WO2001014334A1 - Composes de fluorenone avec substituants de position 7 modifies destines au traitement et a la prevention de lesions medullaires et cerebrales - Google Patents

Composes de fluorenone avec substituants de position 7 modifies destines au traitement et a la prevention de lesions medullaires et cerebrales Download PDF

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WO2001014334A1
WO2001014334A1 PCT/US2000/022990 US0022990W WO0114334A1 WO 2001014334 A1 WO2001014334 A1 WO 2001014334A1 US 0022990 W US0022990 W US 0022990W WO 0114334 A1 WO0114334 A1 WO 0114334A1
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propyl
tetrahydro
dichloro
fluoren
compound
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PCT/US2000/022990
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English (en)
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Edward J. Cragoe, Jr.
Paul J. Marangos
Torsten R. Weimann
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Questcor Pharmaceuticals, Inc.
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Priority claimed from US09/379,656 external-priority patent/US6251898B1/en
Application filed by Questcor Pharmaceuticals, Inc. filed Critical Questcor Pharmaceuticals, Inc.
Priority to AU69240/00A priority Critical patent/AU6924000A/en
Publication of WO2001014334A1 publication Critical patent/WO2001014334A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
    • C07D213/30—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/32—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing keto groups
    • C07C65/40—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing keto groups containing singly bound oxygen-containing groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/04—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D263/06—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hydrocarbon radicals, substituted by oxygen atoms, attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/04—1,3-Oxazines; Hydrogenated 1,3-oxazines
    • C07D265/06—1,3-Oxazines; Hydrogenated 1,3-oxazines not condensed with other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/04—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members

Definitions

  • This invention is in the fields of neurology and pharmacology, and relates to drugs that can minimize brain or spinal cord injury due to various causes, such as traumatic head injury or crises such as stroke, cardiac arrest, or asphyxiation.
  • fluorenone drugs are all within a class of compounds that were first discovered, and recognized to be potentially useful for reducing brain damage, in the 1970's. A great deal of time, effort, and expense were devoted to these drugs, and they were extensively, patented and studied by one of the world's largest pharmaceutical companies, Merck & Company, Inc .
  • Patents in this field which cover certain fluorenone compounds include U.S 4,316,043 (issued in February 1982); 4,317,922 (March 1982); 4,337,354 (June 1982);
  • R, X, and Y 1 and Y 2 are various organic moieties as specified in the prior art patents. These compounds are sometimes called “fluorene” compounds or derivatives, because they are contain a tri-cyclic structure called fluorene, which is shown in the Merck Index and in various articles cited therein which date back to the 1920's.
  • fluorene analog L-644,71 1 shown at the top of Fig. 1 herein, shows the conventional numbering used for the carbon atoms in the three-ring structure of fluorene.
  • Fluorene has no relation to fluorine (the halogen atom) even though both words are pronounced the same.
  • a fluorene compound which bears a double-bonded oxygen attached to one of the three ring structures can be called a "fluorenone" compound.
  • All of the drugs discussed herein are fluorenone compounds, since they bear a double-bonded oxygen attached to the 3-carbon atom.
  • indanone compounds which are bi-cyclic
  • tri-cyclic fluorenone compounds which had increased activity in reducing brain edema without the unwanted systemic side effects produced on body tissues by diuretic agents; this was discussed in Cragoe 1987.
  • L-644,711 was used as a "benchmark" compound in the new research disclosed herein. This new research, which was sponsored and funded by Cypros Pharmaceutical Corporation (Carlsbad, California), identified a number of compounds that are markedly better than L-644,711 as a neuroprotective drug, as measured by appropriate biological assays.
  • the new compounds disclosed herein should be regarded as highly improved fluorenone compounds which perform markedly better than any of the prior art compounds disclosed in any of the patents cited above, or in any other publications that are known to the Inventors herein.
  • glial cells Inside the mammalian central nervous system (CNS, which includes the brain and spinal cord), cells are divided into two major categories: neurons, and glial cells. Neurons are cells which actually receive and transmit nerve signals. By contrast, the term “glial cells” includes a variety of supporting cells which help nourish and protect neurons, but which do not and cannot receive and transmit nerve signals.
  • Glial cells include, but are not limited to: (1) astrocytes, which have cell shapes that resemble a star in certain respects, with a main central portion having various arms projecting outwardly from the central portion; (2) oligodendrocytes, which have several long projecting "dendrites", which usually wrap around certain portions of the neurons, to provide myelin sheaths which surround neuronal dendrites and axons; and (3) microglial cells, which are migratory cells that are part of the immune system inside the brain, and which collect and break down waste products, dead cells, and bacterial cells and viruses inside the brain tissue.
  • glial cells Information on glial cells, and on the interactions between glial cells and neurons inside the central nervous system, is contained in numerous reference books on neurology, such as Principles of Neural Science, 3rd edition, by E. Kandel & J. Schwartz (Elsevier Publishing, New York, 1991), and Encyclopedia ofNeuroscience, edited by G. Adelman (Birkhauser Publishing, Boston, 1987). It should be understood that astrocytes and other glial cells also exist and function in essentially the same manner in a mammalian spinal cord. Accordingly, the drugs disclosed herein are believed to be useful for reducing neuronal damage to a spinal cord as well as to a brain, as further discussed below.
  • Astrocyte cells were selected and used for various tests disclosed herein for a number of reasons, as follows.
  • the complete set of causes and aggravating factors which lead or contribute to astrocyte swelling and edema are not totally understood; however, a sequence of three important cellular reactions are known to be major contributing factors.
  • chloride ions begin entering astrocyte cells in abnormally large quantities by an active process, from surrounding extra-cellular fluids. These ions enter the cells through specialized chloride channels that pass through the astrocyte cell membranes.
  • positively charged sodium ions also begin entering the astrocyte cells in abnormally large quantities by a passive process, due to effect of the excess of negatively charged Cl-ions inside the cells.
  • the third step after the influx of ions into astrocyte cells creates an osmotic imbalance between the intracellular and extracellular fluids, water molecules begin seeping into the astrocyte cells, in an effort to re-establish proper osmotic balances across the cell membranes.
  • the affected astrocyte cells become swollen due to the presence of large quantities of excess water.
  • the medical term for this condition is "edema", which refers to swelling of cells or tissue caused by a combination of (a) entry of too much fluid into the cells or tissue, combined with (b) an inability of the cells or tissue to excrete or otherwise properly manage the excess fluid.
  • astrocyte cells When astrocyte cells become swollen, they begin pressing against the capillaries that provide blood to the brain tissue. Capillary walls inside the brain are very thin and pliable; this is necessary to allow adequate quantities of glucose, oxygen, and other nutrients to permeate out from the blood and through the capillary walls, to provide nourishment to nearby neurons and glial cells.
  • astrocyte edema can severely restrict subsequent blood flow through capillaries that serve an affected region inside the brain. This reduction of capillary blood flow through the brain can quickly become catastrophic, and will lead to severe and possibly lethal brain damage, unless it is relieved quickly. Astrocyte cells can also severely aggravate brain damage after a head injury, due to a second major factor. This factor arises from the fact that in a healthy brain, astrocyte cells help to "mop up" excess quantities of certain types of excitatory neurotransmitters, especially glutamate and aspartate.
  • glutamate and/or aspartate are released by a neuron in order to transmit a nerve impulse to adjacent neurons.
  • a glutamate or aspartate molecule briefly binds to a receptor protein on the surface of the signal-receiving neuron.
  • This interaction between the glutamate or aspartate transmitter molecule and the neuronal receptor provokes a cellular response, which causes ion channels in the signal-receiving neuron to briefly open and allow certain types of ions to enter the neuron.
  • This influx of ions changes the chemical state of the neuron, thereby activating ("depolarizing") the neuron, and causing it to release its own set of neurotransmitter molecules at synapses with other neurons.
  • a neuron As soon as a neuron has been activated (i.e., depolarized), it activates its ion pumps and begins pumping ions back out of the cell, in order to regain its polarized state so it will be ready to receive another nerve impulse. This effort to regain a polarized "ready-to-fire” state requires a neuron to expend substantial amounts of energy. In effect, the "resting state" of a neuron is on a high-energy plateau; it can reach a "ready-to-fire” resting state only by pumping out large quantities of ions.
  • glutamate or aspartate When glutamate or aspartate are used to transmit a nerve impulse, the glutamate or aspartate molecules quickly disengage from the receptor proteins and enter the synaptic fluid again.
  • glutamate and aspartate molecules which have been released from neuronal receptors in this manner are quickly pumped back inside the neurons that released those transmitter molecules, by a cellular transport system which requires energy to run.
  • glutamate and aspartate molecules are not handled properly by this neuronal pumping system, and they diffuse out of the synaptic junctions, in a manner comparable to a slowly dripping faucet. These errant neurotransmitters would pose a serious risk of creating unwanted and possibly destructive nerve impulses, if they were not promptly managed by other mechanisms.
  • astrocyte cells To prevent uncontrolled nerve signals from being triggered by glutamate and aspartate molecules which have gradually leaked out of the synaptic junctions between neurons, astrocyte cells have developed a highly useful "mopping up" function. In simple terms, astrocyte cells will grab any glutamate or aspartate molecules they encounter, and pump those molecules into their cell interiors. Because astrocyte cells do not quickly metabolize and degrade these neurotransmitter molecules, the astrocyte cells gradually accumulate fairly large quantities of glutamate and aspartate molecules. In a healthy brain, this is good and proper; the glutamate and aspartate molecules which are stored inside astrocyte cells do not harm those cells in any way.
  • the stressed astrocytes can begin releasing their stored-up quantities of glutamate and aspartate. If this occurs, the newly-released glutamate and aspartate will begin contacting neurons again, triggering unwanted nerve impulses in uncontrolled ways and at the worst possible time.
  • the neurons will already be under severe stress due to the brain injury which triggered the crisis, and as mentioned above, each time a neuron undergoes a depolarizing event, it immediately begins expending large quantities of energy in an effort to pump out the ions that entered it when the neuron "fired", so it can get ready to receive the next nerve impulse.
  • excitotoxic and "excitotoxin” are used by neurologists to indicate that excitatory neurotransmitters, which play an essential role in a healthy brain, can become deadly neurotoxins in a brain suffering from a crisis.
  • glutamate and aspartate both become excitotoxins, and can kill affected neurons through toxic over-excitation.
  • this invention discloses new compounds which are more potent and effective than any previously known compounds in reducing the release of excitotoxic quantities of glutamate and aspartate by glial cells (including astrocyte cells) following a CNS crisis.
  • glial cells including astrocyte cells
  • correlations between cellular swelling and the release of glutamate and aspartate (excitatory neurotransmitters) inside CNS tissue are discussed in articles such as Bourke et al 1983 and Kimelberg et al 1990.
  • Astrocytes perform various useful functions, including "mopping up” and sequestering glutamate and aspartate (excitatory neurotransmitter) molecules that have improperly leaked out from the synaptic junctions between neurons. This prevents the excitatory neurotransmitters from randomly triggering spurious and harmful nerve impulses.
  • astrocytes can begin releasing their sequestered glutamate and aspartate molecules. If this type of excitatory transmitter release occurs as a result of a head injury, stroke, or other crisis, it can severely aggravate the extent of neuronal damage and death inside the brain or spinal cord.
  • astrocyte cells can become swollen and engorged with water, due to process that involve (i) abnormally high influx of ions into the astrocyte cells, followed by (ii) efforts by the cells to take in more water in order to re-establish proper osmotic balances across the cell membranes.
  • This fluid-swollen condition is called "edema”.
  • astrocyte cells become swollen (edematous), they begin pressing against the walls of the capillaries that supply oxygen and nutrients to the brain. Since capillary walls are very thin, they cannot resist that type of squeezing pressure from swollen astrocyte cells. As a result, astrocyte swelling can further choke off and reduce the supply of fresh blood to affected regions of the brain or spinal cord, thereby severely aggravating cellular death and damage in the affected parts of the brain and spinal cord.
  • This invention discloses new compounds which are much more potent and effective than any previously known compounds in reducing the release of excitotoxic quantities of glutamate and aspartate by glial cells (including astrocyte cells), following a CNS crisis such as a head or spinal injury or stroke. Since these new compounds exert this effect, they are referred to herein as "GERI” compounds, where GERI is the acronym for "Glial Excitotoxin Release Inhibitor”.
  • GERI is the acronym for "Glial Excitotoxin Release Inhibitor”.
  • This activity has been shown using an assay involving the release of radiolabelled aspartate by osmotically-stressed astrocytoma cells, described in detail in the Example Section. This assay is described briefly below, and in greater detail in the examples in the simultaneously-filed application, "Medical Use of Fluorenone Derivatives for Treating and Preventing Brain Injury”.
  • Glial Excitotoxin Release Inhibitors does not imply that their GERI function is their only known useful activity. A correlation was observed during the astrocyte assays, indicating that the potency of various fluorenone analogs in inhibiting excitotoxin release by stressed astrocyte cells apparently correlates with their ability to also reduce edematous swelling by the cells.
  • the GERI class of fluorenone analogs may be extremely useful in preventing or reducing CNS damage caused by various types of crises as discussed below; and, (ii) the D-aspartate release assay may be useful as an easily measured, readily quantifiable indicator of a GERI compound's ability to minimize astrocyte swelling, and possibly relieve and reduce elevations in intracranial pressure as well, following a head trauma or other CNS crisis. If desired, such correlations can be further elucidated by quantitative measurements of edema in astrocytes, using in vitro assays such as described in O'Connor et al 1993.
  • the GERI compounds disclosed herein are believed to be effective and potent neuroprotective compounds, which can be used to reduce and prevent damage to a mammalian brain and/or spinal cord due to any of the following causes and etiologies:
  • stroke including ischemic stroke caused by thrombosis or embolism, regardless of where a blood clot or other embolus originates in the body;
  • hemodynamic shock such as caused by loss of blood due to injury or hemorrhage elsewhere in the body
  • vasculatory damage as can be caused by vascular disease, certain types of bacterial, viral, or other microbial infection, and other comparable causes
  • cerebral or spinal tumors and, (vi) glial cell swelling caused by infections (such as viral, bacterial, or other microbial meningitis, encephalitis, or encephalomyelitis, Reyes syndrome, or AIDS) or other mechanisms, such as hydrocephalus;
  • hypoxic injury to the brain i.e., inadequate oxygen supply
  • hypoxic injury to the brain i.e., inadequate oxygen supply
  • respiratory disruption as occurs during incipient drowning or suffocation, carbon monoxide poisoning, etc.
  • peri-operative or post-operative CNS injury or stress as can be caused by neurosurgery, or by cardiopulmonary bypass for a prolonged period.
  • one object of this invention is to disclose new drugs which can reduce and minimize brain and spinal damage following traumatic injuries.
  • Another object of this invention is to disclose new drugs which can reduce and minimize brain damage following various types of medical crises, such as strokes, cardiac arrest, and infective or inflammatory processes such as meningitis, encephalitis, or encephalomyelitis.
  • Another object of this invention is to disclose a class of drugs which can minimize astrocyte swelling inside the brain, following an injury or infection that affects the head or central nervous system.
  • Another object of this invention is to disclose a class of drugs which can be used to minimize the release, by astrocyte cells, of excitatory neurotransmitters (especially glutamate and aspartate) inside the brain or spinal cord after a head or spinal injury.
  • another object of this invention is to disclose certain drugs that can markedly outperform and improve upon L-644,711, a preferred compound from the "fluorenone" class of drugs which was extensively studied but never commercialized or made available to the public.
  • the compounds of this invention possess greater potency for the treatment of brain and spinal cord injury, and they also enjoy a broad scope of biochemical mechanism of action.
  • Newly-created fluorenone drugs can be used to prevent, treat, or otherwise reduce damage to the brain or spinal cord of a human patient suffering a medical crisis. These newly created drugs are markedly improved.
  • X is a lower alkyl, substituted alkyl, or cycloalkyl group
  • R is selected from certain types of ether, ester, or amide groups
  • Yl and Y2 are halogen, hydrogen, or methyl.
  • One exemplary compound is R(+)-2-[[(5,6-dichloro-2,3,9,9a-tetrahydro-3-oxo-9a-propyl- lH-fluoren-7-yl) oxy]-methyl]-oxazoline.
  • the newly created analogs were more than 30 times more potent than the previously known best lead compound in reducing aspartate release by stressed astrocyte cells.
  • the new analogs can also help reduce swelling in astrocyte cells, and can thereby help reduce brain damage and promote proper blood flow through the brain following a head injury or other crisis.
  • These new analogs have been shown to work with very good efficacy in in vivo animal models of both global and focal brain ischemia. Accordingly, these compounds can reduce brain or spinal cord damage caused by a hypoxic, ischemic, infective, inflammatory, or other injury, crisis, or insult to the brain or spinal cord.
  • FIGURE 1 depicts a "Method A" synthetic pathway used to prepare GERI- El, a fluorenone ether analog with improved biological activity, using a brominated intermediate. This synthesis is described in detail in Example 1.
  • FIGURE 2 depicts a "Method B” synthetic pathway used to prepare several other fluorenone ether analogs with improved biological activity.
  • FIGURE 3 depicts a "Method C” synthetic pathway used to create GERI-E6, a fluorenone ether analog containing a basic heterocyclic (2-oxazolinyl) group in place of the acidic carboxy group of compound GERI-E5.
  • Similar pathways for creating other intermediates with heterocyclic rings and their reactions with hydroxy compound [2] are described in Examples 8 through 11.
  • FIGURE 4 depicts a "Method D" synthetic pathway used to create a basic heterocyclic 7-substituent. This 3 -step synthetic pathway is described in detail in Example 7, and the resulting compound was designated as GERI-E7.
  • FIGURE 5 depicts a "Method E” synthetic pathway which used an alcohol rather than brominated intermediate to create a fluorenone ether analog, designated as GERI-E12. This synthetic pathway is described in Example 12.
  • FIGURE 6 depicts a general "Method A” for creating fluorenone analogs with substituents attached to the 7-position via ester linkages. This process was used to create an ester compound designated as GERI-Estl, described in Example 15. An alternate process for creating ester analogs is also shown as “Method B", which was used in Example 16 to give GERI-Est2.
  • FIGURE 7 depicts a method for creating fluorenone analogs with substituted aminocarbonylmethoxy substituents attached to the 7-position.
  • These amide-ether compounds are designated as "GERI-AmE” compounds, as disclosed in Examples 17-22.
  • FIGURES 8 A and 8B show the 7-substituents for various ether analogs designated as GERI-E1 through El 4, described in Examples 1 through 14.
  • FIGURE 9 shows the 7-(substituted acyloxy) analogs designated as GERI-Estl and Est2, described in Examples 15 and 16.
  • FIGURE 10 shows the 7-(substituted aminocarbonylmethoxy) analogs designated as GERI-AmE 1 through AmE6, described in Examples 17 through 22.
  • FIGURE 11 depicts the results of excitotoxin release assays, described in Example 23, using the fluorenone analog designated as GERI-E7.
  • FIGURE 12 depicts the results of in vivo assays which tested the ability of compound GERI-E7 to protect against brain damage caused by global brain ischemia in gerbils.
  • FIGURE 13 depicts the results of in vivo assays which tested the ability of compound GERI-E7 to protect against brain damage caused by focal brain ischemia in rats.
  • X is lower alkyl, such as methyl, ethyl, propyl, isopropyl, and the like; lower cycloalkyl, such as cyclopropyl, cyclobutyl, cyclopentyl, and the like; and substituted lower alkyl, such as 2-hydroxyethyl, chloroethyl, trifluoromethyl, and the like.
  • R is selected from among R 1 groups which consist of substituted alkyl such as hydroxyalkyl, polyhydroxyalkyl, di(hydroxyalkyl) alkyl, tri(hydroxyalkyl) alkyl, alkoxyalkyl, dialkoxyalkyl, haloalkyl, aminoalkyl, alkylaminoalkyl, dialkylalkyl, substituted alkylamino such as hydroxyalkylaminoalkyl, di(substituted-alkylamino) alkyl such as di(hydroxyalkyl)aminoalkyl; aryl or substituted aryl groups, wherein the aryl group may be substituted by halo, carboxy, amino, alkyl, alkoxy, alkoxycarbonyl, or alkanoyl groups; and aralkyl groups, wherein the alkyl group can be lower alkyl and wherein the aryl group may be substituted as above; heterocyclic, substituted heterocyclic and heterocyclic-alky
  • R may also be alkanoyl or substituted alkanoyl which can be represented by R 2 -C(O)- wherein R 2 is alkyl or substituted alkyl where the substituent is alkoxy, dialkylamino and the like.
  • R may also be represented by R R 4 NC(O)CH 2 - where R 3 can be alkyl (unbranched, branched, unsubstituted) or substituted alkyl wherein the substituent is: amino, dialkylamino, guanidino, hydroxy (such as where R 3 -alkyl is 2, 2-di(hydroxymethyl)propyl or 2, 2-di(hydroxymethyl)-3-hydroxypropyl), alkoxy (such as where R 3 -alkyl is 2,2-di(methoxy)ethyl, 2,2-di(ethoxy)ethyl, or 3,3-di(methoxy)propyl), aryl or substituted aryl (such as where R 3 -alkyl is benzyl, phenethyl, chlorobenzyl, methoxybenzyl, ethoxybenzyl, aminobenzyl, hydroxybenzyl, carboxybenzyl, acetylbenzyl, methylbenzyl, 2-
  • R 4 can be hydrogen, a lower alkyl group, such as a methyl or ethyl group; or generally any other group such as amino which does not generate steric hindrance or interference when coupled to the nitrogen atom along with the R 3 group.
  • the 5-position and 6-position substituents, represented by Y 1 and Y 2 can be halogen, hydrogen, or methyl. Especially preferred are 5,6-dichloro compounds, as illustrated above and in the drawings. Other halogen, hydrogen, or methyl substituents can be incorporated at these positions by methods disclosed in the prior art cited above, or by other methods known to those skilled in the art.
  • the 9a-carbon atom (to which the X-substituent is attached) is chiral, and therefore the compounds of the invention may be racemic.
  • these compounds or their precursors can be resolved so that the pure (or essentially pure) enantiomers can be prepared; thus, the invention includes the pure or essentially pure enantiomers.
  • the racemates possess one enantiomer which is much more active than the other one.
  • the less active enantiomer may possess some level of toxicity, and may depress or interfere with the inhibitory action of the more active enantiomer at the tissue level.
  • the R(+) is the more active enantiomer (i.e. compounds in which X is ethyl or propyl) by a wide margin.
  • the more biologically active enantiomer may be the (-) configuration (e.g., where X is cyclopentyl) and the margin of difference in the biological activity of the two enantiomers may be considerably more narrow.
  • R 5 is dimethylaminoalkyl, hydroxyalkyl, polyhydroxyalkyl, substituted aralkyl, heterocyclic-alkyl, alkanoyl, substituted alkanoyl, alkanoyloxyalkyl, or substituted alkanoyloxyalkyl.
  • R 5 may also be represented by R 6 R 7 NC(O)CH 2 - wherein R 6 is alkyl, hydroxyalkyl, dialkoxyalkyl, aralkyl, substituted aralkyl, or heterocyclic-alkyl, and R 7 is H or lower alkyl or hydroxyalkyl. Also included are the enantiomers of each racemate, and the corresponding esters and salts.
  • 2- ⁇ [(5,6-dichloro-2,3,9,9a-tetrahydro-3-oxo-9a-propyl- lH-fluoren-7-yl)oxy] methyl ⁇ -oxazoline, and the corresponding salts and enantiomers, particularly the R(+) form.
  • R- (+) - 5,6-dichloro-9a-propyl-7-(3-pyridylmethoxy)-2,3,9,9a- tetrahydro-lH-fluoren-3-one are also preferred.
  • R- (+) -5,6-dichloro- 9a-propyl-7- (2-pyridylmethoxy) -2,3,9,9a-tetrahydro-lH-fluoren-3-one are also preferred.
  • the pure or essentially pure enantiomers since, in most instances, one enantiomer is more active biologically than its enantiomer.
  • the pharmaceutically acceptable salts of the novel compounds of this invention since a major medical use of these compounds is solutions of their soluble salts which can be administered parenterally.
  • FIGURE 1 depicts a "Method A" synthetic pathway used to prepare a fluorenone analog with improved biological activity. This synthesis, described in detail in Example 1 , was used to prepare a compound designated as GERI-E1.
  • Example 1 The "Method A" synthesis pathway disclosed in Example 1 and Figure 1 can be modified in ways that will be obvious to those skilled in the art, to create a variety of other fluorenone analogs having 7-substituent groups that are bonded to the fluorene structure via ether linkages.
  • FIGURE 2 depicts a "Method B" synthetic pathway used to prepare another fluorenone analog with improved biological activity, which can be viewed as an analog of L-644,711 bearing an ortho-interphenylene moiety inserted between the carboxy and methylene groups. This synthesis, described in detail in Example 3, was used to prepare a compound designated as GERI-E3.
  • FIGURE 3 depicts a "Method C" synthetic pathway for preparing another fluorenone analog with improved biological activity, which can be viewed as an analog of GERI-E5 bearing a basic heterocyclic (2-oxazolinyl) group in place of an acidic carboxy group.
  • This synthesis is described in detail in Example 6. It should be noted that Methods A, B, and C all use brominated intermediates to form the desired ethers, as shown in Figs. 1 through 3.
  • FIGURE 4 depicts a completely different (“Method D") synthetic pathway to create various 7-heterocyclic-alkyl ethers by cyclization of the appropriate acyclic intermediate.
  • Example 7 An example of this synthesis pathway is described in Example 7, and was used to create compound GERI-E7. Because of its performance in the aspartate release assay, the GERI-E7 analog was selected for testing in animals. It was shown to be very effective in reducing brain damage caused by either global or focal brain ischemia in the animal models used.
  • FIGURE 5 shows yet another synthesis pathway, designated as “Method E”, in which an alcohol intermediate reacts with compound [2]. This pathway was used to create analog GERI- 12, as described in Example 12.
  • Fig. 5 also cites Cragoe et al, J. Med. Chem 29: 825-841 and US patents 4,356,313 (Cragoe et al 1982) and 4,731,471 (Cragoe et al 1988).
  • This prior art teaches how to introduce a selected group (such as a methyl, ethyl, propyl, or other alkyl group, a hydroxyethyl or other hydroxyalkyl group, and the like) to the 9a-position of a fluorenone structure, as indicated by the variable "X" group shown in Fig. 5.
  • the pathway described in the cited article and patents uses an indanone reagent as a starting compound, and generates a fluorenone compound with the general structure as shown. Racemic forms can be resolved by procedures like those described in the cited article and patents.
  • FIGURE 6 illustrates a general "Ester Method A” for preparing esters derived from hydroxy compound [2].
  • This method described in detail in Example 15, was used to generate R-(+) -7-acetoxy-5,6-dichloro-9a-propyl-2,3,9,9a-tetrahydro-lH-fluoren-3-one, designated as GERI-Estl .
  • esters with other "R 2 " groups at the 7-position can be generated, by using a different anhydride reagent having the desired R 2 groups. Since anhydrides tend to be difficult to prepare and work with, preferred R 2 groups for this approach include alkyl, substituted alkyl, aryl, and substituted aryl groups.
  • Example 16 An alternate "Ester Method B” for creating ester linkages at the 7-position is described in Example 16.
  • a reactant bearing a carboxylic acid group such as N,N-dimethylglycine
  • CDI carbonyldiimidazole
  • the hydroxy group from the carboxylic acid is replaced by a 1 -imidazolyl group to generate a potent acylating compound.
  • hydroxy compound [2] the desired ester linkage and a 7-substituent as shown for GERI-Est2 (shown in Fig. 9) is formed.
  • FIGURE 7 illustrates a general method for synthesizing fluorenone analogs bearing a R 3 R 4 NCOCH 2 O- moiety in the 7-position. These compounds are referred to herein as “amide-ether” compounds, and are designated as the "GERI-AmE” series of compounds. Examples 17 through 22 describe the use of this method for generating a number of amide-ether compounds having the 7-substituents shown in Fig. 10.
  • IC 50 values Several exemplary results from the astrocyte cell tests, expressed as IC 50 values, are provided in Table 1. These "inhibitory concentration, 50%" values indicate the micromolar concentration of a tested compound which was effective in suppressing the amount of aspartate released by the stressed astrocyte cells by 50%, when compared to stressed cell populations that were not treated by a test compound. A low IC 50 value indicates that a compound is highly potent.
  • FIG. 11 displays the results obtained from essentially the same type of astrocyte cell test as discussed in connection with Table 1. The results are expressed as both a function of time and as a function of concentration. These results were obtained using three different concentrations of compound GERI-E7, which was selected as a lead compound for testing against global or focal brain ischemia.
  • results from the first round of focal assays indicated that the GERI-E7 analog reduced "infarct volumes" by about 30%.
  • the compounds of this invention can be administered by any technique capable of introducing the compounds into the bloodstream, such as by intravenous, intramuscular, subcutaneous, intraperitoneal, or intracisternal injection, or by oral or rectal administration, or by any other suitable form of administration (such as transdermal, nasal, and the like), so long as any such route of administration provides adequate concentrations of the selected compound in the bloodstream.
  • parenteral injection is used, the active compound must be administered in a suitable pharmaceutical formulation, such as an aqueous carrier vehicle.
  • a suitable pharmaceutical formulation such as an aqueous carrier vehicle.
  • the nature of the carrier vehicle is not crucial to this invention, so long as it does not interfere with the desired pharmacological activity of the active agent.
  • Such formulations may comprise a mixture of one or more active agents, mixed with one or more pharmaceutically acceptable carriers or diluents. Such formulations may also contain one or more compounds to increase the solubility of the active agent in the carrier vehicle, or to increase the extent to which the active agent will permeate through a mammalian blood-brain barrier and contact glial cells within the central nervous system.
  • the drugs of this invention are intended to be used to prevent or reduce brain damage in acute-care crises (such as immediately after a stroke, cardiac arrest, near-suffocation or asphyxiation, or severe blood loss, or in various other medical crises as listed in the Background section), the preferred mode of administration is intravenous injection or infusion.
  • preferred dosage ranges for the compounds disclosed herein will depend on factors which include the nature and severity of the medical crisis, the excitotoxin-release-inhibiting potency of the particular compound being used, and the ability of that compound to readily permeate the blood-brain barrier. In general, if injected intravenously in an initial bolus or within roughly 15 minutes of initial infusion, dosages in the range of about 0.05 to about 50 mg/kg (i.e., milligrams of drug per kilogram of patient body weight) are likely to be useful. These compounds are not intended for over-the-counter sale or use; instead, the preferred dosage for any specific patient can be easily determined by one skilled in the art. Useful pharmaceutical compendia including Remington's Pharmaceutical Sciences by E. M. Martin.
  • infusion dosages for prolonged administration must be determined for a specific patient by a treating physician.
  • Several of the compounds disclosed herein are prone to gradual chemical degradation due to hydrolysis, after they have been mixed with an aqueous carrier solution. Such hydrolysis is generally believed to accumulate gradually, over a span of several days or weeks, in those compounds in which hydrolysis has been observed.
  • a selected compound with otherwise desirable activities suffers from an undesirably high rate of hydrolysis, it can be manufactured using non-aqueous solvents (or, if necessary, the final preparative steps can be carried out using non-aqueous solvents or by taking steps to minimize the duration of any aqueous steps).
  • Such compounds can be packaged in dehydrated form (with a desiccating agent if desired), and mixed with an aqueous carrier liquid (if such a carrier liquid is necessary) shortly before injection or other administration. Manufacturing methods, packaging devices, and reconstitution procedures that are suited for such handling are well-known and conventional in the art.
  • pharmaceutically acceptable as used herein embraces those characteristics which make a drug suitable and practical for administration to humans.
  • a compound must be sufficiently chemically stable under reasonable storage conditions to have an adequate shelf life, and it must be physiologically acceptable and have an adequately low level of toxicity and adverse side effects, when introduced into the body by a suitable route of administration.
  • a compound is intended to be administered by injection, it preferably should have adequate solubility in water to allow it to be dissolved in an injectable aqueous carrier; however, solubilizing agents such as polyhydroxy compounds or dimethyl sulfoxide can be used to increase aqueous solubility, if necessary.
  • enantiomer or salt must be effective in reducing swelling of, or neurotransmitter release by, at least one type of glial cell (such as astrocyte cells) when such cells are stressed (such as by osmotic stress, or by hypoxic or ischemic stress). Any such salt or enantiomer must also be able to reduce neuronal damage in at least one type of scientifically accepted in vivo model of ischemic, hypoxic, or other insult to the brain or spinal cord.
  • the neuroprotective activity and potency of such compounds is likely to correlate with either or both of the following, which can be measured by in vitro assays: (i) suppression of swelling of at least one type of glial cell (such as astrocyte cells), when such cells are subjected to appropriate types of osmotic, hypoxic, or ischemic stress; and, (ii) suppression of glutamate and/or aspartate release by glial cells that are subjected to appropriate types of osmotic, hypoxic, or ischemic stress.
  • glial cell such as astrocyte cells
  • salts can include salts of free acids or free bases.
  • salts made from fluorenone analogs that are acidic include sodium, potassium, ammonium, trimethylammonium, piperazinium, guanidinium, 1-methylpiperazinium, bis- (2-hydroxyethyl)ammonium, N-methylglucosammonium salts, and the like.
  • the invention as it relates to such basic compounds includes pharmaceutically acceptable salts such as the hydrochloride, hydrobromide, isothionate, maleate, sulfate, methanesulfonate, sulfate, acetate, succinate, and citrate salts and the like.
  • any references to compound or reagent [1] refer to the prior art benchmark compound, L-644,711 , which was used as a starting reagent in a number of the syntheses listed below.
  • Compound [1] which is illustrated in Fig. 1, was prepared by the method described in Cragoe et al, J Med. Chem. 29: 825-841 (1986).
  • Compound [2], also shown in Fig. 1, is the hydroxy (or phenol) intermediate that was used to prepare various ether and ester analogs. It was prepared by cleaving the carboxymethyl group from compound [1], as described in Cragoe et al, J Med. Chem. 29: 825-841 (1986).
  • This second intermediate (675 mg, 0.99 mmol) was dissolved in MeOH (15 ml), then water (5 ml) and TFA (5 ml) were added. The reaction was stirred at room temperature until TLC (20% MeOH/CHCl 3 ) indicated a complete reaction. The mixture was evaporated to dryness and the product was isolated by silica gel chromatography (20% MeOH/CHCl 3 ). A total of 205 mg (48%) of the final product was obtained.
  • This compound is prepared as in Example 1 , except that the methyl 3-(bromomethyl)benzoate is replaced by an equimolar amount of N-(2-bromoethyl)phthalimide. Hydrolysis of the first formed 7-[(2-phthalimido)ethoxy] compound provides the desired product, designated as GERI-E2.
  • methyl 2-(bromomethyl)benzoate reagent methyl 2-methylbenzoate (Aldrich Chemical Co., 5.97 g, 40.0 mmol) was treated with N-bromosuccinimide (7.1 g, 40.0 mmol) in the presence of benzoyl peroxide (53 mg) in tetrachloromethane.
  • the reaction mixture was refluxed overnight under N 2 atmosphere, and was monitored by TLC using 5% EtOAc/hexane. After the reaction was completed, the solvent was removed by evaporation and the residue was separated between water and EtOAc.
  • the intermediate (1.2 g, 2.6 mmol) was dissolved in THF/methanol (100 ml, 1:1). NaOH pellets (2 g) were added, and the reaction was stirred at room temperature. When TLC (100% EtOAc) indicated complete reaction, the mixture was evaporated to dryness. The crude residue was purified by silica gel chromatography using 60% EtOAc/toluene, then 100%EtOAc, and finally 100% THF. 360 mg (30%) of the sodium salt product were obtained.
  • This synthesis requires a brominated intermediate, which is prepared by a four-step process, as illustrated in Fig. 3.
  • p-Toluic acid is first converted to N-(2-hydroxyethyl)-4-toluamide, using carbonyldiimidazole (CDI) and ethanolamine.
  • This first intermediate is then converted to a N-[2-methanesulfonyloxy] -4-toluamide, using methanesulfonyl chloride.
  • This second intermediate is then cyclized to form 2-(4-methylphenyl)oxazoline by processes analogous to those described in Example 7, below.
  • This third intermediate is then brominated, using N-bromosuccinimide and benzoyl peroxide as described in Example 3, to produce 2-[(4-bromomethyl)phenyl]-oxazoline.
  • This intermediate is then reacted with hydroxy compound [2], using a procedure similar to that described in Example 3, to give the desired compound, GERI-E6.
  • o-toluic acid or m-toluic acid serves as the starting material.
  • the remaining three synthetic steps are conducted in a manner analogous to that described for the synthesis of GERI-E6.
  • This compound was synthesized in three major steps, as shown in FIG. 3.
  • compound [1] (365 mg, 1.0 mmol) was dissolved in dry THF (2 ml), and CDI (200 mg, 1.2 mmol) was added. The mixture was stirred for 5 mm and ethanolamine (92 mg, 1.5 mmol) was added. After TLC (15%MeOH/CHCl 3 ) indicated complete reaction, EtOAc was added to the reaction mixture and washed with 10% aqueous citric acid solution and then water. The organic phase was dried with sodium sulfate, evaporated, and purified by silica gel chromatography (15% MeOH/CHCl 3 ).
  • This compound designated as GERI-E7, was highly potent as measured by the
  • -2-oxazoline The starting material for this synthesis is R(+)[(5,6-dichloro-2,3,9,9a- tetrahydro-9a-(2-hydroxyethyl)-3-oxo-lH-fluoren-7-yl)oxy]acetic acid, prepared as described in Cragoe et al, J. Med. Chem. 29: 825-841 (1986).
  • This compound has the same structure as the L-644,711 benchmark compound, except that it has a 2-hydroxyethyl group in the 9a-position, rather than a propyl group.
  • This compound is prepared in five steps, beginning with R(+)-N- [2-hydroxyethyl] [(5, 6-dichloro-2, 3,9, 9a-tetrahydro-3-oxo-9a-propyl-lH-fluoren-7-yl)oxy] acetamide, prepared as described in the first step of Example 7.
  • reaction of this compound with ethylene glycol and p-toluene-sulfonic acid gives the corresponding 3 -spiroketal compound.
  • treatment of the spiroketal compound with Lawesson's reagent gives the corresponding thioamide.
  • This compound is prepared by the 3-step process described in Example 7, except an equimolar amount of 3-aminopropanol is used in place of ethanolamine.
  • 2-(2-bromoethyl)-2-(tetrahydropyran-2-yloxymethyl)-l,3-bis-(tetrahydropyran-2-yloxy) propane is prepared in a two-step process starting with 3-bromopropionic acid. Reaction of this acid with CDI followed by ethanolamine gives N-(2-hydroxyethyl)-3-bromopropionamide. Treatment of this compound with triphenylphosphine (PPh 3 ) and DEAD in THF yields 2-(2-bromoethyl) oxazoline.
  • Example 12 Compound GERI-E12: R(+ -2-[T(5.6-dichloro- 2.3.9. 9a-tetrahvdro-
  • TPP Triphenylphosphine (TPP; 254 mg, 0.97 mmol) and diethyl azodicarboxylate
  • GERI-E12 The synthesis of this compound, designated as GERI-E12, serves as an example of a synthetic route that uses an alcohol intermediate rather than a brominated intermediate to generate a final ether product.
  • This compound is prepared as described in the first step of Example 4, except that an equimolar amount of (3-bromo-methyl)pyridine hydrobromide is used instead of methyl 3-(bromomethyl)benzoate, and the molar amount of sodium carbonate is doubled.
  • the 2-pyridylmethoxy (ortho) and 4-pyridylmethoxy (para) isomers can also be prepared by essentially the same method, by using either (2-bromomethyl)pyridine hydrobromide or (4-bromomethyl)pyridine hydrobromide as a starting reagent, in place of (3-bromomethyl)pyridine hydrobromide as described above.
  • This compound is prepared by first reacting N,N-dimethyl-glycine with CDI in THF, to generate l-(dimethylaminoacetyl)-imidazole. This intermediate is then treated with compound [2] under conditions that generate the dimethylaminoacetoxy compound designated as GERI-Est2.
  • This compound was synthesized by dissolving compound [1] (240 mg, 0.65 mmol) in dry CH 2 C 1 2 (2 ml). CDI (317 mg, 1.95 mmol) was added and the reaction was stirred for 10 minutes. Benzylamine (214 ⁇ l, 1.95 mmol) was added dropwise. When TLC (EtO Ac/toluene 20:1) indicated the reaction was complete, dichloromethane was added and the solution was washed with water, 10%) citric acid solution, and water again. The organic phase was dried with sodium sulfate, filtered, and evaporated. Silica gel chromatography (5% MeOH/CH 2 Cl 2 ) was used to purify 265 mg (89% yield) of the product.
  • This compound was synthesized by dissolving compound [1] (0.5 g, 1.36 mmol) in dry THF (5 ml). CDI (235 mg, 1.4 mmol) and l-(3-aminopropyl)imidazole (250 ⁇ l, 2 mmol) were added, and stirring was continued overnight at room temperature. When thin layer chromatography (TLC) using 10% MeOH in CH 2 C1 2 indicated complete reaction, toluene and 10% ethyl acetate were added to the reaction mixture. The reaction mixture was washed with water, the organic phase was dried with magnesium sulfate, filtered, and evaporated to give 442 mg of a slightly yellowish powder (66% yield). This compound was analyzed by nuclear magnetic resonance, and the results were as follows:
  • This compound is prepared by using several steps that were developed and used to synthesize various related compounds of the invention.
  • compound [1] is dissolved in dry THF, and CDI is added. The mixture is stirred for 5 min, and the ethyl ester of sarcosine is added.
  • this ester is hydrolyzed to the corresponding acid by dissolving in dichloromethane (DCM), and adding MeOH, water, and NaOH.
  • DCM dichloromethane
  • MeOH MeOH
  • water MeOH
  • NaOH MeOH
  • the organic layer is washed with water and dried over Na 2 SO 4 .
  • this intermediate is dissolved in DCM and triethylamine. Then methanesulfonyl chloride is added dropwise. After stirring overnight, more methanesulfonyl chloride and a catalytic amount of 4-(dimethylamino)pyridine (DMAP) are added. After TLC indicates complete reaction, the mixture is diluted with EtOAc and washed with water. The organic phase was dried with sodium sulfate, filtered and evaporated. This generates an intermediate wherein the 7- position substituent is CH 3 SO 2 OCH 2 CH 2 NHC(O) CH 2 N(CH 3 ) C (O) CH 2 O-.
  • DMAP 4-(dimethylamino)pyridine
  • this intermediate is dissolved in DCM, and MeOH and water are added. Hydrolysis is started by the addition of a sodium hydroxide pellet. The reaction is stirred for 30 minutes, then washed with water several times. The organic phase is washed with saturated sodium bicarbonate solution and finally with water. It is then dried with Na 2 SO 4 , filtered and evaporated. Finally, the product is purified by silica gel chromatography, to give the compound named above and shown in the figures as GERI-AmE6.
  • the UC11-MG astrocytoma cells were cultured in 75 ml vented flasks at 37 °C inside a humidified incubator in 5% CO2/95% air.
  • the cells were grown in RPMI Medium 1640 supplemented with 10% fetal bovine serum and 200 mg/ml gentamicin.
  • the cells were detached and suspended using a 0.0625%) trypsin solution in Buffer A (137 mM NaCl, 5.37 mM KC1, 5.55 mM glucose, 4.17 mM NaHCO3, and 0.54 mM EDTA disodium salt, pH 7.4).
  • the cells were then plated onto 6-well tissue culture plates at a seeding ratio of 4 plates per flask and 4 ml of cell-media suspension per well.
  • the plated cells were used in experiments the next day at approximately 90% confluence. Plates that appeared to be more than 95% confluent were not used, because higher levels of confluence led to uncontrollable differentials in exposure of the cells to culture media and labelled aspartate. All tests were performed in triplicate. All media collections were done at 5 minute intervals, using pipettes, and were followed by promptly loading fresh buffer into each well before any drying occurred.
  • the next set of normal Buffer B added to that well contained a test compound at a known concentration; each compound was tested over a range of concentrations. Cells were then incubated for two 5 minute intervals with solutions of test compounds in normal Buffer B.
  • DMSO was added to the mixture to increase the solubility of the compound in water. DMSO was also tested without any test drugs, and it was shown to have no effect on aspartate release.
  • hypo-osmotic buffer prepared by diluting Buffer B with an equal volume of distilled and deionized H20, resulting in an osmolarity of about 145 mOsm.
  • This hypo-osmotic solution caused the cells to begin taking in excess water, to try to reestablish the normal osmotic gradients that exist across the cell membranes.
  • the water-induced swelling and stress then began to induce release of aspartate by the cells, and the amount of aspartate released by the swollen astrocyte cells was measured by removing the buffer every 5 minutes and testing it in a scintillation counter.
  • the cells were then washed for 20 minutes using isotonic Buffer B, causing neurotransmitter release to return to near baseline levels.
  • the remaining radioactivity i.e., the quantity of D-aspartate that remained inside the cells despite the entire treatment process
  • the wells were washed with an additional 1 ml of distilled and deionized H2O, which was combined with the cell extract in a 7 ml scintillation vial, for counting.
  • the percent of D-aspartate that had been released at each 5 minute interval was calculated by dividing the radioactivity of each 5-minute sample, by the total radioactivity
  • the concentration of test compound that inhibited neurotransmitter release by 50% was determined for each compound by a method that calculated the total area under the curve (AUC) for each of several concentrations, and analyzing the AUC values as a function of concentrations for each test compound.
  • Each gerbil in a test group was then treated with a test compound at either 2 or 20 mg/kg, dissolved in injectable saline and administered intravenously (IV).
  • Control animals were treated with saline. All treatment and control groups had 10 animals per group.
  • Body temperature was maintained throughout the procedure by a heat lamp and rectal probe. The gerbils were sacrificed 72 hours after surgery, and their brains were
  • the GERI-E7 compound provided a substantial and statistically significant reduction in neuronal damage in both of the brain regions that were evaluated and stained with hematoxylin and eosin, and quantitative cell counts of live and dead neurons were made, using light and fluorescence microscopy, in two different parts of
  • GERI-E7 the brain that are highly sensitive to ischemic damage: (i) the subiculum, a zone of transition between the parahippocampus and the hippocampus; and (ii) the CA1 portion of the hippocampus.
  • Visual evaluation of other brain regions also confirmed that GERI-E7 provided significant reductions.
  • Statistical analysis was performed on all damage scores (non-paired student t test). All evaluations were made using double-blinded methods. Damage results which compare control and treated animals for damage in both brain regions are shown in Fig. 12. As indicated by those graphs, the GERI-E7 compound provided a substantial and statistically significant reduction in neuronal damage in both brain regions that were evaluated.
  • Example 25 In Vivo Tests of Focal Cerebral Ischemia
  • compound GERI-E7 was evaluated for reduction of neuronal damage caused by focal brain ischema, in rats. These tests were also performed in the laboratory of Dr. Claude Wasterlain, at the Sepulveda VA Medical Center in Los Angeles.
  • Focal cerebral ischemia was induced by reversibly clipping the right middle cerebral artery (MCA) and the right common carotid artery (CCA), as described in articles such as Kaplan et al 1991.
  • MCA right middle cerebral artery
  • CCA common carotid artery
  • a tracheal incision approximately 2.5 cm long was made, allowing placement of 4-0 suture silk under the CCA. This allowed rapid manipulation of the artery for clip placement.
  • the temporalis muscle was partially excised and a 2 mm burr hole was drilled 2-3 mm rostral to the point of fusion of the zygoma with the temporal bone. Saline was used throughout this procedure for washing and maintenance of moisture.
  • the MCA was occluded below the rhinal fissure with a Codman Microaneurysm Clip #1. After the MCA was occluded the animal was turned onto its back and the CCA was occluded with a Roboz Microaneurysm Clip RS-5424. A moistened piece of cotton was placed on the head wound and the tracheal region was partially closed by suture prior to placement of the animal in a warm cage with an oxygen tent. After a two-hour occlusion period (shown to produce a consistently measurable but non-lethal infarct), the clips were removed and methoxyflurane was administered via a nose cone. Visual verification was made for both artery occlusion, and post-occlusion reperfusion.
  • each animal was treated with either 20 mg/kg IV or with the saline vehicle. All test and control groups had 10 animals. The wounds were closed and the animals were returned to their home cages for 2 hours. Blood flow was then restored, and the GERJ-E7 compound, was administered. Animals were sacrificed 72 hours later, and the brains were perfusion-fixed with 4% paraformaldehyde. Serial sections of the brain were cut and stained with hematoxylin and eosin.
  • Infarct area was measured with to evaluate the volume of the infarct (i.e., the amount of severely damaged or necrotic tissue), using an image analysis system at 8 to 10 levels between the posterior hippocampus and piriform olfactory cortex, to the level of the rhinal fissure.
  • Infarct volume was determined by multiplying the average infarct area by the length of the brain between the posterior hippocampus and the piriform olfactory cortex.
  • Edema was estimated by subtracting the area of the left (ischemic) hemisphere from the right (non-ischemic) hemisphere. Infarct volume and edema values among groups were compared, using the non-paired Students t test.
  • Kimelberg, H.K., et al "Improved recovery from a traumatic-hypoxic brain injury in cats by intracisternal injection of an anion transport inhibitor," Cent Nerv Syst Trauma 4: 3-14 (1987)
  • Kimelberg, H.K., et al "Astrocytic swelling in traumatic-hypoxic brain injury: Beneficial effects of an inhibitor of anion exchange transport and glutamate uptake in glial cells," Mol Chem Neuropathol 77/ 1-31 (1989)

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Abstract

L'invention concerne des médicaments neuroprotecteurs se présentant sous la forme d'analogues de certains composés de fluorénone. Ces composés sont représentés par la formule structurale (I), dans laquelle X est un alkyle inférieur, un alkyle substitué ou un groupe cycloalkyle, R est choisi parmi certains types d'éther, d'ester ou de groupes amide, et Y1 et Y2 sont halogène, hydrogène ou méthyle. Ces nouveaux composés inhibent efficacement la sécrétion indésirable d'excitotoxines par les astrocytes à la suite d'une lésion ou d'une agression du cerveau ou de la moelle épinière. A titre d'exemple, ces nouveaux analogues sont 30 fois plus puissants que le principal composé utilisé précédemment pour réduire la sécrétion d'aspartate par les astrocytes soumis à des tensions. Ces nouveaux analogues peuvent également contribuer à réduire le gonflement des astrocytes, ce qui favorise la circulation du sang dans le cerveau à la suite d'une lésion à la tête ou d'une autre crise. Ces nouveaux analogues agissent avec efficacité chez des spécimens animaux in vivo présentant une ischémie cérébrale locale ou générale. Par conséquent, ces composés permettent de réduire les lésions du cerveau ou de la moelle épinière résultant des crises hypoxiques, ischémiques, infectieuses ou inflammatoires, entre autres, ou des agressions subies par le cerveau ou la moelle épinière.
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