OA10901A - Benzamide treatment of dementia associated with aids virus (hiv-1) infection - Google Patents

Abstract

Benzamide-based compositions are disclosed to have activity as therapeutic and prophylactic agents in the treatment of conditions associated with HIV-1 virus infection, referred to in advanced stages as dementia associated with HIV infection or HIV Dementia.

Description

01 CS CI

BENZAMIDE TREATMENTOF DEMENTIA ASSOCIATED WITHAIDS VIRUS (HIV-1) INFECTION

Field of the Invention

5 This invention relates to the treatment of dementia associated with AIDS virus (HIV-1) infection. More particularly it concems compositions andmethods for prophylactically or therapeutically treating this condition.

Background Information

This Background Information section is divided into two parts. The first 10 provides information on the condition being treated by this invention, thedementia associated with AIDS virus infection. The second providesinformation conceming benzamides and their use as médicaments, benzamidesbeing the active agents employed in the methods and compositions of thisinvention. 15 HIV Dementia (AIDS Dementia Complex)

Acquired Immune Deficiency syndrome (AIDS) is often accompanied by neurological complications at Iater States of the disease. Approximately onethird of adults and one half of children with AIDS eventually hâve thesecomplications. These neurological conditions involve a complex set of 20 cognitive, motor and behavioral dysfunctions which hâve been grouped underthe names "AIDS Dementia Complex" (ADC) or more properly "HIV-associated dementia" or "HIV dementia". As many as 50% of infected childrenhâve neurological déficits manifested as delayed developmental milestones.Neurological diseases associated with HIV infection include myelopathy, 25 peripheral neuropathy and myopathy. The neuropathological alterations that 1 010901 accompany HIV infection in the CNS include myelin pallor, increasedastrogliosis, neuronal loss, and loss of dendritic arborization as well as adecrease in the presynaptic area. Resulting neurologie dysfunction can impairdaily function, work productivity and in severe cases mandate expensive 5 institutional care. Although early losses in mental capacity are not consideredfull-blown dementia, they nevertheless reflect neuronal damage associated withHIV-1. At présent there are no effective thérapies for HIV-dementia. Themédicaments described herein should minimize the neuronal damage andprevent the progression of neuronal damage thus allowing extended functional 10 capabilities of the affected individuals and hence considérable savings tosociety.

In the United States alone, over 1 million individuals are infected withHIV and approximately one third of this group hâve AIDS. Thus, the potentialtarget population for an anti-HIV dementia therapeutic treatment is currently 15 greater than 100,000 patients/year and the target population which wouldacutely benefit from a prophylactic HIV dementia treatment some ten timesthat. The need for treatments of HIV dementia is expected to grow as moreeffective thérapies allow persons with AIDS to live longer.

There is no known cure for AIDS available at the présent time and in 20 the absence of an effective treatment to completely eliminate the virus fromafflicted individuals il is unlikely that any completely effective treatment forHIV dementia is available. Zidovudine (AZT) has been used extensively totreat the AIDS infection. Although there is now doubt as to the long termeffectiveness of this treatment because of high mutational frequency of the virus 25 there is no doubt that AZT has been effective in treating HIV dementia on ashort-term basis. The neurological symptoms associated with HIV dementiahâve been treated with certain drugs. For instance, the psychosis associatedwith HIV dementia has been treated with haloperidol and thioridazine.Molindone has been used for psychotic and delirious HIV dementia patients. 2 010901 > l

Methylphenidate has been used for treatment of dépréssion associated with HIVdementia. Electro-convulsive therapy has been used for HIV-induced stupor.

Ail of these treatments serve to ameliorate symptoms of HIV dementia. Nonetreat HIV dementia, itself. 5 The envelope glycoprotein of HIV, gpl2O, has been implicated in the pathogenesis of HIV dementia. This protein which is shed abundantly byinfected cells has been found to be neurotoxic to neurons in culture at extremelylow concentrations, to impair leaming, to induce cytokines, and to reducecérébral glucose utilization. Hill et al. (.Hill, J.M., Mervis, R.R., Avidor, R., 10 Moody, T.W. and Brenneman, D.E. (1993) Brain Res., 603:222-233.) hâveshown that in néonatal rats, administration of gpl20 causes morphologicaldamage to the brain as well as retardation of the development of complex motorbehaviors.

No approved treatments are available. Use of calcium channel 15 antagonists and NMDA antagoniste hâve been proposed as possible thérapies byLipton. Numerous calcium channel antagonists are available on the market, eg,nimodipine, but NMDA antagonists are still being studied clinically by manycompanies, primarily for acute use in stroke or chronic use in epilepsy andParkinson’s disease. Amantadine, which is on the market as an anti-viral, is 20 now known to possess NMDA antagonist properties. A doser cogener of amantadine, memantidine, is on the market in Europe and has been proposed byLipton as a possible candidate for treatment of HIV dementia. Another agentwhich is available for testing is nitroglycerin. Under certain circumstances, theNO generated front the nitroglycerin can protect neurons front overstim dation 25 of the NMDA receptors with the resulting calcium and glutamate excitotoxicity.However, cardiovascular effects and the extremely erratic pharmacokinetics ofnitroglycerin make this approach seem problematic.

In work related to the présent invention, together with Robert Floyd, Idiscovered that certain nitrone compounds exhibited activity as agents against 3 01CS01 r HIV-dementia. This separate invention is covered in another patent applicationfiled simultaneously herewith.

Benzamides as Médicaments

This invention’s approach to mitigating HIV dementia employs a family5 of benzamide analogues as the active agent. Commonly owned United States patent number 5,472,777 describes certain benzamides and their use in treatingneurological conditions. Commonly owned Patent Coopération Treatyapplication PCT/US96/04538 describes the compounds employed herein anddescribe their use as pharmaceutical compositions for conditions not specifically 10 including HIV-dementia.

References

Other references of interest include:

Lipton, SA, Gendelman, HE (1995) Dementia associated with theacquired immunodeficiency syndrome, New England Journal of Medicine, 15 332(14): 934-940.

Simpson, DM, Tagliati, M (1994) Neurologie manifestations of HIVinfection, Ann lntem Med, 121(10): 769-785.

Lipton, SA (1994) Neuronal injury associated with HIV-1 and potentialtreatment with calcium-channel and NMDA antagonists, Dev Neurosci, 16(3-4): 20 145-151

Danysz, W, Parsons, CG, Bresink, I, Quack, G (1995) Glutamate inCNS disorders, Drug News and Perspectives, 8: 261-277.

Lipton, SA, Choi, YB, Pan, ZH, Lei, SZ, Chen, HSV et al. (1993) Aredox-based mechanism for the neuroprotective and neurodestructive effects of 25 nitric oxide and related nitroso-compounds, Nature, 364: 626-632. 4 010901

S

Dawson, VL, Dawson, TM, Uhl, GR, Synder, SH (1993) Humanimmunodeficiency virus type 1 coat protein neurotoxicity mediated by nitricoxide in primary cortical cultures, Proc Natl Acad Sci, 90: 3256-3259.

Mollace, V, Colasanti, M, Persichini, Bagetta, G, Lauro, GM, Nistico,

5 G (1993) HIV gpl20 glycoprotein stimulâtes the inducible isoform of NO synthase in human cultured astrocytoma cells, Biochem Biophys Res Comm 194:439-445.

Schultz, JB, Henshaw, R, Siwek, D, Jenkins, BG, Ferrante, RJ,Cipolloni, PB, Kowall, NW, Rosen, BR and Beal, MF (1995) Involvement of 10 free radicals in excitotoxicity in-vivo. J. Neurochem. 64: 2239-2247.

Winrow, VR, Winyard, PG, Morris, CJ, Blake, DR (1993) Freeradicals in inflammation: Second messengers and mediators of tissuedestruction, Br Med Bull 49: 506-522.

Lafon-Cazal, M, Pietri, S, Culcasi, M, Bockaert, J (1993) 15 NMDA-dependent superoxide production and neurotoxicity, Nature, 364: 535-537.

Olanow, CW (1992) An introduction to the free radical hypothesis inparkinson’s disease, Armais of Neuroloçy, 32 (supplément): 53-59.

Floyd, R.A. and Camey, J., Nitrone radical traps (NRTs) protect in20 experimental neurodegenerative diseases, in Neuroprotective approaches to the treatment of Parkinson’s disease and other neurodegenerative disorders (Olanow, C.W. Jenner, P. and Youssim, Eds.) Academie Press, New York,New York, in press.

Cao, X. and Phillis, J.W. (1994) a-Phenyl-N-tert-butyl-nitrone Reduces25 Cortical Infarct and Edema in Rats Subjected to Focal Ischemia. Brain Res. 644: 267-272

Zhao, Q., Pahlmark, K., Smith, M.-J., and Siesjo, B. (1994) Delayedtreatment with the spin trap aphenyl-n-tert-butyl nitrone (PBN) reduces infarct 5 010901 size following transient middle cérébral artery occlusion in rats. Acta Physiol.Scad. 152: 349-350.

Oliver, CN, Starke-Reed, PE, Stadtman, ER, Camey, JM and Floyd, RA (1990) Oxidative damage to brain proteins, loss of glutamine synthetaseactivity and production of free radicals during ischemia induced injury to gerbilbrain. Proc. NatlAcad. Sci. USA 87: 5144-5147.

Camey, JM, Starke-Reed, PE Oliver, CN, Landrum, RW, Cheng, MS,Wu, JF and Floyd, RA (1991) Reversai or age-related increase in brain proteinoxidation in enzyme activity, and loss in temporal and spatial memory bychronic administration of the spin-trapping compound N-tert-butyl-a-phenylnitrone. Proc. Natl. Acad. Sci., 88: 3633-3636.

McKechnie, K, Furman, BL, Paratt, JR (1986) Modification by oxygenfree radical scavengers of metabolic and cardiovascular effects of endotoxininfusion in conscious rats, Circulatory Shock, 19: 429-439.

Hamburger, SA, McCay, PB (1989) Endotoxin-induced mortality in ratsis reduced by nitrones, Circulatory Shock, 29: 329-384.

Pogrebniak; HW, Merino, MJ, Hahn, SM, Mitchell, JB, Pass, H1(1992) Spin trap salvage from endotoxemia: The rôle of cytokine downrégulation, Surgery, 112: 130-139.

Edamatsu, R, Mon,A., Packer, L (1995) The spin trapN-tert-a-phenyl-butylnitrone prolongs the life span of the senescence acceleratedmouse, Biochem Biophys Res Comm 211; 847-849.

Achim, C, Heyes, MP, Wiley, CA (1993) Quantitation of humanimmunodefficiency virus, immune activation facyors, and quinolinic acid inAIDS brains, J Clin Invest 91: 2769-2775.

Wesselingh, SL, Power, C, Glass, JD, Tyor, WR et al. (1993)Intracereberal cytokine messenger RNA expression in aquiredimmuniodeficiency syndrome dementia, Annals of Neurology, 33: 576-582. 6 01 C 9 01

Gelbard, HA, Dzenko, KA, DiLoreto, D, delCero, C, delCerro, M,Epstein, LG (1994) Neurotoxic effects of tumor necrosis factor alpha inprimary human neuronal cultures are mediated by activation of the glutamateAMPA receptor subtype: Implications for AIDS neuropathogenesis, DevNeurosci, 15: 417-422.

Selmaj, K, Raine, CS, Farocq, M, Norton, WT, Brosnan, CF (1991)Cytokine cytotoxicity against oligodendrocytes. Apoptosis induced byLymphotoxins, J bnmunol, 147: 1522-1529.

Yeung, MC, Pulliam, L., Lau, AS (1995) The HIV envelope proteingpl20 is toxic to human brain-cell cultures through the induction ofinterleukin-6 and tumor necrosis factor-α, AIDS, 9:137-143.

Pulliam L., Berens, ME, Rosenblum, ML 1988. A normal human braincell aggregate mode! for neurobiological studies, J Neurosci Res 21 :521-530.

Pulliam, L, West, D, Haigwood, N, Swanson, RA (1993) HIV-1envelope gpl20 alters astrocytes in human brain cultures, AIDS Research andHuman Retroviruses, 9: 439-444.

Pulliam, L, Hemdier, B, McGrath, MS (1991) Purified trichosanthin(GLQ223®)) exacerbation of indirect HIV-associated neurotoxicity in vitro,AIDS, 5: 1237-1242.

Robinson, C (1995) N-acetylcysteine, Drugs of the Future, 20(6):559-563.

Sandstrom, PA, Roberts, B, Folks, TM, Buttke.TM (1993) HIV geneexpression enhances T-cell susceptibility to hydrogen peroxide inducedapoptosis, AIDS Res Hum Retroviruses, 9: 1107-1113.

Staal, FJ, Roederer, M, Raju, PA, Anderson, MT et al. (1993)Antioxidants inhibit simulation of HIV transcription, AIDS Res HumRetroviruses, 9: 299-306.

Floyd, RA, Watson,JJ, Wong, PK (1984) Sensitive assay of hydroxylfree radical formation utilizing high pressure liquid chromatography and 7 010901 electrochemical détection of phénol and salicylate hydroxylation products, JBiochem Biophys Methods, 10: 221-235.

Floyd, RA, Henderson, R, Watson, JJ, Wong, PK (1986) Use ofsalicylate with high pressure liquid chromatography and elecrochemical 5 détection (LCED) as a sensitive measure of hydroxyl free radicals inadriamycin treated rats, Free Radical Biol Med, 2: 13-18.

Statement of the Invention

It has now been found that certain benzamide compounds hâve activityin the treatment of AIDS Dementia Complex (HIV dementia). 10 This discovery can take the form of benzamide-based pharmaceutical compositions having activity against HIV-dementia. These compositionsinclude one or more of the acetamidobenzamide, aminobenzamide ornitrobenzamide compounds of Formula I as active agent in a pharmaceuticallyacceptable carrier. (R),

CONHR' I 15 In Formula I R’ is a saturated alkyl of from 3 to 5 carbon atoms, each R is independently -NH-CO-CH3, -NO2 or -NH2, and n is 1 or 2, with the followingprovisos: 1) when n is 1 and R is -NO2 at the 4 position of the ring, R’ is notrerr-butyl, «o-butyl, or propyl; 2) when n is 1 and R is -NO2 at the 2 positionof the ring, R’ is not /jo-butyl or propyl; and 3) when n is 2 and R’ is tert- 8 010901 butyl and both Rs are -NOz, the R groups are not at the 3 and 5 positions of thering. The carrier is preferably an oral carrier but can be an injectable carrieras well. These pharmaceutical compositions can be in bulk form but moretypically are presented in unit dosage form.

In another aspect this invention provides a therapeutic method fortreating a patient suffering from HIV-dementia. This method involvesadministering to the patient an effective HIV-dementia-treating amount of oneor more of the pharmaceutical compositions just described.

In another aspect this invention provides a prophylactic method forprotecting a patient susceptible to HIV-dementia. This method involvesadministering to the patient an effective HIV-dementia prophylactic amount ofone or more of the pharmaceutical compositions just described.

Brief Description of the Drawines

The invention will be further described with référencé being made to thedrawings in which

Fig. 1 is a bar graph showing the protective effect of a benzamide in aHIV-dementia related cell culture test.

Fig. 2 is a bar graph showing the protective effect of a benzamide in aHIV-dementia related cell culture test.

Fig. 3 is a bar graph showing apoptosis response observed in a cellaggregation test with a benzamide.

Fig. 4 is a plot of bioavailability of benzamide as a fonction of time.

Detailed Description of the Invention

The Benzamides

The treatment of this invention employs one or more benzamides as itsactive agent. This invention employs certain acetamidobenzamides,aminobenzamides and nitrobenzamides as active pharmaceutical agents. The 9 . 010901 benzamides are described by Formula I. In this formula, R’ is a saturated alkylof from 3 to 5 carbon atoms and n is 1 or 2.

The acetamido, amino or nitro group (or groups) may be foundanywhere on the ring. Preferred embodiments include when n is 1 and theacetamido group is at the 2, 3 or 4 position of the ring and when n is 2 and theacetamido groups are at the 2 and 3, 2 and 4, 2 and 5, 2 and 6, 3 and 4, or 3and 5 positions of the ring.

With respect to the alkyl substituents, R’, compounds wherein R’ is analkyl which does not hâve a hydrogen on the alpha carbon, that is, the carbonwhich bonds to the nitrogen of the ring, are preferred. Examples of thesepreferred R’ groups are rerr-butyl and rerr-amyl.

Acetamidobenzamides of Formula I of particular interest are:N-rerf-butyl-4-acetamidobenzamide, N-wo-propyl-4-acetamidobenzamide, N-rerr-amyI-4-acetamidobenzamide, N-rerr-butyl-3-acetamidobenzamide, andN-methylcyclopropyl-4-acetamidobenzamide. N-rerf-butyl-4-acetamidobenzamide is the most preferredacetamidobenzamide.

The aminobenzamides and nitrobenzamides employed as active agentsare described by Formula I when R is an amino or nitro group. In thisformula, R’ is a saturated alkyl of from 3 to 5 carbon atoms and n is 1 or 2subject to the same preferences for substituents and their positions set forth withréférencé to the acetamidobenzamides and further subject to the provisos that 1)when n is 1 and R is -NO, at the 4 position of the ring, R’ is not ren-butyl, iso-butyl, or propyl; 2) when n is 1 and R is -NO, at the 2 position of the ring, R’is not tso-butyl or propyl; and 3) when n is 2 and R’ is rerr-butyl and both Rsare -NO,, the R groups are not at the 3 and 5 positions of the ring. 10 010901

Aminobenzamides and nitrobenzamides of Formula I of particular interest asactive agents are: N-iso-propyl-4-nitrobenzamide, N-zm-butyl-3-nitrobenzamide, N-rert-butyl-2-nitrobenzamide, N-n-butyl-4-nitrobenzamide, N-n-propyl~4~nitrobenzamide, N-rerf-butyl-3,5-dinitrobenzamide, N- l-methylpropyl-4-nitrobenzamide, N-zm-butyl-4-aminobenzamide andN-rerr-butyl-3-aminobenzamide.

When the benzamide compound contains an amino group, such as is thecase with N-rerr-butyi-3-aminobenzamide and N-zert-butyl-4-aminobenzamide,the amine functionality can be présent as such or as a sait. In the sait form theamino is protonated to the cation form in combination with a pharmaceuticallyacceptable anion, such as chloride, bromide, iodide, hydroxyl, nitrate,sulfonate, methane sulfonate, acetate, tartrate, oxalate, succinate, or palmoate.When these aminobenzamides are referred to it is to be understood that thesesalts are included as well.

Commonly owned United States Patent number 5,472,983, referred toabove, discloses several benzamides useful in treating neurodegenerativediseases based on their protective action in the MPTP mouse model ofParkinson’s disease. The compound N-rerr-butyl-4-acetamidobenzamide of theprésent invention is an in vivo biotransformation product of one of these.benzamides (N-rerr-butyl-4-nitrobenzamide) which has been found in the bloodof rats and mice to which N-fcrr-butyI'4-nitrobenzamide has been administeredorally.

Mixtures of two or more of these materials may be employed, ifdesired. 11 010901

Pharmaceutical Compositions

The benzamide compound(s) is formulated into pharmaceuticalcompositions suitable for oral or parentéral, e.g. intravenous or intramuscularinjection administration.

The compositions for oral administration can take the form of liquidsolutions or suspensions, powders, tablets, capsules or the like. In suchcompositions, the nitrone or its sait is usually a minor component (0.1 to say50% by weight) with the remainder being various vehicles or carriers andProcessing aids helpful for forming the desired dosing form. A liquid formmay include a suitable aqueous or nonaqueous vehicle with buffers, suspendingdispensing agents, colorants, flavors and the like. A solid form may include, for example, any of the followingingrédients, or compounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, or corn starch; a lubricantsuch as magnésium stéarate; a glidant such as colloïdal Silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent such aspeppermint, sugar, methyl salicylate, or orange flavoring.

In the case of injectable compositions, they are commonly based uponinjectable stérile saline or phosphate-buffered saline or other injectable carriersknown in the art. Again the active nitrone is typically a minor component,often being from about 0.05 to 10% by weight with the remainder being theinjectable carrier and the like.

These components for orally administrable or injectable compositions aremerely représentative. Other materials as well as processing techniques and thelike are set forth in Part 8 of Remington’s Pharmaceutical Sciences. 17th 12 010901 édition, 1985, Mack Publishing Company, Easton, Pennsylvania, which isincorporated by référencé.

One can also administer the compounds of the invention in sustainedrelease forms or from sustained release drug delivery Systems. A description ofreprésentative sustained release materials can be found in the incorporatedmaterials in Remington’s Pharmaceutical Sciences-

Conditions Treated and Treatment Regimens

The conditions treated with the benzamide-containing compositionsgenerally include HIV dementia and the various symptoms which fall within theHIV dementia définition. The benzamide-containing formulations can beadministered to achieve a therapeutic effect and slow or counteract theprogression of HIV dementia or they can be administered prophylactically, topatients not yet exhibiting HIV dementia but exposed to the HIV-1 virus. Thebenzamide-containing composition is administered in manners designed to getthe drug into the patient’s bloodstream and across the blood-brain barrier intothe patient’s brain. One excellent mode for accomplishing this is intravenousadministration. Intravenous dose levels for treating these conditions range fromabout 0.01 mg/kg/hour to about 10 mg/kg/hour, ail for from about 1 to about120 hours and especially 1 to 96 hours. A preloading bolus of from about 10to about 500 mg may also be administered to achieve adéquate steady statelevels. Other forms of parentéral administration, such as intramuscularinjection can be used, as well. In this case, similar dose levels are employed.

While parentéral administration is attractive from a drug delivery pointof view, it should be recognized that the course of HIV infection can stretchover many months or even years so oral dosing may be preferred for patientconvenience and tolérance. With oral dosing, one to three oral doses per day,each from about 0.02 to about 50 mg/kg are called for with preferred doses 13 010901 being from about 0.04 to about 10 mg/kg. These same dosing levels andregimens would be used for prophylactic treatment as well.

In any treatment regimen, the health care professional should assess thepatient’s condition and détermine whether or not the patient would benefït from 5 benzamide treatment. Some degree of expérimentation to détermine an optimaldoing level and pattern may be called for. A positive dose-response relationship has been observed. As such andbearing in mind the severity of the side effects and the advantages of providingmaximum possible protection or amelioration, it may be desired in some 10 settings to administer large amounts of benzamide such as those describedabove.

Methods of Préparation of Compounds

The benzamide compounds employed herein can be prepared usingcommonly available starting materials and readily achievable reactions. 15 One représentative préparation route, which is illustrated with rerf-butyl amine, but which may be used with any alkyl amine, involves the followingreactions: (A) (NO,).

(NO,),

III

14 where X is halo such as I, Br, F or Cl. (B)

Hz XII - (NHj)b

CONHC(CHj)j

IV (C) COClCSjIV - (NHCOCHj). CONHC(CS,)j

In step (A) the Ν-ίεζΐ-butyl nitrobenzamides (III) are formed. Thisreaction should be canried out at températures below 10°C.

This step (A) yields as benzamides III, the compounds of the invention5 where R is -NO2.

In step (B) the nitro groups in the mono- or di-nitro benzamide III aresubjected to réduction. This is commonly carried out with a reducing agentsuch as hydrazine and an appropriate catalyst such as a heterogeneous platinum,iron oxide hydroxide, palladium or nickel catalyst, typically on a support, or 10 ' with hydrogen gas and a catalyst.

This step (B) yields as benzamides IV, the compounds of the inventionwhere R is NH2. 15 010901

In step (C) the amino-benzamides IV are converted toacetamidobenzamides V by reaction with an acetyl halide such asacetylchloride. This reaction is carried out in the presence of a mild base andat low to ambient températures such as - 20 °C to + 20 °C. This yields the 5 compounds of the invention where R is acetamido.

Altemate synthetic schemes may also be used to préparé the compounds.Examples of these altemate routes are set forth below using N-rerr-butyl-4-acetamidobenzamide as the représentative compound. Other compounds maybe prepared using these altemate methods by starting with appropriate starting 10 materials, such as 2- or 3- amino- or nitro-benzonitrile or 2,3-, 2,4-, 2,5-, 2,6-,3,4- or 3,5- diamino- or dinitro-benzonitrile and the appropriate alcohol(Altemate Route 1) or similarly substituted toluene compounds and theappropriate alkyl amine (Altemate Route 3).

Altemate Route 1 15 This route begins with acétylation of, for example, 4-aminobenzonitrile (A) to compound (B) using standard methods. Acid hydrolysis of rerr-butanolin the presence of 4-acetamidobenzonitrile (B), provides a feasible syntheticpathway to N-rerr-butyl-4-acetamidobenzamide.

CN

HaO H·

HjC' NI

H 16 010901

Alteraate Route 2

Acétylation, using standard methods, of the inexpensive starting materialPABA (C) affords a cheap method to produce 4-acetamidobenzoic acid (D).Conversion of (D) to the acid chloride (E) using standard methods (e.g., SOC12) 5 and subséquent amidation using standard methods, such as those describedpreviously, produces N-zerf-butyl-4-acetamidobenzamide from inexpensive rawmaterials.

. Altemate Route 3

Another method for the préparation of the compounds begins with10 acétylation, using standard methods, of, for example, paratoluidine (F) to 4- acetamidotoluene (G). The synthetic intermediate (G) may be converted to 4-acetamidobenzoic acid (D) with common oxidizing agents (e.g., KMnO4) and 17 010901 (* subsequently transformai to N-rerr-butyl-4-acetamidobenzamide as outlined inAltemate Route 2.

F G D

Examples

The invention will be further described by the following Examples. 5 These are provided to il lustrale several preferred embodiments of the inventionbut are not to be construed as limiting its scope which is, instead, defined bythe appended daims. Examples 1 to 19 demonstrate the préparation ofacetamidobenzamides, as well as nitro- and aminobenzamides, which arereprésentative of the benzamide compounds employed in the compositions and 10 methods of this invention. Examples 20 to 24 demonstrate the préparation ofpharmaceutical compositions based on the compounds. Thereafter biologicaltest results illustrating the activity of the compositions of the invention areprovided.

Example 1 15 Préparation of N-rerr-butyl-4-aminobenzamide rerr-Butyl amine (14.6 g, 0.200 mole) was stirred in ethyl acetate (150 mL, purified by washing with 5% sodium carbonate solution, saturatedsodium chloride solution, drying over anhydrous magnésium sulfate, andfiltering through fluted filter paper) and cooled to 5° C with an ice bath. 4- 20 nitrobenzoyl chloride (18.6 g, 0.100 mole) in purified ethyl acetate (75 mL) 18 010901 was added dropwise at such a rate to maintain the température below 10° C.

The ice bath was removed upon complété addition of benzoyl chloride solutionand the reaction stirred for 4 hours. The reaction mixture was then filtered ona Büchner funnel, the filtrate washed three times with 5% HCl, once withsaturated sodium chloride, dried over anhydrous magnésium sulfate, filteredthrough fluted fïlter paper, and the solvent stripped off leaving white crystallineproduct. The product was dried in a vacuum oven at 24 mm and 45° C for14 hours. This procedure produced 17.13 g of crystals of N-rerf-butyl-4-nitrobenzamide (77% yield), mp 162-163° C. Proton nuclear magneticrésonance (89.55 MHz in CDClj) showed absorptions at 8.257 ppm (d, 8.8 Hz,2H; 3,5-aryI H); 7.878 ppm (d, 8.8 Hz, 2H; 2,6-aryl H); 6.097 ppm (bs, 1H;N-H); 1.500 ppm (s, 9H; /erf-butyl H).

Palladium on carbon (5 %, 75 mg) was added to N-rerr-butyl-4-nitrobenzamide (5 g, 22.5 mmole) in 95% éthanol at 55°C. A solution ofhydrazine (1.2 mL) in 95% éthanol (10 mL) was added dropwise over 30 min.and more Pd/C added (75 mg). The reaction was refluxed 3 hours, hydrazine(0.5 g) in 95% éthanol (5 mL) was added and the reaction was refluxed foranother hour. The reaction was filtered on a buchner funnel, the volume ofsolvent reduced under vacuum, and extracted with dichloromethane. Thecombined extracts were dried over magnésium sulfate and solvent stripped,leaving 3.90 g of N-wrr-butyl-4-aminobenzamide (90% yield), melting point125 - 127 °C. 90 MHz proton NMR (in CDC13) showed absorbances at 7.290ppm (2H, d, 8.8 Hz; 2,6 aryl H); 6.368 ppm (2H, d, 8.8 Hz; 3,5 aryl H); 5.45ppm (1 H, bs; NHC=O); 3.727 ppm (2H, bs; aryl-NH2); 1.186 ppm (9 H, s; t-butyl H). 19 010901

Example 2

Préparation of N-ze/7-butyl-4-acetamidobenzamide

Acetyl chloride (0.45 g, 5.7 mmole) in ethyl acetate (25 mL) was addeddropwise to N-ierf-butyl-4-aminobenzamide (1.0 g, 5.2 mmole) and triethylamine (0.58 g, 5.7 mmole) in ethyl acetate at 3° C at such a rate to maintainthe température below 10° C. The reaction was allowed to warm to roomtempérature, stirred 1 hour, and washed with 5% HCl. Recrystallization fromacetone gave 1.08 g N-ierf-butyl-4-acetamidobenzamide (89% yield), meltingpoint 119 - 121 °C. 90 MHz proton NMR (in DMSO-d6) showed absorbancesat 9.726 ppm (1H, bs, N-H); 7.715 ppm (4H, dd, 4.4 Hz; aryl H); 7.295 ppm(1 H, bs; NH); 2.844 ppm (3H, s; CHjCO); 1.448 ppm (9 H, s; t-butyl H).

Example 3

Préparation of N-terf-butyl-3-nitrobenzamide N-rgrr-butyl-3-aminobenzamide and N-terr-butyl-3-acetamidobenzamide

The amidation procedures of Example 1 were followed using 3-nitrobenzoyl chloride instead of 4-nitrobenzoyl chloride. This gave N-tert-butyl-3-nitrobenzamide in 92% yield, melting point 123-125 °C. Proton NMR(in CDClj) showed absorptions at 8.517 ppm (2-aryl H, s, 1H); 8.337 ppm (4-aryl H, d, 8.8 Hz, 1H); 8.121 ppm (6-aryl H, d, 6.4 Hz, 1H); 7.618 ppm (5-aryl H, m, 1H); 6.032 ppm (N-H, bs, 1H); 1.484 ppm (t-butyl H, s, 9 H).

Iron (III) oxide hydroxide catalyzed hydrazine réduction produced N-re/T-butyl-3-aminobenzamide in 53% yield, melting point 118-120 °C. ProtonNMR (in CDCI3) showed absorbances at 7.088 ppm (4-6 -aryl H, m, 3 H);6.794 ppm (2-aryl H, s, 1H); 5.902 ppm (N-H, bs, 1H); 3.145 ppm (aryl N-H,bs, 2H); 1.458 ppm (t-butyl H, s, 9 H).

Acétylation of N-rerr-butyl-3-aminobenzamide as described in Example 2gave N-rert-butyl-3-acetamidobenzamide in 75% yield, melting point 194- 20 . 010901 195°C. Proton NMR (in CDC13) showed absorptions at 7.778 ppm (4-6 -arylH, m, 3 H); 7.392 ppm (2-aryl H, s, 1H); 6.08 ppm (N-H, bs, 1H); 2.174 ppm(acetyl CH3, s, 9 H); 1.500 ppm (t-butyl H, s, 9 H).

Examole 4

Préparation ofN-rerr-butyl-2-nitrobenzamide and N-rgrr-butyl-2-acetamidobenzamide

The method of Example 3 is repeated using 2-nitrobenzoyl chloride inthe amidation step. This yields N-im-butyl-2-nitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-rerr-butyl-2-aminobenzamide.

Acétylation of the aminobenzamide yields N-zerf-butyl-2-acetamidobenzamide.

Example 5

Préparation ofN-iso-propyl-4-nitrobenzarnid& and N-isp-propyl-4-acetamidobenzamide

The method of Example 3 is repeated using 4-nitrobenzoyl chloride andwo-propyl amine in the amidation step. This yields N-z'so-propyl-4-nitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-ûo-propyl-4-aminobenzamide.

Acétylation of the aminobenzamide yields N-iJP-propyl-4-acetamidobenzamide.

Example 6

Préparation ofN-rezT-amyl-4-nitrobenzamide and N-ferr-amyl-4-acetamidobenzamide 21 010901

The method of Example 3 is repeated using 4-nitrobenzoyl chloride andfe/T-amyl amine in the amidation step. This yields N-rerr-amyl-4-nitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-rerr-amyl-4-aminobenzamide.

Acétylation of the aminobenzamide yields N-rerr-amyl-4-acetamidobenzamide.

Example 7

Préparation of N-w>-butvl-4-acetamidobenzamideThe method of Example 3 is repeated using 4-nitrobenzoyl chloride and «o-butyl amine in the amidation step. This yields N-«o-butyl-4- nitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-«o-butyl-4-aminobenzamide.

Acétylation of the aminobenzamide yields N-iso-butyl-4-acetamidobenzamide.

Example 8

Préparation of N-n-butyl-4-nitrobenzamide and N-n-butyl-4-acetamidobenzamide

The method of Example 3 is repeated using 4-nitrobenzoyl chloride andn-butyl amine in the amidation step. This yields N-n-butyl-4-nitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-n-butyl-4-aminobenzamide.

Acétylation of the aminobenzamide yields N-n-butyl-4-acetamidobenzamide. 22 010901

Example 9

Préparation of N-n-propvl-4-nitrobenzamide and N-n-propyl-4-acetamidobenzamide

The method of Example 3 is repeated using 4-nitrobenzoyl chloride and5 n-propyl amine in the amidation step. This yields N-n-propyl-4- nitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-n-propyl-4-aminobenzamide.

Acétylation of the aminobenzamide yields N-n-propyl-4-10 acetamidobenzamide.

Example 10

Préparation of N-1.2-dimethvlpropy1-4-nitrobenzamide and N-1 .2-dimethylpropyI-4-acetamidobenzamideThe method of Example 3 is repeated using 4-nitrobenzoyl chloride and 15 1,2-dimethylpropyl amine in the amidation step. This yields N-1,2-dimethylpropyl-4-nitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-1,2-dimethylpropyl-4-aminobenzamide.

Acétylation of the aminobenzamide yields N-l,2-dimethylpropyl-4-20 acetamidobenzamide.

Example 11

Préparation of N-n-pentyl-4-nitrobenzamide and N-n-pentyl-4-acetamidobenzamide

The method of Example 3 is repeated using 4-nitrobenzoyl chloride and25 n-pentyl amine in the amidation step. This yields N-n-pentyl-4-nitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-n-pentyl-4-aminobenzamide. 23 . U10901

Acétylation of the aminobenzamide yields N-n-pentyl-4-acetamidobenzamide.

Example 12

Préparation of N-2-methylbutyl-4-nitrobenzamide and N-2-methylbutyl-4-acetamidobenzamideThe method of Example 3 is repeated using 4-nitrobenzoyl chloride and 2-methylbutyl amine in the amidation step. This yields N-2-methylbutyl-4-nitrobenzamide. Réduction of the nitrobenzamïde with hydrazine yields N-2-methylbutyl-4-aminobenzamide.

Acétylation of the aminobenzamide yields N-2-methylbutyl-4-acetamidobenzamide.

Example. ,13

Préparation of N-w-pentyl-2-nitrobenzamide and N-fl-pentyl-2-acetamidobenzamide

The method of Example 3 is repeated using 2-nitrobenzoyl chloride andn-pentyl amine in the amidation step. This yields N-rt-pentyl-2-nitrobenzamide. Réduction of the nitrobenzamïde with hydrazine yields N-n-pentyl-2-aminobenzamide.

Acétylation of the aminobenzamide yields N-n-pentyl-2-acetamidobenzamide.

Example 14

Préparation of N-rert-butyl-2.3-diacetamidobenzamideThe method of Example 3 is repeated using 2,3-dinitrobenzoyl chloride in the amidation step. This yields N-rerr-butyl-2,3-dinitrobenzamide. 24 010901 Réduction of the nitrobenzamide with hydrazine yields N-rezr-butyl-2,3-diaminobenzamide

Acétylation of the aminobenzamide yields N-zm-butyl-2,3-diacetamidobenzamide.

Example 15

Préparation of N-/err-amvl-2.4-diacetamidobenzamideThe method of Example 3 is repeated using 2,4-dinitrobenzoyI chloride and rerr-amyl amine in the amidation step. This yields N-zerr-amyl-2,4-dinitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-rezT-amyl-2,4-diaminobenzamide.

Acétylation of the aminobenzamide yields N-rerr-amyl-2,4-diacetamidobenzamide.

Example 16

Préparation of N-rerr-butyl-2.5-diacetamidobenzamideThe method of Example 3 is repeated using 2,5-dinitrobenzoyl chloride in the amidation step. This yields N-rerr-butyl-2,5-dinitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-rerr-butyl-2,5- diaminobenzamide.

Acétylation of the aminobenzamide yields N-rm-butyl-2,5-diacetamidobenzamide.

Example 17

Préparation of N-rg/Î-butyl-2.6-diacetamidobenzamideThe method of Example 3 is repeated using 2,6-dinitrobenzoyl chloride in the amidation step. This yields N-rezï-butyl-2,6-dinitrobenzamide. 25 010901 Réduction of the nitrobenzamide with hydrazine yields N-rerr-butyl-2,6-diaminobenzamide.

Acétylation of the aminobenzamide yields N-rerf-butyl-2,6-diacetamidobenzamide.

Example 18

Préparation of N-re?r-butyl-3.4-diacetamidobenzamideThe method of Example 3 is repeated using 3,4-dinitrobenzoyl chloride in the amidation step. This yields N-/err-butyl-3,4-dinitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-fert-butyl-3,4- diaminobenzamide.

Acétylation of the aminobenzamide yields N-rerr-butyl-3,4-diacetamidobenzamide.

Example 12

Préparation of N-rerr-butyl-3.5-diacetamidobenzamide

The method of Example 3 is repeated using 3,5~dinitrobenzoyl chloridein the amidation step. This yields N-f<?rr-butyl~3,5-dinitrobenzamide. Réduction of the nitrobenzamide with hydrazine yields N-rerr-butyl-3,5-diaminobenzamide.

Acétylation of the aminobenzamide yields N-rm-butyl-3,5-diacetamidobenzamide.

Préparation of Pharmaceutical Compositions

Example 20

The compound of Example 1 is admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnésium stéarate is added as a lubricant. The mixture is formed into 240-270 mg tablets (80-90 mg of active benzamide) in a tablet press. If these 26 010901 tablets were administered to a patient suffering from HIV dementia on a daily,twice daily or thrice daily regimen they would slow the progress of the patient’sdisease.

Example 21

The compound of Example 2 is admixed as a dry powder with a starchdiluent in an approximate 1:1 weight ratio. The mixture is filled into 250 mgcapsules (125 mg of active benzamide). If these capsules were administered toa patient susceptible to coming down with HIV dementia on a daily, twice dailyor thrice daily regimen they would slow or prevent the onset of the HIVdementia. E-xampls.22

The compound of Example 3 is suspended in a sweetened flavoredaqueous medium to a concentration of approximately 50 mg/mL. If 5 mLs ofthis liquid material was administered to a patient suffering from HIV dementiaon a daily, twice daily or thrice daily regimen they would slow the progress ofthe patient’s disease.

Example 23

The compound of Example 4 is admixed as a dry powder with a drygelatin binder in an approximate 1:2 weight ratio. A minor amount ofmagnésium stéarate is added as a lubricant. The mixture is formed into 450-900 mg tablets (150-300 mg of active benzamide) in a tablet press. If thesetablets were administered to a patient suffering from HIV dementia on a daily,twice daily or thrice daily regimen they would slow the progress of the patient’sdisease. 27 010901

Example 24

The compound of Example 14 is dissolved in a buffered stérile salineinjectable aqurous medium to a concentration of approximately 5 mg/ml. If 50mLs of this liquid matériel was administered to a patient suffering from HIVdementia on a daily, twice daily or thrice daily regimen this dose would slowthe progress of the patient’s disease.

It will be appreciated that any of the compounds of Formula I could beemployed in any of these représentative formulations, and that any of theseformulations could be administered in any of these manners so as to treat any ofthe HIV dementia conditions described in this spécification.

Biological Testing

These tests utilized two neural cell culture Systems for determining theefficacy of N-rezt-butyl-4-acetamidobenzamide ("Compound Γ) in reversingneurotoxicity which mimic that observed with HTV dementia. In both assays,human neural cell cultures were used either as a bilayer (neurons on anastrocyte layer) or a three dimensional model (brain cell aggregates). TNF-oc(100 pg/ml) was used as the neurotoxin and the length of incubation was 72hours. A considérable body of evidence supports the notion that TNF-<x is oneof the neurotoxins responsible for HIV dementia. Brain concentrations of TNF-« are elevated in deep grey matter from AIDS patients with mild HTVdementia. Achim, C, Heyes, MP, Wiley, CA (1993) Quantitation of humanimmunodefficiency virus, immune activation facyors, and quinolinic acid inAIDS brains, J Clin Invest 91: 2769-2775. The distribution of messenger RNAexpressing TNF-oc in the brain follows a similar pattern. Wesselingh, SL,Power, C, Glass, JD, Tyor, WR et al. (1993) Intracereberal cytokinemessenger RNA expression in aquired immuniodeficiency syndrome dementia,Armais of Neurology, 33: 576-582. Gelbard et al. hâve shown that HTV-1infected monocytes in culture with astroglial cells produce concentrations 28 C1C9C1 (>200 pg/ml) of TNF-« sufficient to cause neurotoxicity. Gelbard, HA,Dzenko, KA, DiLoreto, D, delCero, C, delCerro, M, Epstein, LG (1994)Neurotoxic effects of tumor necrosis factor alpha in primary human neuronalcultures are mediated by activation of the glutamate AMPA receptor subtype:Implications for AIDS neuropathogenesis, Dev Neurosci, 15: 417-422. TNF-ais reported to cause its neurotoxicity by inducing apoptosis. Selmaj, K, Raine,CS, Farocq, M, Norton, WT, Brosnan, CF (1991) Cytokine cytotoxicity againstoligodendrocytes. Apoptosis induced by Lymphotoxins, J Immunol, 147:1522-1529. Recently, it was shown that gpl20 exerts toxic effects throughinduction of IL-6 and TNF-a. Yeung, MC, Pulliam, L., Lau, AS (1995) TheHTV envelope protein gpl20 is toxic to human brain-cell cultures through theinduction of interleukin-6 and tumor necrosis factor-a, AIDS, 9:137-143.

Brain Aggregate Procedure

Brain cell aggregates were prepared from second trimester abortion(issue as previously described. Pulliam L., Berens, ME, Rosenblum, ML1988. A normal human brain cell aggregate model for neurobiological studies, J Neurosci Res 21 :521-530. Briefly, human brain tissue between 16 and 18weeks gestation are gently dissociated through nylon screens to obtain singlecells. Approximately 4 X 107 cells within 4 ml DME supplemented with 0.6%dextrose, 50 mg/ml gentamicin and 10% FCS are distributed into 25 mlDeLong flasks. Aggregates are constantly rotated and incubated at 37°C in anatmosphère of 10% CO2. After 2-3 days, aggregates are transferred to 50 mlflasks and 5 ml of DME supplemented with 15% FCS (exchange medium)added. Each flask contains several thousand aggregates that can be sampledover time. Five ml of medium is exchanged every other day in culture. After10-12 days in culture samples are taken for histology and trypan blue exclusionis performed to détermine viability. Samples are screened for HIV, HepatitisA, B, C and mycoplasma. Aggregates remain viable for approximately 40 days 29 010901 in culture. Brain cell aggregates are differenüated at the time of sampling inthat they express neural cell markers for identification. Brain cell aggregatescontain ail the cells of the CNS- approximately 40% neurons, 40% astrocytes,10% oligodendrocytes with myelin and 10% microglia. Neural cell 5 apoptosis/death was measured by DNA fragmentation Elisa technique accordingto manufactures directions (Boehringher Mannheim).

Neural Cell Bilaver Procedure

Brain aggregates were prepared as described above. Several aggregatesare placed in each well of a mulü-well chamber slide (Nunc) coated with Cell 10 TAK (Collaborative Research) at a concentration of 20 ug/ml. Cells migratefrom the brain aggregates within 3 days. Astrocytes form a monolayer withneurons on top and rare microglia ( < 1 %)/oligodendrocytes ( < 1 %). Thesecultures are confluent within 1 week. Monolayers can be maintained for up tothree weeks. Characterization of cell types is determined by using

15 immunohistochemistry and the antibodies neuron spécifie enolase (NSE, Dako)for neurons and glial fibrillary acidic protein (GFAP, Dako) for theidentification of astrocytes. Confocal microscopy was used to visualize andidentify neurons and astrocytes by size and shape. Neuronal viability wasdetermined by exposing chambers with and without different treatments to AO 20 and ethidium bromide (EtBr). Neurons and total cell counts were determinedby AO staining with visual confirmation by phase microscopy. Enumération ofcell viability by computerized software was performed at the time ofmicroscopy; in addition, a visual printout of the fields observed alwaysaccompanied the data. 30 010901

Experimental Design

Expérimenté System 1 Neural CellBilayers 2 Neural CellBilayers 3 Brain

Aggregate TNF-oc (pg/ml)0 0 100 100 0 100 100 0 0 100 100

Compound1 (μΜ)0 100 0 100 0 0 100 0 100 0 100

Test compound is N-rerf-butyl-4-acetamidobenzamide.

Results

Experiment 1 (Figure 1): This was a human neural cell bilayerexperiment. N-wrr-butyl-4-acetamidobenzamide ("Compound Γ) showed sometoxicity relative to the control. The TNF-a treatment produced a high degreeof cell death, over 61 %. N-rerr-butyl-4-acetamidobenzamide treatmentproduced substantial protection.

Experiment 2 (Figure 2): This experiment was a repeat of experiment 1using a different brain préparation. Results essentially duplicated those fromthe first experiment, except the TNF- oc treatment gave less neuronal toxicity. 31 010901

Experiment 3 (Figure 3): This experiment utilized human brainaggregates. In this experiment, apoptosis/cell death was measured by animmunoassay for quantitation of cytoplasmic histone-associated DNAfragments. In this experiment, N-re/ï-butyl-4-acetamidobenzamide treatments 5 gave substantial protection both with and without the TNF-« treatments. Thebars in Figure 4 represent the mean of duplicate experiments. The errer bars inthis figure express the individual values.

Physical/Chemical Parameters N-/ezr-butyI-4-acetamidobenzamide was studied to détermine 10 physical/chemical properties which suggest its suitability for this application.

The following results were obtained: N-rezr-butyl-4- acetamidobenzamide t1/2(min) in Aqueous 3000 15 HCI Solution (pHl)

Octanol-Water Partition 31

This shows that N-rert-butyl-4-acetamidobenzamide is lipophilie andslowly cleared from the body. N-iert-butyl-4-acetamidobenzamide is acompound of particular interest for HIV dementia because, at least in the rat, it 20 shows excellent brain distribution, bioavailability and pharmacokinetic profile.N-ferr-butyl-4-acetamidobenzamide is also significantly stable at a pHcommonly observed in the stomach. 32 010901

Brain pénétration of N-rgrr-butyl-4-acetamidobenzamide

Following a 30 mg/kg oral dose, blood and brain samples from the same animais were analyzed for N-ierr-butyl-4-acetamidobenzamide at 4 and 8 hourspost-dose with the following results:

Time Post-Dose Mean Brain Concentration Mean Blood Concentration (hours) Oxg/g) +/- SEM (/ig/ml) +/- SEM 4 8.9 +/- 3.2 43 +/- 7.9 8 9.1 +/- 1.7 39 +/- 4.2

Absolute Bioavailability of N-rerr-butyl-4-acetamidobenzamide Oral Suspension

The absolute bioavailability of N-zerr-butyl-4-acetamidobenzamide in ratswas determined by comparing the area under the curve following a 20 mg/kgdose of the benzamide dissolved in 1 % methyl cellulose. Blood concentrationswere determined at either 0, 0.083, 0.15, 0.5, 1, 2, 4, 8 and 24 hours post-dose (IV) or 0, 0.5, 1, 2, 4 and 8 hour post-dose (oral), and the AUCsdetermined. Four animais were dosed orally and 4 animais were dosed IV.

Route Mean AUC +/- SEM(/xg hr ml'1) Absolute Bioavailability IV 252 +/- 73 Oral 130 +/- 33 52%

The pharmacokinetic profile of a 30 mg/kg dose to Sprague Dawley ratscan be found in Figure 4. The apparent t,y2 for N-zerz-butyl-4-acetamidobenzamide in this experiment was 8 hours, a very long tI/2 for a drugin rat- a good predictor of once-a-day dosing if N-zerr-butyl-4-acetamidobenzamide would ever be dosed in man. Such a dosing regimenwould be a significant therapeutic advantage in the clinic. 33 010901

Further Brain Aggregation Studies

Further studies were conducted as follows:

Experiment # gpl20(ng/ml) TNF-a(ng/ml) Compound* (μΜ) 4 - 0 0 - 1 0.1 - 1 0.3 - 1 3.0 5 0 - 0 1 3 10 _ 1 Test compound is N-rm-butyl-4-acetamidobenzamide.

As shown in the following Table, Experiment 4 showed that atunexpectedly low concentrations, N-rm-butyl-4-acetamidobenzamide providedcomplété protection in human brain aggregates from DNA fragmentation, a 15 measure of apoptosis, induced by 1 ng TNF-α. Some degree of dose proportionality was found. The results at ail test compound concentrations arestatistically significant at p <0.05 by Student t-test from the TNF only group,but, of the compound treated groups, only the TNF + 0.3 μΜ test compoundgroup is statistically significant from the other two treatment groups. 34 010901

Table Experiment 4 Results Experiment DNA Fragmentation % P: (Absorbance + SF.n=3) roteci Control 0.663 + 0.048 - TNF Only 1.592 ± 0.156 -- TNF + 0.1 μΜ Compound1 0.955 ± 0.101 78 TNF + 0.3 μΜ Compound1 0.835 ± 0.051 87 TNF + 3.0 μΜ Compound1 0.801 ± 0.123 90 1 Test compound is N-zert-butyl-4-acetamidobenzamide.

The data above suggests that protection from apoptosis can be achievedat concentrations of approximately 1 μΜ and below. A ΙμΜ concentration of N-zerz-butyl-4-acetamidobenzamide is in theorder of 0.2 pg/ml. To achieve this concentration in rat brain would require ablood concentration of only 1 Mg/ml based on the brain/blood ratio datapresented previously. If some degree of dose proportionality is found withlower doses of N-zezr-butyl-4-acetamidobenzamide, a 6 mg/kg dose to ratsshould achieve this concentration even at 24 hours post-dose (trough value).Using liver blood flow différences to scale the clearance of drug in rats to thatin man as described in Pulliam, L, Hemdier, B, McGrath, MS (1991) Purifiedtrichosanthin (GLQ223®)) exacerbation of indirect HIV-associated neurotoxicityin vitro, AIDS, 5: 1237-1242, a dose of 1.5 mg/kg to man would be predictedto achieve at 24 hours post-dose the 1 μΜ target concentration in the brain forprotection from apoptosis. 35 010901

Consistent with the results above, N-zm-butyl-4-acetamidobenzamidealso provided complété protection in human brain aggregates from toxicityinduced by 1 ng TNF-at, although the concentration of the benzamide neededwas considerably higher than that found to prevent DNA fragmentation. These 5 data are as follows: Experiment LDH Release % Protection Absorbance ± SD (n=) Control 0.875 ± 0.022 — TNF 1.071 ± 0.036 - 10 TNF + 0.1 μΜ Compound* 1.114 ± 0.023 0 TNF + 0.3 μΜ Compound* 1.103 ± 0.034 0 TNF + 3.0 μΜ Compound* 0.864 ± 0.028 100 1 Test compound is N-zerr-butyl-4-acetamidobenzamide. 15 Experiment 5:

In this experiment, N-zerr-butyl-4-acetamidobenzamide providedsignificant protection in human brain aggregates from cell toxicity induced by 1ng gpl20. The différence in absorbance was statistically significant for ailgroups at p< 0.003. 36 010901

Experiment LDH Release % Absorbance ± SD(n=) Protection Control 0.328 ± 0.011 gpl20 0.575 ± 0.008 gpl20 + 3.0 μΜ Compound1 0.427 ± 0.034 60% 1 Test compound is N-rm-butyl-4-acetamidobenzamide.

There was no evidence in this experiment for DNA fragmentationinduced at this concentration of gpl20.

Experiment 6:

Using procedures essentially the same as those described above fordetermining LDH release induced by TNF, programmed cell death (PCD)analysis was performed by ELISA using standardized kits (BoehrigerMannheim). The results were as follows:

Experiment 6A Control PCD 0 ± 0.359 TNF-a 1.18 ± 0.759 TNF-α + 10.0 μΜ Compound*TNF-α + 10.0 μΜ Compound2TNF-or + 10.0 μΜ Compound3 1.15 ± 0.125 1.021 ± 0.099 0.34 ± 0.029

Test compound is N-rerr-butyl-4-acetamidobenzamide.Test compound is N-reri-butyl-4-aminobenzamide.Test compound is N-rm-amyl-4-acetamidobenzamide. 37 010901 PCD0 ± 0.691.16 ± 0.0881.05 + 0.0430.567 + 0.0260.671 ± 0.043

Experiment 6B

ControlTNF-α TNF-α + 10.0 μΜ Compound1TNF-α + 10.0 μΜ Compound2TNF-α + 10.0 μΜ Compound3

Test compound is N-rerf-butyl-4-acetamidobenzamide.Test compound is N-ierr-butyl-4-aminobenzamide,Test compound is N-re/ï-amyl-4-acetamidobenzamide.

Experiment 6C PCD

Control 0 ± 0.032 TNF-a 0.674 + 0.058 TNF-a + 10.0 μΜ Compound4 0.565 ± 0.042

Test compound is N-isopropyl-4-acetamidobenzamide.

Experiment 6D PCD

Control 0 ± 0.018 TNF-a 0.531 ± 0.034 TNF-a + 10.0 μΜ Compound4 0.016 + 0.03 4 Test compound is N-isopropyl-4-acetamidobenzamide.

The data from Experiments 6A-D demonstrate that various benzamidesof this invention provided protection in human brain aggregates from toxicityinduced by 1 ng TNF-α as measured by PCD analysis. 38 010901

In vivo Tests

In order to détermine the effectiveness of this approach for treatingADC, a sériés of in vivo biological tests were carried out.

In vivo Test A

Material and Methods Used

Sodium N-methyl D-glucamine dithiocarbamate (MGD) and the nitrone,PBN, were obtained from OMRF Spin Trap Source, Oklahoma City,

Oklahoma. gpl2O was obtained from Intracel Corporation, Cambridge,Massachusetts. These materials were used in the following preliminary test:

Treatment of Animais: Sprague-Dawley néonatal rats (sixteen siblings)were divided into four groups. Starting at day one after birth until day six, theneonates received 60 μ! subcutaneous injections of the following treatments.Group 1: phosphate buffer-saline (PBS), Group 2: 5 ng gpl20 in PBS, Group3: 5 ng gpl20 plus PBN (50 mg/kg) in PBS, and Group 4: PBN (50 mg/kg) inPBS. Rats were weighed daily and the amount of PBN injected was adjustedaccordingly.

Behavioral Assessments: Time required to perform two developmentalmilestones were measured to détermine the adverse effects of gpl20administration on behavioral development as reported by Hill et al. and todétermine the possible protective action of PBN on these parameters.

Behavioral parameters studied were surface righting (animal placed head downon 45° inclined screen will tum around and climb up.) These two tests hâvebeen shown to be the most sensitive tests for assessment of the neurologicaldisorder caused by gpl20 treatment. Furthermore, they can be examined earlyenough in the life of the animal (day 3 for surface righting and day 6 fornégative geotaxis) that their détermination will not interfère with NO trappingin the brain which we performed at the end of the first week of the life of theanimal. Animais were tested for the time required for surface righting on day 3 39 010901 and day 4 after birih, immediately prior to receiving the injections on thosedays, and on day 6 (2 hrs after the last injection that the animais received) aswell as day 7 (70 hrs following the last injections) for the time required toperform négative geotaxis. The angle chosen for the setting used for négative 5 geotaxis was decreased from 45° (the angle used by Hill et al) to 35° sinceunder the experimental setting employed, animais were not able to stay on thescreen set at 45° and would slide down before being able to make an attempt totum upward.

In vivo Test B 10 Protection by N-zw-butyl-4-acetamidobenzamide from gpl20-induced behavioral changes

The striking results obtained with PBN prompted preliminaryexperiments with N-zerz-butyl-4-acetamidobenzamide in the same model. Theresults are suggestive that N-zerz-butyl-4-acetamidobenzamide is effective as 15 demonstrated by the data shown below obtained on neonates that had beenadministered gpl20 at 10 ng per dose starting on 3 day old animais. N-zerf-butyl-4-acetamidobenzamide was given at an oral dose of 35 mg/kg 2 hoursprior to administering the gpl20. Treatment with N-zerz-butyl-4-acetamidobenzamide and gpl20 continued daily. The négative geotaxis test was 20 conducted on day 6.

Treatment Négative Geotaxis (sec) 3 h Post-Last Dose gpl20 (Day 6) Vehicle 8.89 ± 3.74 gpl20 18.0 ± 13.8 gpl20 + Compound1 8.39 ± 3.94 Compound1 8.56 ± 5.11

Test compound is N-zerz-butyl-4-acetamidobenzamide. 40 010901

The data suggests N-œri-butyl-4-acetatnidobenzamide had a protective effect. 41

Claims (20)

1. Πί'901 WHAT IS CLAIMED IS:

   1. A pharmaceutical composition for treating HIV dementiacomprising a benzamide compound of the formula: (R)n

   CONHR' I wherein R’ is a saturated alkyl of from 3 to 5 carbon atoms, each R is5 independently -NO2 or -NH2 or NHCOCH3, and n is 1 or 2, with the following provisos: 1) when n is 1 and R is -NO2 at the 4 position of the ring, R’ is notierf-butyl, «obutyl, or propyl; 2) when n is 1 and R is -NOj at the 2 positionof the ring, R’ is not âo-butyl or propyl; and 3) when n is 2 and R’ is tert-butyl and both Rs are -NC^, the R groups are not at the 3 and 5 positions of the 10 ring, in a pharmaceutical!y acceptable carrier.
2. 2. The pharmaceutical composition of Claim 1 wherein thebenzamide compound is an acetamidobenzamide of the formula: (NHCOCH3)n CONHR'

   42 CHSC/i where R’ is a saturated alkyl of from 3 to 5 carbon atoms and n is 1 or 2.
3. 3. The pharmaceutical composition of Claim 2 wherein n is 1.
4. 4. The pharmaceutical composition of Claim 3 wherein R’ is rerr-butyl.
5. 5. The pharmaceutical composition of Claim 3 wherein R’ is rerr-amyl.
6. 6. The pharmaceutical composition of Claim 3 wherein thebenzamide compound is N-rerr-butyl-4-acetamidobenzamide.
7. 7. The pharmaceutical composition of Claim 1 wherein the10 carrier is an oral carrier.
8. 8. The pharmaceutical composition of Claim 1 wherein thecarrier is an injectable carrier.
9. 9. A method for treating HIV dementia comprisingadministering to a patient in need of such treating an effective HIV dementia 15 complex-treating amount of a composition of Claims 1-8.
10. 10. The method of Claim 9 wherein the administering is oral.
11. 11. The method of Claim 9 wherein the administering is parentéral. 43 010901 *
12. 12. The method of Claim 11 wherein the administering is byinjection.
13. 13. The method of Claim 9 wherein the treating is therapeutic.
14. 14. The method of Claim 9 wherein the treating is prophylactic.
15. 15. The use of a benzamide compound of the formula: (R),

    CONHR' I wherein R' is a saturated alkyl of from 3 to 5 carbon atoms, eachR is independently -NO2 or -NH2 or NHCOCH,, and n is 1 or 2,with the following provisos: 1) when n is 1 and R is -NO2 at the 4position of the ring, R' is not te/Y-butyl, z'50-butyl, or propyl; 2) 10 when n is 1 and R is -NO2 at the 2 position of the ring, R' is not z'5<9-butyl or propyl; and 3) when n is 2 and R' is reri-butyl andboth Rs are -NO2, the R groups are not at the 3 and 5 positions ofthe ring, in the manufacture of a pharmaceutical composition forthe treatment of HIV dementia. 15
16. 16. The use of Claim 15 wherein the benzamide compound is anacetamidobenzamide of the formula: (NHCOCH3)n CONHR'

    44 r 010901 where R' is a saturated alkyl of from 3 to 5 carbon atoms and n is 1 or 2.
17. 17. The use of Claim 16 wherein n is 1.
18. 18. The use of Claim 17 wherein R' is rerf-butyl.
19. 19. The use of Claim 17 wherein R' is rezr-amyl.
20. 20. The use of Claim 17 wherein the benzamide compound is N-ierr-butyl-4-acetamidobenzamide. 45