di-hydroxylated analog with hydroxyl moieties bonded to both carbon atoms) .
  • 8- OH-EHNA or 9-OH-EHNA or a di-hydroxylated analog with hydroxyl moieties bonded to both carbon atoms
  • EHNA analogs intended for therapeutic use in humans should have suitable potency as a reversible ADA inhibitor, preferably with a binding affinity for ADA that provides a Ki value in the range of about 10 '7 to about 10 '10 .
  • EHNA analogs with Ki values in this desired range can inhibit the ADA enzyme reversibly, without permanently poisoning enzyme molecules, and without causing the types of toxic side effects that have been caused in some patients or test animals by highly potent "suicide inhibitors" such as deoxycoformycin.
  • the Ki value for any analog of EHNA can be determined by assays such as the spectrophotometric assay described in Harriman et al 1992 (described in more detail in Example 3) .
  • lipophilic drugs tend to be taken into cells more readily and in greater quantities than hydrophilic drugs, because of two chemical factors.
  • lipophilic drugs like droplets of oil in water, lipophilic drugs generate surface tension between themselves and water molecules; they are not "comfortable” floating in the watery liquid that surrounds cells, and they seek configurations that minimize the area of their surface contact with water. This surface tension causes lipophilic molecules to attach and adhere to any lipophilic surfaces they encounter, including the surfaces of cells, to minimize the area of their surface contact with water.
  • hydrophobic drugs can be difficult to administer to a patient via conventional routes such as injection or ingestion, and once inside the body, they often tend to sequester themselves in lipid vesicles or globules, or they tend to cling to various membranes, plaque deposits, or particulates, either in the intestines or inside blood vessels.
  • the lipophilic 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 referred to as P , where "o/w" refers to oil and water; this value is usually referred to by a base 10 logarithm, comparable to pH values; a high log P 0/w value indicates a high degree of oil solubility, and a low (or negative) value refers to a high degree of water solubility.
  • octanol-water partition coefficients can be estimated using commercially available computer software (such as the ACD/LogP software program, sold by Advanced Chemistry Development, Inc. of Toronto, Canada) . This software was used to calculate the octanol-water partition coefficients listed in Table 1. A description of the methods used to calculate and estimate partition coefficients, based on their chemical structures, is described in Bodor et al 1989.
  • EHNA analogs which have a level of lipophilic solubility close to or greater than the chloro- or phthalimido- analogs described below are likely to be preferable to hydroxylated analogs and other more hydrophilic (water-soluble) analogs, for protecting against ischemic or hypoxic damage or for increasing the half-lives of drugs that are degraded by the ADA enzyme.
  • the third primary desirable trait for an EHNA analog intended for use as described herein involves therapeutic utility, either in mammalian patients or in laboratory studies which provide good models of therapeutic utility against certain types of cell damage or drug degradation.
  • the two primary and most urgent uses for the EHNA analogs described herein are: (a) for reducing the amount of damage caused by ischemia or hypoxia in vulnerable tissues, especially in heart muscle or brain tissue; and (b) for prolonging the half-lives and increasing the therapeutic benefits of drugs that are being used to treat patients suffering from cancer, viral infection, or other disease conditions, by reducing the rate of degradation of such drugs by the ADA enzyme.
  • Various other uses may also be currently available, or they may be discovered in the future after these compounds are announced and made available to scientific and medical researchers.
  • This invention also discloses a method of synthesizing analogs of EHNA in which a moiety (such as a hydroxy group or halide atom) has been added to the side chain.
  • This method comprises the following steps: a. reacting an epoxide reagent having a desired chiral orientation with an alkyl halide reagent having an unsaturated bond between two selected carbon atoms, under conditions which cause said reagents to create an unsaturated aliphatic compound comprising a first portion having a desired chiral orientation and a second portion having an unsaturated bond; b.
  • the unsaturated aliphatic compound reacting the unsaturated aliphatic compound with at least one third reagent, under conditions which cause the third reagent to modify the unsaturated aliphatic compound by adding at least one hydroxyl group (or other desired group or atom) to at least one of the carbon atoms involved in the unsaturated bond, thereby creating a hydroxylated (or otherwise modified) saturated aliphatic compound; c. if a hydroxyl group was bonded to the EHNA side chain, the hydroxyl group can be replaced by or converted into a different moiety, as discussed herein.
  • any additional processing is carried out to complete the synthesis of the desired analog, such as removal of benzyl or other protective groups; such groups are commonly used during synthesis to prevent undesired reactions involving a protected constituent.
  • the final de-protected analog is then purified by any suitable means, such as chromatography, gel electrophoresis, or isoelectric focusing.
  • each major starting reagent or intermediate is referred to by a bracketed number.
  • bracketed number is then used to refer to that compound in subsequent processing steps.
  • Example 1 describes in detail the reagents and reactions used to synthesize the EHNA analog which has a hydroxyl group bonded to the #9 carbon atom on the side chain.
  • This compound referred to herein as 9-hydroxy-EHNA or as 9-0H- EHNA, is designated as Compound [10].
  • Its full chemical name is 9-[2 (S) ,9-dihydroxy-3(R)-nonyl]adenine, and its synthesis is depicted in FIG. 1.
  • the full chemical name includes the term
  • dihydroxy because this analog has two hydroxy groups on the side chain. One hydroxy group is attached to the #2 chiral carbon atom, in the same "S" orientation that occurs in unmodified EHNA; the other hydroxy group was added to the #9 carbon atom, to create the 9-OH-EHNA analog.
  • Example 2 describes the reagents and reactions used to synthesize the EHNA analog which has a hydroxyl group bonded to the #8 carbon atom on the side chain.
  • This compound referred to herein as 8-hydroxy-EHNA or as 8-OH-EHNA, is designated as Compound [23].
  • Its full chemical name is 9-[2(S) ,8-dihydroxy- 3(R)-nonyl]adenine, and its synthesis is depicted in FIG. 2.
  • Example 2 also describes the synthesis of Compound [14], which is a di-hydroxylated EHNA analog with hydroxy groups added to both the 8' and 9' carbon atoms (in addition to the standard hydroxy group on the #2 carbon atom) . Its synthesis is depicted in FIG. 3.
  • the 9-OH analog was identified as the preferred candidate, and it was used as a starting reagent for synthesizing other analogs (more precisely, a benzyl-protected precursor of the 9-OH analog, designated as Compound [9] in Example 1, was used; the benzyl group protected the hydroxy group attached to the #2 carbon atom) .
  • a benzyl-protected precursor of the 9-OH analog designated as Compound [9] in Example 1
  • the benzyl group protected the hydroxy group attached to the #2 carbon atom
  • either the 8- hydroxy or the 8,9-dihydroxy analogs could be used instead, to create comparable lipophilic analogs with any desired moieties coupled to the #8 carbon atom instead of (or in addition to) the #9 carbon atom, using the same general procedures and reagents described herein.
  • hydroxyl route should be recognized as a potentially useful route for synthesizing a large number of analogs that can be generated by substituting or derivatizing hydroxyl groups, such as carboxylic acid groups, esters, and ethers, all of which can be created using techniques such as disclosed in the examples, or other techniques known to those skilled in the art of chemical synthesis.
  • hydroxyl groups such as carboxylic acid groups, esters, and ethers, all of which can be created using techniques such as disclosed in the examples, or other techniques known to those skilled in the art of chemical synthesis.
  • hydroxide groups can be converted to numerous other groups by known methods.
  • a hydroxyl group can be converted into an azide group by reacting the hydroxyl with p-toluenesulfonyl chloride (TsCl) to create an 0-tosyl group (abbreviated as OTs in the figures; tosyl refers to toluenesulfonyl) , then reacting the O-tosyl compound with sodium azide (NaN 3 ) , which displaces the O-tosyl group and leaves an N 3 group attached to the carbon chain.
  • TsCl p-toluenesulfonyl chloride
  • NaN 3 sodium azide
  • a hydroxyl group can be converted to a halide group (such as a chlorine, fluorine, bromine, or iodine atom) by methods such as in Example 5 relating to Compounds [28] and [29].
  • a halide group such as a chlorine, fluorine, bromine, or iodine atom
  • analogs that can be synthesized as described herein include, but are not limited to, analogs in which the chemical moiety bonded to the #8 or #9 carbon atom on the nonyl side chain consists of a halide; a nitrogen-containing moiety such a ⁇ an amine, amide, azide, imide, or lactam; a carboxylic acid or salt thereof; or a moiety which is coupled to the #8 or #9 carbon atom via an ester or ether linkage.
  • any such analog must display the traits that can make such analogs therapeutically useful as disclosed herein (i.e., the resulting analog should have a Ki within the desired range of about 10 '7 to about 10" 10 ; it must be pharmacologically acceptable; and it must be therapeutically useful in protecting tissue against ischemic or hypoxic damage, or as an adjunct with one or more anti-cancer, anti-viral, or other drugs) .
  • Epoxide [1] was synthesized as described in Abushanab et al 1984 and 1988. It controls the orientation of the substituents on the two chiral carbon atoms in the final EHNA analog, which are provided by the #3 and #4 carbons in the epoxide. To synthesize different stereoisomers of any of the EHNA analogs discussed herein, different epoxide stereoisomers having any desired chiral configuration can be used as the starting reagent.
  • the benzyl group (-CH 2 C 6 H 5 ) which was attached via an oxygen atom to the #3 carbon in the starting epoxide served as a protective group for the oxygen atom.
  • the benzyl group was displaced by hydrogen to create a hydroxyl group on the #2 carbon of the side chain. That #2 hydroxyl group is part of the normal EHNA molecule.
  • that hydroxyl group can be eliminated by using a starting epoxide without a protected oxygen atom, or it can be modified during synthesis to provide a halide, carboxylic, ester, ether, azide, or other group, as described above.
  • a moiety is desired at the #1 carbon atom in the final EHNA analog, it can be provided by using a starting epoxide having the desired moiety or a precursor at the #4 carbon atom of the epoxide.
  • the synthesis reactions described herein also offer a method of derivatizing (i.e., bonding moieties to) the #4, #5, #6, or #7 carbon atoms on the nonyl side chain.
  • those carbon atoms were provided by the reagent 1-pentenylmagnesium bromide, which has a structure as shown in FIG. 1 in the reaction that converts epoxide [1] into compound [2] .
  • the 1-pentenyl notation indicates that the unsaturated double bond is positioned between the #1 and #2 carbon atoms in 1-pentenylmagnesium bromide; those carbon atoms ultimately become the #8 and #9 carbon atoms in the EHNA analogs of this invention.
  • the location of a hydroxyl (or other desired) group on the side chain of an EHNA analog can be controlled by using a pentenylmagnesium bromide (or similar) compound having a double bond in any desired location.
  • a 2-pentenyl compound would have a double bond between its #2 and #3 carbon atoms, which become the #8 and #7 carbon atoms in the final EHNA analog.
  • a 3-pentenyl reagent (having a double bond between its #3 and #4 carbon atoms) would generate hydroxyl groups attached to the #7 or #6 carbons in the EHNA analog.
  • FIG. 2 also depicts a halogenated analog, Compound [21] .
  • the halogen (chlorine) atom was substituted into the adenine ring structure.
  • chlorine atom was substituted by an amine group during the synthesis of compound [22]
  • that particular reaction could be omitted if desired, so that the halogen moiety would remain after removal of the benzyl protective group.
  • the method used to create the adenyl structure in the EHNA analogs described herein offers a general method for making various changes in the adenine group.
  • the adenyl structure was provided by supplying and then manipulating a heterocyclic compound, 5-amino-4, 6-dichloropyrimidine (ADCP) , which is shown in FIG. 1 in the reaction that generated compound [6]; this same reagent was also used to generate compound [20] shown in FIG. 2.
  • ADCP 5-amino-4, 6-dichloropyrimidine
  • the ADCP was coupled to the side chain by displacing one of the chlorine atoms on the ADCP with an amine group that was coupled to the side chain.
  • the five-member ring in the adenine structure was then closed by forming a carbon bond between two proximal nitrogen atoms.
  • alternate heterocyclic reagents could be used instead of ADCP, to create analogs of EHNA with modified adenine structures, either as moieties attached to one of the rings, or as differing atoms incorporated into either of the rings.
  • Cristalli et al 1988 and 1991 report that certain analogues of EHNA with modified adenine structures (such as a 3-deaza-EHNA derivative) are active as ADA inhibitors. Such modifications to the adenyl structure could be incorporated into the analogs of this invention, which have modified side chains.
  • Ki values are within a desired range, which covers about 10 '7 to about lO '10 .
  • ADA inhibitors having Ki values lower than about 10 '10 run the risk of "poisoning" the enzyme by binding to it so tightly that the reaction is, for all practical purposes, irreversible.
  • ADA inhibitors having Ki values higher than about 10 '7 tend to be insufficiently potent to accomplish the desired level of ADA inhibition; they would need to be administered in relatively large quantities, and even in large quantities they might not be adequately potent.
  • the desired range of Ki values is relatively broad, since candidate compounds can be administered to a patient in any desired quantity, by various routes.
  • An analog having a Ki value in the range of about 10 '9 should be administered in relatively low dosages, such as up to about 10 milligrams per kilogram of body weight per day if injected intravenously, and up to about 50 mg/kg/day if administered orally.
  • a less potent analog having a Ki value in the range of about 10 "7 could be administered in higher dosages, such as up to about 25 mg/kg/day if administered orally or injected in response to a major crisis, or up to 20 mg/kg/day if injected intravenously. Since the metabolic problems caused by ADA deficiency tend to accumulate slowly, short-term dosages can be rather large.
  • the hydroxylated EHNA analogs were tested for protection against ischemic damage to hearts, using procedures described in Example 4. Briefly, these tests involved hearts that were removed from laboratory rats, hooked up to perfusion equipment and given electrical stimulation to sustain the heartbeat, treated with the candidate drugs, subjected to a period of ischemia, and then reperfused, to evaluate how well the hearts could recover their pumping functions. In these initial assays, 9-OH-EHNA provided a higher level of protection than unmodified EHNA in a particular parameter involving reduction of unwanted muscle stiffness after ischemia.
  • Example 5 describes, and Figure 4 depicts, the synthesis of several other analogs, using the benzyl-protected precursor (compound [9]) of the 9-OH analog as a starting reagent.
  • These analogs include two relatively lipophilic analogs, referred to herein as 9-chloro-EHNA (Compound [29]) and 9-phthalimido-EHNA (Compound [26]). These two analogs have shown the best therapeutic results observed to date, in protecting both heart muscle and brain tissue against ischemic damage. Some additional analogs were also created by Cypros
  • Ki values and oil/water solubility values that were gathered or calculated on the final (deprotected) analogs listed in Examples 1, 2, or 5 are compiled in Table 1.
  • these analogs are provided with simple names that indicate what type of modifying group was added to the side chain, and which carbon atom it was bonded to.
  • Complete chemical names are provided in Examples 1, 2, and 5, correlated with bracketed compound numbers.
  • Ki values in Table 1 used extra-cellular ADA enzyme, and did not reflect the apparent ability of lipophilic analogs to enter cells more readily and in greater quantities.
  • Example 6 describes the testing of various analogs to evaluate their ability to protect heart muscle against ischemia.
  • Example 7 describes the results of cell culture tests to evaluate the ability of EHNA and several analogs both (1) to enter human cells, and (2) inhibit ADA activity inside the cells. These tests did not stress the cells, or test any analogs against ischemic damage; instead, they evaluated the ability of various analogs to reach the intracellular enzyme molecules and inhibit their activity. These tests used both red blood cells (which are easy to work with) , and human astrocytoma cells
  • EHNA analogs which are brain cells that can reproduce in cell culture; these were used to provide an indication of whether EHNA analogs can help reduce ischemic damage in brain tissue.
  • Example 8 describes the results of cell culture tests which used several different methods to generate ischemic damage in either brain cells or blood cells. Some of these tests used toxins such as 2-deoxyglucose or sodium azide to interfere with respiration and glycolysis. Other tests used culture media containing no free oxygen, obtained by bubbling nitrogen gas rather than oxygen gas through the cell culture media. In all of these tests, the cells were subjected to a period of oxygen deprivation (usually lasting several minutes) , then the oxygen supply was reestablished. After a brief period to allow the cells to reestablish equilibrium, selected metabolic indicators were evaluated to determine how close the cells had come to regaining their proper metabolic rates. The results indicated that some EHNA analogs (especially the lipophilic analogs) can indeed protect brain cells against ischemic damage.
  • toxins such as 2-deoxyglucose or sodium azide to interfere with respiration and glycolysis.
  • Other tests used culture media containing no free oxygen, obtained by bubbling nitrogen gas rather than oxygen gas through the cell culture media. In all of these tests, the
  • Example 9 describes several assays that can be used to test EHNA analogs to quantify their ability to protect intact mammalian brain tissue against ischemia. Rather than using isolated cultured brain cells, as in Example 7, these tests use intact slices of brain tissue, from the hippocampal regions of sacrificed rats. The hippocampal region is used because it is highly vulnerable to ischemic damage, and the use of intact hippocampal slices that can still generate brain waves in response to electrical stimulation offers a better assurance of overall tissue functioning than the metabolic rates of isolated cells. These tests are currently underway.
  • Analogs that show promising results in the hippocampal slice tests described in Example 9 will be tested further, in in vivo tests on intact animals. These tests can use artery clamping, neck tourniquets, or other methods to induce either local or global ischemia in the brains of test animals, as described in articles such as Nellgard and Wieloch 1992, Buchan and Pulsinelli 1990, Michenfelder et al 1989, and Lanier et al 1988.
  • EHNA analogs described herein have a useful and previously unknown therapeutic benefit in protecting heart muscle and brain cells against ischemic damage.
  • the benefits provided by the relatively lipophilic analogs exceed and surpass the benefits provided by unmodified EHNA or hydroxylated EHNA analogs.
  • agents useful for the purposes described herein include any isomers (including "threo" isomers) , analogs, or salts of the compounds described herein, provided that such isomers, analogs, and salts are functionally effective as ADA inhibitors, are pharmacologically acceptable, and are therapeutically effective in either reducing ischemic or hypoxic damage or in slowing the degradation of anti-cancer, anti-viral, or other drugs.
  • the potency of any candidate isomer, analog, or salt in inhibiting ADA activity can be tested using methods such as described in Example 3.
  • the therapeutic efficacy of any candidate isomer, analog, or salt against ischemic or hypoxic damage can be tested using methods such as described in Examples 4, 6, and 7.
  • any candidate isomer, analog, or salt in slowing the degradation of anti-cancer, anti ⁇ viral, or other drugs can be measured by methods known to those skilled in the art, such as by administering an EHNA analog to animals (or humans) that have received the drug of interest, and after an appropriate period (which will usually be in the range of 2 to 24 hours later, depending on the drug) , measuring the quantities of the drug that are present in the blood or tissue of the test animals (or humans, if blood tests are used) , and comparing that quantity to the quantity of the same drug in animals or humans that have not been treated with an EHNA analog.
  • Acceptable salts can include alkali metal salts as well as addition salts of free acids or free bases.
  • acids which are widely used to form pharmacologically acceptable acid-addition salts include inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, and organic acids such as maleic acid, succinic acid and citric acid.
  • Alkali metal salts or alkaline earth metal salts could include, for example, sodium, potassium, calcium or magnesium salts. All of these salts may be prepared by conventional means. The nature of the salt is not critical, provided that it is non-toxic and does not substantially interfere with the desired activity.
  • analog is used herein in the conventional pharmaceutical sense, to refer to a molecule that structurally resembles a referent molecule (EHNA, 9-OH-EHNA, or 8-OH-EHNA, in this case) but which has been modified in a targeted and controlled manner to replace a specific substituent of the referent molecule with an alternate substituent, other than hydrogen (since replacement of the #9 hydroxyl group on 9-OH- EHNA with a hydrogen atom would give unmodified EHNA rather than a true analog of 9-OH-EHNA.
  • a chemical analog requires an "offspring" type of relationship, wherein an analog is created by chemical modification of a known compound (often called a parent or referent compound) .
  • the hydroxylated compounds [10], [14], and [23] are analogs of EHNA, but EHNA is not regarded as an analog of those hydroxylated compounds.
  • a substitution which converts a known molecule into a new analog may be inserted into or coupled to any location in the molecule, such as in one of the rings in the adenyl structure, in the attached side chain, or in one of the pendant groups attached to the ring structure or side chain.
  • Administration of the compounds of this invention to humans or animals can be by any technique capable of introducing the compounds into the bloodstream, including oral administration or via intravenous or intramuscular injections.
  • the active compound is usually administered in a pharmaceutical formulation, such as in a liquid carrier for injection, or in a capsule, tablet, or liquid form for oral ingestion.
  • Such formulations may comprise a mixture of one or more active compounds mixed with one or more pharmaceutically acceptable carriers or diluents.
  • lipophilic drugs When lipophilic drugs are formulated for injection, they are usually mixed with water, a buffer compound (such as a mixture of a carboxylic acid and a salt thereof) , and an organic compound having a plurality of hydroxyl groups; propylene glycol, dextran compounds, and cyclodextrin compounds are often used for such purposes.
  • a buffer compound such as a mixture of a carboxylic acid and a salt thereof
  • an organic compound having a plurality of hydroxyl groups propylene glycol, dextran compounds, and cyclodextrin compounds are often used for such purposes.
  • an injectable ingestible formulation which contains a suitable EHNA analog as described herein.
  • a mixture of an anti-cancer or anti ⁇ viral nucleoside analog, with an EHNA analog, can be very useful, since the EHNA analog can prolong the half-life and efficacy of the nucleoside analog in the blood, by suppressing degradation of the nucleoside analog by ADA enzymes.
  • tissue-protecting efficacy of any analog will depend on a combination of factors, rather than on any factor in isolation.
  • unmodified EHNA has a log P 0/H coefficient that is roughly the same as for 9-chloro-EHNA or 9-phthalimido-EHNA
  • 9-hydroxy-EHNA has a Ki value which is roughly the same as for 9-chloro-EHNA or 9-phthalimido-EHNA.
  • an analog should have both (a) a Ki value for adenosine deaminase inhibition which is less than about 5 x 10 *9 , and (b) an octanol/water partition coefficient of at least about 2. Neither unmodified EHNA nor any of the hydroxyated analogs created to date have this combination of traits.
  • the 9-benzoyloxy-EHNA analog had the best combination of low Ki value and high P 0/w value out of all the analogs listed in Table 1. In the future, it will be tested in both cell culture and intact tissue tests. In the assays carried out to date, it was not tested, due to concerns that the benzoyloxy group would likely be cleaved off from the EHNA molecule by various mammalian enzymes, thereby converting it into 9-OH-EHNA, which has relatively low efficacy for tissue protection.
  • EXAMPLE 1 SYNTHESIS OF 9-OH-EHNA ANALOG This example describes how various intermediate and final compounds were synthesized. For convenience, bracketed numbers which correspond to the subheadings below and to the callout numbers in the drawings are used to refer to each compound. Unless otherwise indicated, all organic solutions were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. Ratios of chromatography solvents are expressed in v/v. Silica gel (Davison, grade H, 230-425 mesh) suitable for flash column chromatography was purchased from Fisher Scientific.
  • a Chromatotron (centrifugally accelerated, preparative thin-layer, radial chromatograph) Model 5 7924T was used to complete various separations.
  • the 1.0 and 2.0 mm plates used were coated with silica gel PF254 containing CaS0 4 .
  • the starting epoxide [1] was synthesized as described in 0 Abushanab et al 1984 and 1988. This epoxide determines the orientation of the substituents on the two chiral carbon atoms in the final EHNA analog, which are provided by the #3 and #4 carbons in the epoxide.
  • different epoxide 5 stereoisomers having any desired chiral configuration can be used as the starting reagent.
  • a benzyl group attached to the #3 carbon atom via an oxygen atom was used to protect the oxygen atom during synthesis.
  • COMPOUND 4 (2S.3R ⁇ -3-Azido-2-Q-benz ⁇ l-2-nona-8-en-ol Sodium azide (1.027g, 15.8 mmol) was added to a stirred solution of [2] (4.0g, 13.2 mmol) in anhydrous DMF (20 mL) .
  • COMPOUND 6 5-Amino-6-chloro-4[2 (S)-O-benzyl-3 CR) -nona-8- enyl]aminopyrimidine
  • COMPOUND 9 9-[2 ( S)-0-Benzyl-9-hvdroxy-3 fR)-nonyl]adenine
  • the reaction was done under nitrogen atmosphere. The mixture was permitted to stir for additional hours at RT to continue the completion of the reaction. Water (0.05 mL) was added slowly and the mixture was allowed to stir at RT until hydrogen no longer evolved. The flask was immersed in an ice bath and 3 molar NaOH
  • COMPOUND 10 9-T2 (S) .9-dihvdrox ⁇ -3 (Ri-nonylladenine
  • This compound is the 9-hydroxy analog of EHNA, which was tested as described in Examples 4 and 6.
  • COMPOUND 11 6-Chloro-9-r2 fS)-O-benz ⁇ l-8.9-epoxy-3 (R) -nona-8- enyl]purine
  • COMPOUND 12 6-Chloro-9 [ 2 (S) -O-benzyl-8.9-dihvdroxy-3 fR) -nonyll purine
  • COMPOUND 13 9-f2 (S) -O-Benzyl-8.9-dihvdroxy-3 fR ⁇ -nonylladenine Compound [12] was obtained from [11] according to the procedure described for the preparation of [8], in 90% yield.
  • COMPOUND 14 9-T2fSi . 8.9-trihvdroxy-3 fR) -nonylladenine
  • Triol [14] was prepared in 90% yield by debenzylating [13] using the procedure described for [10].
  • This compound is the 8,9-dihydroxy analog of EHNA, which was tested as described in Example 3. Although it was shown to be a moderately effective inhibitor of ADA activity, its potency was lower than the 9-OH-EHNA analog, and it was not tested further.
  • COMPOUND 16 2S.3S)-2-0-Benzyl-3-0-tosyl-2.3.8-nonenetriol
  • a solution of aluminum hydride (30 mL, 15 mmol, 1 M solution in THF), was added to epoxide [15] (3.143g, 7.52 mmol) in dry ether (100 mL) at 0°C.
  • the mixture was stirred at RT for 1 h and decomposed slowly by the addition of water (25 mL) and the aqueous solution was extracted with ether (4 x 50 mL) .
  • the combined ether extracts were dried (MgS0 4 ) and concentrated to furnish [16] (3g, 95%) .
  • An analytical sample was obtained by silica gel column chromatography with hexane-EtOAc (7:3) as eluent.
  • COMPOUND 17 f2S.3S> -2-0-Benzyl-3-0-tosyl-8-0-tetrahvdropyranyl- 2.3.8-nonanetriol
  • COMPOUND 18 2S,3R) -3-Azido-2-0-benzyl-8-0-tetrahydr ⁇ pyranyl- 2.8-nonanediol
  • Compound [18] was prepared from [17] following the procedure described for the formation of [4].
  • COMPOUND 20 5-Amino-6-chloro-4 r 2fS)-O-benzyl-8-O- tetrahvdrop ⁇ ran ⁇ l-3fR)-2.8-dihydroxynon ⁇ llaminop ⁇ rimidine
  • Compound [20] was prepared from 19, by following the procedure described for the formation of [6]. The residue, obtained after solvent removal, was chromatographed over silica gel (EtOAc-hexanes 1:5) to give [20] (28%).
  • COMPOUND 21 6-Chloro-9-f2fS)-O-benzyl-2.8-dihvdroxy-3fR)- nonyll urine
  • COMPOUND 23 9- r 2 (S) .8-Dihvdroxy-3fR)-nonyl1adenine
  • Diol [23] was prepared in 90% yield by debenzylating [22] using the procedure described for [10] .
  • This compound is the 8-hydroxy analog of EHNA which was tested as described in Example 3. It was shown to inhibit ADA activity in the desired range, but its potency was lower than the 9-OH-EHNA analog, so it was not tested further.
  • EXAMPLE 3 TESTING FOR ADA INHIBITION
  • Compound [10] (the 9-hydroxy analog) , compound [23] (the 8- hydroxy analog), and compound [14] (the 8,9-dihydroxy analog) were tested for ADA inhibition activity, using calf intestinal ADA (Type III, Sigma Chemical Company) measured at 30°C by direct spectrophoto etric assays at 265 nm, as described in Harriman et al 1992. These tests used extra-cellular enzyme preparations, and did not require any of the analogs to enter cells in order to reach the enzyme.
  • Table 1 compiles ADA inhibition data and octanol-water partitiion coefficients (an index of lipophilicity) for all of the final (deprotected) analogs listed in Examples 1, 2, or 5.
  • EXAMPLE 4 TESTING OF 9-OH-EHNA FOR PROTECTION AGAINST ISCHEMIC DAMAGE TO TISSUE After synthesis of the 9-hydroxy and 8-hydroxy analogs of
  • EHNA EHNA
  • samples were provided by the Applicant to Dr. Robert Rodgers of the Department of Pharmacology and Toxicology at the University of Rhode Island. There were sufficient quantities of 9-OH-EHNA, while quantities of 8-OH-EHNA were smaller. Accordingly, most tests used 9-OH-EHNA and compared it to unmodified EHNA and to disulfiram, an unrelated compound that is known to have certain protective anti-ischemic effects in cardiovascular tissue.
  • Dr. Rodgers used a widely-used protocol known as a "working heart" preparation. These tests involved removing intact hearts from sacrificed lab animals (male Sprague-Dawley rats were used) , and perfusing the hearts with liquids containing controlled quantities (or deficits) of oxygen and glucose for fixed periods of time. The procedures used in these experiments are described in detail in Davidoff and Rodgers, Hypertension 15: 633-642 (1990), with certain minor modifications. The left atrium was filled at 15 cm H 2 0 pressure, and the left ventricle ejected into a buffer-filled column against a pressure which equated to 72 mm Hg, except during ischemic periods.
  • the perfusate was Krebs-Henseleit buffer with HC0 3 ' (25 mM) , Ca ++ (2.2 mM) , and glucose (10 mM) .
  • HC0 3 ' 25 mM
  • Ca ++ 2.2 mM
  • glucose 10 mM
  • the pH of the perfusate was 7.4 ⁇ 0.2.
  • Perfusate and ambient temperatures were held at 37°C, and the hearts were allowed to beat spontaneously.
  • the isolated hearts were allowed to stabilize for 10 minutes, then they were treated for 10 minutes with one of the test drugs (unmodified EHNA, 9-OH-EHNA, or disulfiram) or buffered saline containing either dilute ethyl alcohol (used to increase the solubility of EHNA or 9-hydroxy- EHNA) or dilute dimethyl sulfoxide (used to increase the solubility of disulfiram) .
  • one of the test drugs unmodified EHNA, 9-OH-EHNA, or disulfiram
  • buffered saline containing either dilute ethyl alcohol used to increase the solubility of EHNA or 9-hydroxy- EHNA
  • dilute dimethyl sulfoxide used to increase the solubility of disulfiram
  • LVPP - left ventricular pulse pressures time-dependent pressures, calculated as peak pressure minus diastolic pressure, in mm Hg, millimeters of mercury column
  • LVEDP - left ventricular end diastolic pressure i.e, time-dependent pressures as the ventricle relaxed during diastolic filling, in mm Hg
  • ECG - electrocardiogram surface potential, in mV
  • 9-OH-EHNA was more potent as an ADA inhibitor than 8-OH-EHNA, and because 9-OH-EHNA appeared to have an additional useful effect in reducing heart muscle stiffness, subsequent research used 9-OH-EHNA, compound [10], or its benzyl-protected precursor, compound [9], as starting compounds for synthesizing other analogs.
  • This analog which has benzoyloxy groups coupled to the #2 carbon and a benzyloxy group at the #9 carbon atom, was prepared by adding n,n-diisopropylazo-dicarboxylate (DIAD, 202 mg, 1 mmol) to a stirred solution of compound [9] (314 mg, 0.82 mmol), benzoic acid (BzOH, 122 mg, 1 mmol) , and triphenyl phosphine (PPh 3 , 262 mg, 1 mmol) in THF (5 ml) . The mixture was stirred at room temperature for 24 hr and precipitated triphenyl phosphine oxide was filtered out.
  • DIAD n,n-diisopropylazo-dicarboxylate
  • BzOH benzoic acid
  • PPh 3 triphenyl phosphine
  • This alcohol which has a benzoyloxy group coupled to the #9 carbon atom, was created by treating compound [24] (200 mg) in ethanol (EtOH, 18 ml) and cyclohexene (6 ml) with 20% palladium hydroxide on charcoal (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 at reduced pressure. The residue was chromatographed over silica (EtOAc and MeOH at 9:1) to give pure [25] , 130 mg (80%) .
  • COMPOUND 26 9-[2 fS)-0-benzyl-9-phthalimido-3 R)-nonyl]adenine
  • compound [26] was prepared from compound [9] in 87% yield in the same way as compound [24], except that phthalimide (1 mmol, 147 mg) was used in place of benzoic acid. Analytically pure compound could not be obtained as it always contained traces of triphenyl phosphine oxide and dihydro-DIAD. It was used as a reagent in the following step, to create compound [26].
  • COMPOUND 27 9-[2fS)-hvdroxy-9-phthalimido-3 fR)-nonyl]adenine
  • COMPOUND 29 9-r9-chloro-2(S)-hvdroxy-3 R)-nonyl1adenine
  • This analog with a halide moiety at the 9 carbon and an alcohol group at the #2 carbon atom, was prepared in 82% yield using [28] as the starting reagent and using the same palladium on charcoal (PdOH 2 /C) catalytic procedure used to create [25].
  • Other halide analogs with fluorine, bromine, or iodine atoms can be created, if desired, by proper selection of reagents containing such atoms.
  • the synthetic procedure described for Compound [28] could be modified by using CBr 4 or CI 4 instead of CC1 4 .
  • a 9-fluoro-EHNA analog could be produced by reacting compound [9] (the benzyl- protected 9-OH-EHNA analog) with the well known fluorination agent diethylaminosulfur trifluoride (DAST) .
  • DAST diethylaminosulfur trifluoride
  • COMPOUND 30 Methyl-7fR)-adenine-9-yl)-8fS)-O-benzyl-nonoate Analog [30], with an ester group at the #9 carbon and a benzyl group at the #2 carbon, was created by adding pyridinium dichromate (PDC, 2.755 g, 7.3 mmol) to a solution of [9] (1.369 g, 3.4 mmol) in dimethyl formamide (DMF, 2 ml) . The mixture was stirred at room temperature for 24 hours, then diluted with ethyl acetate and passed through a mixture of silica gel and Na 2 S0 4 (1:1) to give the corresponding acid (220 mg, 15.7% yield) .
  • PDC pyridinium dichromate
  • DMF dimethyl formamide
  • COMPOUND 31 Meth ⁇ l-7fR)-adenine-9-yl)-8 S)-hvdroxy-nonoate Analog [31], with an ester group at the #9 carbon and a hydroxy group at the #2 carbon, was created in 82% yield using the same palladium on charcoal (PdOH 2 /C) catalytic procedure used to create [25], using the benzyl analog [30] as the starting reagent.
  • Ester 31 can also be referred to as 9-[2(s)- hydroxy-9-carboxymethyl-3(R)-nonyl]adenine.
  • Unsaturated analog [32] was created by using the unsaturated benzyl-protected analog [7], shown in FIG. 3 and described in Example 1, as the starting reagent.
  • Compound [7] 200 mg, 0.55 mmol
  • toluene (10 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 was neutralized with NH 4 C1 and methanol and evaporated to dryness. The compound was then extracted with CH 2 C1 2 and the extracts were dried over Na 2 S0 4 and evaporated.
  • the silicon-containing moiety was chosen for two reasons: (1) calculations indicated that it had a very high level of lipophilicity (with a log P 0/w value in the range of 9, compared to 2 or 3 for the chloro and phthalimido analogs) and could provide a potentially useful compound to help evaluate high-level lipophilicity; and, (2) this moiety could be added to the #9 atom in a de-protected 9-OH-EHNA molecule without disturbing the unprotected hydroxyl group on the #2 carbon atom of the side chain.
  • compound [10] (30 mg, 0.102 mmol) was dissolved in 0.5 ml of DMF and added to a solution of 50 ⁇ l of diisopropylethylamine and 15 mg of dimethylaminopyridine contained in 1 ml of CH 2 C1 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 was evaporated and the product was purified by chromatography over silica gel (10% CH 3 0H/CHC1 3 ) to give 26 mg (48%) of 9-t-BDPSi-EHNA [compound 33] .
  • EXAMPLE 6 PROTECTION OF HEART MUSCLE AGAINST ISCHEMIA
  • Several of the analogs described in Example 5 were tested to evaluate their ability to protect heart muscle against ischemia, using a so-called "Langendorf heart preparation”. Briefly, male Sprague-Dawley rats weighing between 250 and 350 grams were anaesthetized with sodium heparin and sacrificed with C0 2 . The heart was rapidly excised via thoracotomy and placed in physiological salt solution (PSS) un contraction ceased.
  • PSS physiological salt solution
  • the heart was then mounted via the aortic aot to a cannula and retrogradely perfused with PSS containing (in mM) : NaCl (118) , KCl (4.7), CaCl 2 (2.2), KH 2 P0 4 (1.18), MgS0 4 (1.17), NaHC0 3 (25), dextrose (11) at 80 mm Hg at 37 degrees C.
  • PSS containing
  • the catheter was connected to a pressure transducer and was used to measure left ventricular hemodynamic performance, i.e. left ventricular systolic pressure (LVSP) , left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP) , +dP/dt ma ⁇ (the maximum rate at which pressure developed in the left ventricle during each contraction) , -dP/dt
  • LVSP left ventricular systolic pressure
  • LVEDP left ventricular end-diastolic pressure
  • LVDP left ventricular developed pressure
  • +dP/dt ma ⁇ the maximum rate at which pressure developed in the left ventricle during each contraction
  • ra ⁇ the maximum rate at which left ventricular pressure declined following each contraction
  • heart rate pulmonary artery was cannulated to collect coronary effluent for measurement of coronary flow.
  • PSS for 20 min at a pressure of 80 mm Hg. Measurements were again taken at 5 minute intervals during the reperfusion period.
  • KYAMPT.TC 7 CELL CULTURE TESTS. UNST ESSED CKT.T.g
  • EHNA and its analogs were evaluated for their ability to inhibit ADA activity in two different types of cells: human red blood cells (which are relatively easy to work with) , and human astrocytoma cells (which are brain cells that can reproduce in cell culture; these were used to provide an indication of whether EHNA analogs can help reduce ischemic damage in brain tissue) .
  • a first set of tests was carried out, using "normoxic" conditions (i.e., the cells had not been stressed by hypoxia or by simulated ischemia, using deoxyglucose or sodium azide) , to determine an IC 50 value for EHNA and several analogs.
  • the IC 50 TABLE 4 VENTRICDLAR PRESSURE INCREASE RATES DURING CONTRACTIONS (maximum dP/ t)
  • IC 50 value indicates the concentration of drug which was required to inhibit half of the ADA activity in the cells; this value reflects both the ability of a drug to enter cells and reach the ADA enzyme, and the potency of a drug in binding to and inhibiting the ADA enzyme inside the cells.
  • a low IC 50 value indicates that a drug is a potent ADA inhibitor and can enter cells readily.
  • cell populations were preincubated with EHNA or an EHNA analog, in varying concentrations, for l hour.
  • the cells were then incubated for 30 minutes with 10 uM 5- iodotubercidin, which inhibits the activity of a different enzyme called adenosine kinase, which adds phosphate groups to adenosine.
  • This step ensured that adenosine levels would not be altered by a phosphorylation pathway which can consume adenosine in intact cells.
  • the cells were then incubated for 30 minutes with 100 uM radiolabelled adenosine.
  • concentrations of radiolabelled inosine and hypoxanthine (the molecules that are created when the ADA enzyme degrades adenosine) were measured in cell medium after separation using cellulose thin layer chromatography.
  • concentrations of EHNA or an EHNA analog were used in each set of tests, and an IC 50 for each compound was calculated based on the dose-response curve for that compound.
  • EXAMPLE 8 CELL CULTURE TESTS FOR ISCHEMIC PROTECTION
  • Adenosine release is a normal and proper metabolic function of cells during ischemia or hypoxia, and the quantity of adenosine released by EHNA-treated or analog-treated cells was compared to the quantity of adenosine released by control cells, which had been identically stressed by the same toxin without any treatment by EHNA or an analog.
  • the results, in Tables 6 and 7, are expressed in percentages of adenosine release by treated cells, compared to untreated control cells.
  • astrocytoma cells were subjected to actual hypoxia for 2 hours, in an anaerobic chamber while nitrogen gas (rather than oxygen) was bubbled through the cell culture medium. After the hypoxic period release of radiolabelled adenosine into the culture medium (TABLE XXX) .
  • EHNA and its 9-phthalimido analog elevate adenosine release in both simulated and unsimulated hypoxia, and that the phthalimido analog offers a higher level of protection.
  • This ability to help sustain a normal metabolic function, in brain cells, in the face of ischemic insult indicates that the phthalimido analog can help protect brain tissue against ischemic damage.
  • EXAMPLE 9 ADDITIONAL ASSAYS FOR PROTECTION OF BRAIN TISSUE
  • Various assays are currently underway to quantify the ability of several EHNA analogs to reduce hypoxic or ischemic damage in intact brain tissue, as distinct from cultured cells. These assays are being sponsored and funded by the Applicant, Cypros Pharmaceutical Corporation, and are being carried out at the UCLA Medical Center in Los Angeles by an independent investigator. Based upon the results of other tests already completed (including the heart tests and the cultured brain cell tests) , it is anticipated that at least some of the EHNA analogs will display a substantial ability to reduce hypoxic or ischemic damage in intact brain tissue.
  • this example describes experimental protocols that can be used to carry out such evaluative tests, using intact tissue slices in relatively simple, rapid and inexpensive tests.
  • the in vitro assays currently being carried out use intact slices of hippocampal tissue from the brains of sacrificed rats, for two reasons. First, tissue from the hippocampal region of the brain is known to be highly vulnerable to ischemic damage. And second, tests using perfused intact tissue slices are often preferred in neurological research, since intact cohesive tissue often provides a better model of in vivo cell and tissue behavior than either broken cell fragments, or isolated cells cultured under artificial conditions (especially when such cells are cancerous or have been genetically altered to increase their replication rates in artificial cell culture conditions) .
  • CAl° neurons in hippocampal tissue slices will generate electrophysiological responses (comparable to brain waves in living animals) which can be measured quickly and easily, using a device comparable to an electroen ⁇ ephalograph, for several hours. Accordingly, a candidate neuroprotective drug can be tested to find out whether it can prolong, restore, or otherwise increase the ability of neurons in perfused hippocampal tissue slices to generate electrical spikes having normal and desirable amplitudes and frequencies (as distinct from seizure-inducing convulsant drugs, which induce spikes having abnormal amplitudes or frequencies) .
  • CSF cerebrospinal fluid
  • This fluid contains (in mM) NaCl, 126; KCl, 5 4.0; KH 2 P0 4 , 1.4; MgS0 4 , 1.3; CaCl 2 , 2.4; NaHC0 3 , 26; and glucose, 4.0, with a pH of 7.4, saturated with a gas mixture of 95% 0 2 and 5% C0 2 .
  • the chilled brain tissue is then sliced to provide hippocampal tissue slices, which are placed in recording wells with the temperature of the surrounding bath thermostatically
  • PS orthodromic CAl population spike
  • tissue slices are submerged in artificial CSF fluid that dose not contain any EHNA analogs or other neuroprotective drugs. Test samples are treated
  • EHNA or an EHNA analog as described herein.
  • the assays involve subjecting hippocampal tissue slices to hypoxic conditions for limited periods of time, and then measuring the ability of the CAl cells to respond to electrical
  • paired hippocampal slices i.e., two tissue slices from the same animal
  • the perfusion fluid to both wells is changed to artificial CSF containing no free oxygen; the CSF fluid is saturated with 95% N 2 (instead of 35 0 2 ) and 5% CO...
  • One slice in each pair additionally receives exposure to EHNA or an EHNA analog, beginning 30 minutes prior to the onset of hypoxia, and continued until 15 minutes after the termination of hypoxia.
  • the period of hypoxic deprivation is variable for different slices; during hypoxia, each control tissue slice (i.e., each slice that had not been treated by EHNA or an EHNA analog) is monitored to ensure that a "hypoxic injury potential" (HIP; see Fairchild et al 1988) is still present.
  • the hypoxic deprivation for paired slices is terminated by adding oxygen to the perfusion medium 5 minutes after the disappearance of the HIP in the untreated slice.
  • the two paired slices are then monitored for an additional 90 minutes, and brain wave amplitudes are recorded.
  • Final CAl orthodromic PS amplitude is then compared to the original CAl orthodromic PS amplitude which was measured prior to treatment. Antidromic PS amplitude is also assessed before hypoxia, and after 90 minutes of recovery.
  • hippocampal tissue slices are given electrical stimulation every 30 seconds throughout the entire perfusion period, including the hypoxic period.
  • ongoing periodic stimulation imposes additional metabolic demands, which aggravates the excitotoxic injury and provides an even more rigorous test.
  • EHNA analogs described herein offer substantially improved levels of neuroprotection against ischemia, compared to unmodified EHNA or hydroxylated EHNA.
  • Any specific EHNA analog which substantially reduces hypoxic or ischemic damage in these relatively simple and inexpensive in vitro tests can then be tested in intact animals, using in vivo studies as described in articles such as Nellgard and Wieloch 1992 (surgically-induced ischemia in rats) , Buchan and Pulsinelli 1990 (surgical ischemia in gerbils) , Michenfelder et al 1989 W

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AU6297294A (en) * 1993-02-03 1994-08-29 Gensia, Inc. Adenosine deaminase inhibitor therapies

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CA2199615A1 (en) 1997-08-14
AU5522996A (en) 1997-08-28
WO1997028803A1 (en) 1997-08-14

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