CA2252403A1 - 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

CA 022~2403 1998-10-16 BENZAMIDE TREATMENT
OF DEMENTIA ASSOCIATED WITH
AIDS VIRUS (HIV-1) INFECTION

Field of the Invention This invention relates to the treatment of dementia associated with AIDS
virus (HIV-1) infection. More particularly it concerns compositions and methods 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, the dementia associated with AIDS virus infection. The second provides information concerning benzamides and their use as medicaments, benzamides being the active agents employed in the methods and compositions of this invention.

HIV Dementia (AIDS Dementia Complex) Acquired Immune Deficiency syndrome (AIDS) is often accompanied by neurological complications at later states of the disease. Approximately one third of adults and one half of children with AIDS eventually have these complications. These neurological conditions involve a complex set of 20 cognitive, motor and behavioral dysfunctions which have been grouped under the names "AIDS Dementia Complex" (ADC) or more properly "HIV-associated dementia" or "HIV dementia". As many as 50% of infected children have neurological deficits manifested as delayed developmental milestones.
Neurological dise~es associated with HIV infection include myelopathy, 25 peripheral neuropathy and myopathy. The neuropathological alterations that accompany HIV infection in the CNS include myelin pallor, increased astrogliosis, neuronal loss, and loss of dendritic arborization as well as a decrease in the presynaptic area. Resulting neurologic dysfunction can impair daily function, work productivity and in severe cases mandate expensive 5 institutional care. Although early losses in mental capacity are not considered full-blown dementia, they nevertheless reflect neuronal damage associated with HIV-l. At present there are no effective therapies for HIV-dementia. The medicaments described herein should minimi7~ the neuronal damage and prevent the progression of neuronal damage thus allowing extended functional 10 capabilities of the affected individuals and hence considerable savings to society.
In the United States alone, over 1 million individuals are infected with HIV and approximately one third of this group have AIDS. Thus, the potential target population for an anti-HIV dementia therapeutic treatment is currently greater than 100,000 patients/year and the target population which would acutely benefit from a prophylactic HIV dementia treatment some ten times that. The need for treatments of HIV dementia is expected to grow as more effective therapies allow persons with AIDS to live longer.
There is no known cure for AIDS available at the present time and in 20 the absence of an effective treatment to completely elimin~te the virus from afflicted individuals it is unlikely that any completely effective treatment forHIV dementia is a~ailable. Zidovudine (AZT) has been used extensively to treat the AIDS infection. Although there is now doubt as to the long term effectiveness 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 a short-term basis. The neurological symptoms associated with HIV dementia have been treated with certain drugs. For instance, the psychosis associated with HIV dementia has been treated with haloperidol and thioridazine.
Molindone has been used for psychotic and delirious HIV dementia patients.

CA 022~2403 l998- l0- l6 Methylphenidate has been used for treatment of depression associated with HIV
dementia. Electro-convulsive therapy has been used for HIV-induced stupor.
All of these treatments serve to ameliorate symptoms of HIV dementia. None treat HIV dementia, itself.
S The envelope glycoprotein of HIV, gpl20, has been implicated in the pathogenesis of HIV dementia. This protein which is shed abundantly by infected cells has been found to be neurotoxic to neurons in culture at extremely low concentrations, to impair learning, to induce cytokines, and to reduce cerebral glucose utilization. Hill et al. (.Hill, J.M., Mervis, R.R., Avidor, R., Moody, T.W. and Brenneman, D.E. (1993) Brain Res., 603:222-233.) have shown that in neonatal rats, administration of gpl20 causes morphological damage to the brain as well as retardation of the development of complex motor behaviors.
No approved treatments are available. Use of calcium ch~nnçl antagonists and NMDA antagonists have been proposed as possible therapies by Lipton. Numerous calcium channel antagonists are available on the market, eg, nimodipine, but NMDA antagonists are still being studied clinically by many co,..panies, primarily for acute use in stroke or chronic use in epilepsy and Parkinson's disease. Amantadine, which is on the market as an anti-viral, is 20 now known to possess NMDA antagonist properties A closer cogener of amantadine, memantidine, is on the market in Europe and has been proposed by Lipton as a possible candidate for treatment of HIV dementia. Another agent which is available for testing is nitroglycerin. Under certain circumstances, the NO generated from the nitroglycerin can protect neurons from overstimulation 25 of the NMDA receptors with the resulting calcium and glutamate excitotoxicity.
However, cardiovascular effects and the extremely erratic pharmacokinetics of nitroglycerin make this approach seem problematic.
In work related to the present invention, together with Robert Floyd, I
discovered that certain nitrone compounds exhibited activity as agents against CA 022~2403 1998- lo- 16 wo 97l38684 PCT/US97/06351 HIV-dementia. This separate invention is covered in another patent application filed simultaneously herewith.

Benzamides as Medicaments This invention's approach to miti~ting HIV dementia employs a family of benzamide analogues as the active agent. Commonly owned United States patent number 5,472,777 describes certain benzamides and their use in treating neurological conditions. Commonly owned Patent Cooperation Treaty application PCT/US96/04538 describes the compounds employed herein and describe their use as pharm~eutical compositions for conditions not specificallyincluding ~IV-dementia.

References Other references of interest include:
Lipton, SA, Gendelman, HE (1995) Dementia associated with the acquired immunodeficiency syndrome, New England Journal of Medicine, 332(14): 934-940.
Simpson, I~M, Tagliati, M (1994) Neurologic manifestations of HIV
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Lipton, SA (1994) Neuronal injury associated with HIV-l and potential treatment with calcium-channel and NMDA antagonists, Dev Neurosci, 16(3-4):

Danysz, W, Parsons, CG, Bresink, I, Quack, G (1995) Glut~m~t~- in CNS disorders, Drug News and Perspectives, 8: 261-277.
Lipton, SA, Choi, YB, Pan, ZH, Lei, SZ, Chen, HSV et al. (1993) A
redox-based mechanism for the ne~,lop,otective and neurodestructive effects of nitric oxide and related nitroso-compounds, Nature, 364: 626-632.

CA 022~2403 1998-10-16 Dawson, VL, Dawson, TM, Uhl, GR, Synder, SH (1993) Human immunodeficiency virus type l coat protein neurotoxicity meAi~ted by nitric oxide in primary cortical cultures, Proc Natl Acad Sci, 90: 3256-3259.
Mollace, V, Colasanti, M, Persichini, Bagetta, G, Lauro, GM, Nistico, G (1993) HIV gpl20 glycoprotein stimulates the inducible isoform of NO
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439-445.
Schultz, JB, Henshaw, R, Siwek, D, Jenkins, BG, Ferrante, RJ, Cipolloni, PB, Kowall, NW, Rosen, BR and Beal, MF (1995) Involvement of free radicals in excitotoxicity in-vivo. J. Neurochem. 64: 2239-2247.
Winrow, VR, Winyard, PG, Morris, CJ, Blake, DR (1993) Free radicals in inflammation: Second messçngers and me~ rs of tissue destruction, Br Med Bllll 49: 506-522.
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535-537.
Olanow, CW (1992) An introduction to the free radical hypothesis in parkinson's disease, Annals of ~Veurology, 32 (supplement): 53-59.
Floyd, R.A. and Carney, J., Nitrone radical traps (NRTs) protect in experimental neurodegenerative (lis~cps~ in Neuroprotective approaches to the treatment of Parkinson's disease and other neurodegenerative disorders (Olanow, C.W. Jenner, P. and Youssim, Eds.) Academic Press, New York, New York, in press.
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644: 267-272 Zhao, Q., Pahlmark, K., Smith, M.-J., and Siesjo, B. (1994) Delayed treatment with the spin trap aphenyl-n-tert-butyl nitrone (PBN) reduces infarct CA 022~2403 1998-10-16 size following transient middle cerebral artery occlusion in rats. Acta Physiol.Scad. 152: 349-35().
Oliver, CN, Starke-Reed, PE, Stadtman, ER, Carney, JM and Floyd, RA (1990) Oxidative damage to brain proteins, loss of glutamine synthetase activity and production of free radicals during ischemia induced injury to gerbil brain. Proc. Natl ~cad. Sci. USA 87: 5144-5147.
Carney, JM, Starke-Reed, PE Oliver, CN, Landrum, RW, Cheng, MS, Wu, JF and Floyd, RA (1991) Reversal or age-related increase in brain protein oxidation in enzyme activity, and loss in temporal and spatial memory by chronic ~-lminictration of the spin-trapping compound N-tert-butyl-~-phenylnitrone. Proc. Natl. Acad. Sci., 88: 3633-3636.
McKechnie, K, Furman, BL, Paratt, JR (1986) Modification by oxygen free radical scavengers of metabolic and cardiovascular effects of endotoxin infusion in conscious rats, Circulatory Shock, 19: 429-439.
Hamburger, SA, McCay, PB (1989) Endotoxin-induced mortality in rats is reduced by nitrones, Circulatory Shock, 29: 329-384.
Pogrebniak; HW, Merino, MJ, Hahn, SM, Mitchell, JB, Pass, Hl (1992) Spin trap salvage from endotoxemia: The role of cytokine down regulation, Surgery, 112: 130-139.
F~m~t~u, R, Mori,A., Packer, L (1995) The spin trap N-tert-~-phenyl-butylnitrone prolongs the life span of the senescence accelerated mouse, Biochem Biophys Res Comm 211: 847-849.
Achim, C, Heyes, MP, Wiley, CA (1993) Quantitation of human immunodefficiency virus, immune activation facyors, and quinolinic acid in AIDS brains, J Clin Invest 91: 2769-2775.
Wesselingh, SL, Power, C, Glass, JD, Tyor, WR et al. (1993) Intracereberal cytokine messenger RNA expression in aquired immuniodeficiency syndrome dementia, Annals of Neurology, 33: 576-582.

CA 022~2403 1998-10-16 W O 97/38684 PCTrUS97/~6351 Gelbard, HA, Dzenko, KA, DiLoreto, D, delCero, C, delCerro, M, Epstein, LG (1994) Neurotoxic effects of tumor necrosis factor alpha in primary human neuronal cultures are mediated by activation of the glut~m~e AMPA receptor subtype: Implications for AIDS neuropathogenesis, Dev Neurosci, 15: 417-422.
Selmaj, K, Raine, CS, Farocq, M, Norton, WT, Brosnan, CF (1991) Cytokine cytotoxicity against oligodendrocytes. Apoptosis induced by Lymphotoxins, J lmmunol, 147: 1522-1529.
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Pulliam, L, West, D, Haigwood, N, Swanson, RA (1993) HIV-1 envelope gpl20 alters astrocytes in human brain cultures, AIDS Research and Human Retroviruses, 9: 439-444.
Pulliam, L, Herndier, B, McGrath, MS (1991) Purified trichosanthin (GLQ223''g)) exacerbation of indirect HlV-associated neurotoxicity in vitro, AIDS, 5: 1237-1242.
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559-563.
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Floyd, RA, Watson,JJ, Wong, PK (1984) Sensitive assay of hydroxyl free radical formation utilizing high pressure liquid chromatography and , CA 022~2403 l998- l0- l6 electrochemical detection of phenol and salicylate hydroxylation products, J
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Floyd, RA, Henderson, R, Watson, JJ, Wong, PK (1986) Use of salicylate with high pressure liquid chromatography and elecrochemic~l S detection (LCED) as a sensitive measure of hydroxyl free radicals in adriamycin treated rats, Free Ra~ical Biol Med, 2: 13-18.

Statement of the Invention It has now been found that certain ben_amide compounds have activity in the treatment of AIDS Dementia Complex (HIV dementia).
This discovery can take the form of ben_amide-based pharmaceutical compositions having activity against HIV-dementia. These compositions include one or more of the acetamidobem~mide, aminobenzamide or nitroben_amide compounds of Formula I as active agent in a pharm~-~eutic~lly acceptable carrier.

(R)n <~CON~ 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 following provisos: 1) when n is 1 and R is -NO2 at the 4 position of the ring, R' is not tert-butyl, iso-butyl, or propyl; 2) when n is 1 and R is -NO2 at the 2 positionof the ring, R' is not iso-butyl or propyl; and 3) when n is 2 and R' is te~t-CA 022~2403 l998- l0- l6 butyl and both Rs are -NO2, 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 more typically are presented in unit dosage form.
S In another aspect this invention provides a therapeutic method for treating a patient suffering from HIV-dementia. This method involves administering to the patient an effective HIV-dementia-treating amount of one or more of the pharmaceutical co.~positions just described.
In another ~spect this invention provides a prophylactic method for protecting a patienL susceptible to HIV-dementia. This method involves administering to the patient an effective HIV-dementia prophylactic amount of one or more of the pharmaceutical compositions just described.

Brief Description of the Drawings The invention will be further described with reference being made to the drawings in which Fig. 1 is a bar graph showing the protective effect of a benzamide in a HIV-dementia related cell culture test.
Fig. 2 is a bar graph showing the protective effect of a benzamide in a HIV-dementia related cell culture test.
Fig. 3 is a bar graph showing apoptosis response observed in a cell aggregation test wi~h a benzamide.
Fig. 4 is a plot of bioavailability of benzamide as a function of time.

Detailed Description of the Invention The Benzamides The treatment of this invention employs one or more benzamides as its active agent. This invention employs certain acetamidobenzamides aminobenzamides and nitrobenzamides as active pharmaceutical agents. The CA 022~2403 1998-10-16 benzamides are described by Formula I. In this formula, R' is a saturated alkyl of from 3 to 5 carbon atoms and n is 1 or 2.
The acetamido, amino or nitro group (or groups) may be found anywhere on the ring. Preferred embodiments include when n is 1 and the ~cet~mido group is at the 2, 3 or 4 position of the ring and when n is 2 and the~cet~mido groups are at the 2 and 3, 2 and 4, 2 and 5, 2 and 6, 3 and 4, or 3 and 5 positions of the ring.
With respect to the alkyl substituents, R', compounds wherein R' is an alkyl which does not have a hydrogen on the alpha carbon, that is, the carbon which bonds to the nitrogen of the ring, are preferred. Examples of these preferred R' groups are tert-butyl and tert-amyl.
Acet~midobenzamides of Formula I of particular interest are:
N-tert-butyl-4-~cet~midobenzamide, N-iso-propyl-4-~çet~midobenzamide, N-tert-amyl-4-~cet~midobenzamide, N-tert-butyl-3-~ret~midobenzamide, and N-methylcyclopropyl-4-acetamidobenzamide .
N-tert-butyl-4-acetamidobenzamide is the most preferred ~et~midobenzamide.
The aminobenzamides and nitrobenzamides employed as active agents are described by Formula I when R is an amino or nitro group. In this formula, R' is a saturated alkyl of from 3 to 5 carbon atoms and n is 1 or 2 subject to the same preferences for substituents and their positions set forth with reference to the ~et~midobenzamides and further subject to the provisos that 1) when n is 1 and R is -NO2 at the 4 position of the ring, R' is not tert-butyl, iso-butyl, or propyl; 2) when n is 1 and R is -NO2 at the 2 position of the ring, R'is not iso-butyl or propyl; and 3) when n is 2 and R' is tert-butyl and both Rs are -NO2, the R groups are not at the 3 and 5 positions of the ring.

CA 022~2403 1998-10-16 Aminoben_amides and nitroben7~mides of Formula I of particular interest as active agents are:
N-iso-propyl-4-nitroben_amide, N-tert-butyl-3 -nitrobenzamide, S N-tert-butyl-2-nitroben_amide, N-n-butyl-4-nitroben7~mide, N-n-propyl -4-nitroben_amide, N-tert-butyl-3,5-dinitrobenzamide, N- 1 -methylpropyl-4-nitroben_amide, N-tert-butyl-4-aminobenzamide and N-tert-butyl-3 -aminoben_amide .
When the ben_amide compound contains an amino group, such as is the case with N-tert-butyl-3-aminoben_amide and N-tert-butyl-4-aminoben_amide, the amine functionality can be present as such or as a salt. In the salt form the amino is protonated to the cation form in combination with a pharm~ eutically acceptable anion, such as chloride, bromide, iodide, hydroxyl, nitrate, sulfonate, methane sulfonate, acetate, tartrate, oxalate, succinate, or palmoate.
When these aminoben_amides are referred to it is to be understood that these salts are included as well.
Commonly owned United States Patent number 5,472,983, referred to above, discloses several bçn7~mides useful in treating neurodegenerative rlice~cec based on their protective action in the MPTP mouse model of Parkinson's disease. The compound N-tert-butyl-4-acetamidoben_amide of the present invention is an in vivo biotransformation product of one of these ben7~mi(1es (N-tert-butyl-4-nitroben_amide) which has been found in the blood of rats and mice tb which N-tert-butyl-4-nitroben_amide has been ~dminic~çred orally.
Mixtures of two or more of these materials may be employed, if desired.

CA 022~2403 1998-10-16 Pharmaceutical Compositions The benzamide compound(s) is formulated into pharmaceutical compositions suitable for oral or parenteral, e.g. intravenous or intramuscular injection administration.
S The compositions for oral administration can take the form of liquid solutions or suspensions, powders, tablets, capsules or the like. In such compositions, the nitrone or its salt is usually a minor col-lponent (0.1 to say50% by weight) with the remainder being various vehicles or carriers and processing aids helpful for forming the desired dosing form. A liquid form may include a suitable aqueous or nonaqueous vehicle with buffers, suspending dispensing agents, colorants, flavors and the like.
A solid form may include, for example, any of the following ingredients, or compounds of a similar nature: a binder such as microcrystallinecellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricantsuch as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, sugar, methyl salicylate, or orange flavoring.
In the case of injectable compositions, they are commonly based upon injectable sterile saline or phosphate-buffered saline or other injectable carriers known in the art. Again the active nitrone is typically a minor coll,ponent, often being from about 0.05 to 10% by weight with the remainder being the injectable carrier and the like.
These components for orally administrable or injectable compositions are merely representative. Other materials as well as processing techniques and the like are set forth in Part 8 of Remington's Pharmaceutical Sciences, 17th CA 022~2403 1998-10-16 WO 97/38684 rCT/US97/06351 edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is incorporated by reference.
One can also administer the compounds of the invention in sustained release forms or from sustained release drug delivery systems. A description of 5 representative s-lst~ined release materials can be found in the incorporated materials in Remington's Pharmaceutical Sciences.

Conditions Treated and Treatment Regimens The conditions treated with the benzamide-containing compositions generally include HIV dementia and the various symptoms which fall within the 10 HIV dementia definition. The benzamide-containing formulations can be admini~tered to achieve a therapeutic effect and slow or counteract the progression of HIV dementia or they can be administered prophylactically to patients not yet exhibiting HIV dementi~ but exposed to the HIV-1 virus. The benzamide-cont~ining composition is adminictered in manners decigned to get 15 the drug into the patient's bloodstream and across the blood-brain barrier into the patient's brain. One excellent mode for accomplishing this is intravenous administration. Intravenous dose levels for treating these conditions range fromabout 0.01 mg/kg/hour to about 10 mg/kg/hour, all for from about 1 to about 120 hours and especially 1 to 96 hours. A preloading bolus of from about 10 20 to about 500 mg may also be adminictered to achieve adequate steady state levels. Other forms of parenteral administration, such as intramuscular injection can be used, as well. In this case, similar dose levels are employed.
While parenteral administration is attractive from a drug delivery point of view, it should be recognized that the course of HIV infection can stretch 25 over many months or even years so oral dosing may be p,efe,led for patient convenience and tolerance. With oral dosing, one to three oral doses per day, each from about 0.02 to about 50 mg/kg are called for with l~r~felled doses CA 022~2403 l998- l0- l6 WO 97/38684 PCT/US97/063~1 being from about 0.04 to about 10 mglkg. These same dosing levels and regimenc would be used for prophylactic treatment as well.
In any treatment regimen, the health care professional should assess the patient's condition and determine whether or not the patient would benefit from 5 bçn7~mide treatment. Some degree of experimentation to determine an optimal doing level and pattern may be called for.
A positive dose-response relationship has been observed. As such and bearing 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 ~-lminicter large amounts of benzamide such as those described above.

Methods of Preparation of Compounds The benzamide compounds employed herein can be prepared using commonly available starting materials and readily achievable reactions.
lS One representative preparation route, which is illustrated with tert-butyl amine, but which may be used with any alkyl amine, involves the following reactions:

tN~2) ,. (~~2) ~
COX + NH2C ( C~3~ 3 - ~CONHC ( C~I3) 3 III

where X is halo such as I, Br, F or Cl.

(B) (N~2) H2 ,1~
~CON~IC ( C~3 ) 3 IV
(C) (N~C2C~3~ ~
C~C1C~3 ~CON~C (C~3~ 3 ~V _ In step (A) the N-tert-butyl nitrobenzamides (III) are formed. This reaction should be carried out at te"lp~ldtures below 10~C.
This step (.4) yields as benzamides III, the compounds of the invention S where R is -NO2.
In step (B) the nitro groups in the mono- or di-nitro benzamide III are subjected to reduction. This is commonly carried out with a re~ucing agent such as hydrazine and an apl)lop,iate 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 invention where R is NH2-In step (C) the amino-benzamides IV are converted to ~ret~midobenzamides V by reaction with an acetyl halide such as acetylchloride. This reaction is carried out in the presence of a mild base and at low to arnbient te",pel~tures such as - 20~C to + 20~C. This yields the S compounds of the invention where R is ~cet~mido.
Alternate synthetic schemes may also be used to prepare the compounds.
Examples of these alternate routes are set forth below using N-ter~-butyl-4-acetamidobenzamide as the representative compound. Other compounds may be prepared using these alternate methods by starting with applo~liate starting 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 ap~lupliate alcohol (Alternate Route 1) or similarly substituted toluene compounds and the applol,liate alkyl amine (Alternate Route 3).

Alternate Route 1 This route begins with acetylation of, for example, 4-aminobenzonitrile (A) to compound (B) using standard methods. Acid hydrolysis of tert-butanol in the presence of 4-~cet~midobenzonitrile (B), provides a feasible synthetic pathway to N-~ert-butyl-4-~et~midobenzamide.

H~ J~CN ~C~c~

A B

H~ NJ~ C l~

Alternate Route 2 Acetylation, using standard methods, of the inexpensive starting material PABA (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 subsequent amidation using standard methods, such as those described previously, produces N-tert-butyl-4-~eet~midQbenzamide from inexpensive raw materials.

~ CO~ ~3~CO2H

H~ JJ3 H H
E

Alternate Route 3 Another method for the preparation of the compounds begins with 10 acetylation, using standard methods, of, for example, paratoluidine (F) to 4-~-~et~midotoluene (~). The synthetic intermediate (G) may be converted to 4-~cePmidobenzoic acid (D) with common oxidizing agents (e.g., KMnO4) and ,, CA 022~2403 1998-10-16 WO 97138684 PCT/tlS97/06351 subsequently transformed to N-te~-butyl-4-acetamidobenzamide as outlined in Alternate Route 2.

H~ H,CJ~ H, o ~CO~

F G D

Examples The invention will be further described by the following Examples.
These are provided to illustrate several preferred embodiments of the invention but are not to be construed as limiting its scope which is, instead, defined by the appended claims. Examples 1 to 19 demonstrate the preparation of acetamidobenzamides, as well as nitro- and aminobenzamides, which are representative of the benzamide compounds employed in the compositions and methods of this invention. Examples 20 to 24 demonstrate the preparation of pharmaceutical compositions based on the compounds. Thereafter biological test results illustrating the activity of the compositions of the invention are provided.

Example 1 Preparation of N-tert-butyl-4-aminobenzamide tert-Butyl amine (14.6 g, 0.200 mole) was stirred in ethyl acetate (150 mL, pùrified by washing with 5% sodium carbonate solution, saturated sodium chloride solution, drying over anhydrous magnesium sulfate, and filtering through fluted filter paper) and cooled to 5~ C with an ice bath. 4-nitrobenzoyl chloride (18.6 g, 0.100 mole) in purified ethyl acetate (75 mL) CA 022~2403 1998-10-16 was added dropwise at such a rate to maintain the temperature below 10~ C.
The ice bath was removed upon complete addition of benzoyl chloride solution and the reaction stirred for 4 hours. The reaction mixture was then filtered on a Ruchner funnel, the filtrate washed three times with 5% HCl, once with S saturated sodium chloride, dried over anhydrous magnesium sulfate, filtered through fluted filter paper, and the solvent stripped off leaving white crystalline product. The product was dried in a vacuum oven at 24 mm and 45~ C for 14 hours. This procedure produced 17.13 g of crystals of N-tert-butyl-4-nitrobenzamide (77% yield), mp 162- 163~ C. Proton nuclear magnetic resonance (89.55 MHz in CDCl3) showed absorptions at 8.257 ppm (d, 8.8 Hz, 2H; 3,5-aryl H); 7.878 ppm (d, 8.8 Hz, 2H; 2,6-aryl H); 6.097 ppm (bs, lH;
N-H); 1.500 ppm (s, 9H; tert-butyl H).
Palladium on carbon (5%, 75 mg) was added to N-te~-butyl-4-nitrobçn7~mide (5 g, 22.5 mmole) in 95% ethanol at 55~C. A solution of hydrazine (1.2 mL) in 95% ethanol (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% ethanol (5 mL) was added and the reaction was refluxed for another hour. The reaction was filtered on a buchner funnel, the volume of solvent reduced under vacuum, and extracted with dichloromethane. The 20 combined extracts were dried over magnesium sulfate and solvent stripped, leaving 3.90 g of N-tert-butyl-4-aminobenzamide (90% yield), melting point 125 - 127 ~C. 90 MHz proton NMR (in CDCI3) showed absorbances at 7.290 ppm (2H, d, 8.8 Hz; 2,6 aryl H); 6.368 ppm (2H, d, 8.8 Hz; 3,5 aryl H); 5.45 ppm (1 H, bs; NHC=O); 3.727 ppm (2H, bs; aryl-NH2); 1.186 ppm (9 H, s; t-25 butyl H).

CA 022~2403 1998-10-16 Example 2 Preparation of N-tert-butyl-4-acetamidobenzamide Acetyl chloride (0.45 g, 5.7 mmole) in ethyl acetate (25 mL) was added dropwise to N-tert-butyl-4-aminoben_amide (1.0 g, 5.2 mmole) and triethyl amine (0.58 g, 5.7 mmole) in ethyl acetate at 3~ C at such a rate to m~int~in the ~ dture below 10~ C. The reaction was allowed to warm to room ~e.,.p~lature, stirred 1 hour, and washed with 5% HCl. Recryst~lli7~tion from acetone gave 1.08 g N-tert-butyl-4-acet~midobenzamide (89% yield), melting point 119 - 121 ~C. 90 MHz proton NMR (in DMSO-d6) showed absorbances at 9.726 ppm (lH, bs, N-H); 7.715 ppm (4H, dd, 4.4 Hz; aryl H); 7.295 ppm (1 H, bs; NH); 2.~44 ppm (3H, s; CH3CO); 1.448 ppm (9 H, s; t-butyl H).

Example 3 Preparation of N-tert-butyl-3-nitrobenzamide N-ter~-butyl-3-aminobenzamide and N-tert-butyl-3-acetamidobenzamide The amidation procedures of Example l 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 CDC13) showed absorptions at 8.517 ppm (2-aryl H, s, lH); 8.337 ppm (4-aryl H, d, 8.8 Hz, lH); 8.121 ppm (6-aryl H, d, 6.4 Hz, lH); 7.618 ppm (5-aryl H, m, lH); 6.032 ppm (N-H, bs, lH); 1.484 ppm (t-butyl H, s, 9 H).
Iron (III) oxide hydroxide catalyzed hydrazine reduction produced N-tert-butyl-3-aminobenzamide in 53% yield, melting point 118-120 ~C. Proton NMR (in CDC13) showed absorbances at 7.088 ppm (4-6 -aryl H, m, 3 H);
6.794 ppm (2-aryl H, s, lH); 5.902 ppm (N-H, bs, lH); 3.145 ppm (aryl N-H, bs, 2H); 1.458 pprn (t-butyl H, s, 9 H).
Acetylation of N-tert-butyl-3-aminoben_amide as described in Example 2 gave N-tert-butyl-3-acetamidoben7~mide in 75% yield, melting point 194-CA 022~2403 l998- l0- l6 195~C. Proton NMR (in CDCl3) showed absorptions at 7.778 ppm (4-6 -aryl H, m, 3 H); 7.392 ppm (2-aryl H, s, lH); 6.08 ppm (N-H, bs, lH); 2.174 ppm (acetyl CH3, s, 9 H); 1.500 ppm (t-butyl H, s, 9 H).

Example 4 Preparation of N-tert-butyl-2-nitrobenzamide and N-tert-butyl-2-acetamidobenzamide The method of Example 3 is repeated using 2-nitrobenzoyl chloride in the amidation step. This yields N-~ert-butyl-2-nitrobenzamide.
Reduction of the nitrobenzamide with hydrazine yields N-tert-butyl-2-aminobenzamide.
Acetylation of the aminobenzamide yields N-tert-butyl-2-acet~midobenzamide.

Example 5 Preparation ofN-iso-propyl-4-nitrobenzamide and N-iso-propyl-4-acetamidobenzamide The method of Example 3 is repeated using 4-nitrobenzoyl chloride and iso-propyl amine in the amidation step. This yields N-iso-propyl-4-nitroben7~mide.
Reduction of the nitroben7~mide with hydrazine yields N-iso-propyl-4-aminobenzamide.
Acetylation of the aminobenzamide yields N-iso-propyl-4-acetamidobenzamide.

Example 6 Preparation ofN-tert-amyl-4-nitrobenzamide and N-tert-amyl-4-acetamidobenzamide CA 022~2403 1998-10-16 The method of Example 3 is repeated using 4-nitrobenzoyl chloride and tert-amyl amine in the amidation step. This yields N-tert-amyl-4-nitrobenzamide .
Reduction of the nitrobenzamide with hydrazine yields N-tert-amyl-4-S aminobenzamide.
Acetylation of the aminobenzamide yields N-tert-amyl-4-~cet~midobenzamide.

Example 7 Preparation of N-iso-butyl-4-acetamidobenzamide The method of Example 3 is repeated using 4-nitrobenzoyl chloride and iso-butyl amine in the amidation step. This yields N-iso-butyl-4-nitroben7~mide.
Reduction of the nitrobenzamide with hydrazine yields N-iso-butyl-4-aminobenzamide .
Acetylation of the aminobenzamide yields N-iso-butyl-4-~cet~midobenzamide.

Example 8 Preparation of N-n-butyl-4-nitrobenzamide and N-n-butyl-4-acetamidobenzamide The method of Example 3 is repeated using 4-nitrobenzoyl chloride and n-butyl amine in the amidation step. This yields N-n-butyl-4-nitrobenzamide.
Reduction of the nitrobenzamide with hydrazine yields N-n-butyl-4-aminobenzamide .
Acetylation of the aminobenzamide yields N-n-butyl-4-acetamidobenzamide.

CA 022~2403 1998-10-16 Example 9 Preparation of N-n-propyl-4-nitrobenzamide and N-n-propyl-4-acetamidobenzamide The method of Example 3 is repeated using 4-nitrobenzoyl chloride and 5 n-propyl amine in the amidation step. This yields N-n-propyl-4-nitrobenzamide .
Reduction of the nitrobenzamide with hydrazine yields N-n-propyl-4-aminoben7~mide.
Acetylation of the aminobenzamide yields N-n-propyl-4-10 a~et~midobenzamide.

Example lO
Preparation of N-1.2-dimethylpropyl-4-nillubenzamide and N- 1 ~2-dimethylpropyl-4-acetamidobenzamide The 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 .
Reduction of the nitrobenzamide with hydrazine yields N-1,2-dimethylpropyl-4-aminobenzamide .
Acetylation of the aminobenzamide yields N-1,2-dimethylpropyl-4-20 ~cet~midobenzamide.

Example 1 l Preparation of N-n-pentyl-4-nitrobenzamide and N-n-pentyl-4-acetamidobenzamide The method of Example 3 is repeated using 4-nitrobenzoyl chloride and 25 n-pentyl amine in the amidation step. This yields N-n-pentyl-4-nitrobenzamide.
Reduction of the nitrobenzamide with hydrazine yields N-n-pentyl-4-aminobenzamide .

.

CA 022~2403 l998- l0- l6 Acetylation of the aminobenzamide yields N-n-pentyl-4-~midobenzamide.

Example 12 Preparation of N-2-methylbutyl-4-nitrobenzamide and N-2-methylbutyl-4-acetamidobenzamide The 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.
Reduction of the nitrobenzamide with hydrazine yields N-2-methylbutyl-104-aminobenzamide.
Acetylation of the aminobenzamide yields N-2-methylbutyl-4-~cet~midoben7~mide.

Example 13 Preparation of N-n-pentyl-2-nitrobenzamide and 15N-n-pentyl-2-acetamidobenzamide The method of Example 3 is repeated using 2-nitrobenzoyl chloride and n-pentyl amine in the amidation step. This yields N-n-pentyl-2-ni~rvb~n~mide.
Reduction of the nitrobenzamide with hydrazine yields N-n-pentyl-2-aminobenzamide.
20Acetylation of the aminobenzamide yields N-n-pentyl-2-acetamidobenzamide .

Example 14 Preparation of N-te~-butyl-2,3-diacetamidobenzamide The method of Example 3 is repeated using 2,3-dinitrobenzoyl chloride 25in the amidation step. This yields N-tert-butyl-2,3-dinitrobenzamide.

CA 022~2403 1998-10-16 ~ eduction Qf the nitroben7~mide with hydrazine yields N-tert-butyl-2,3-diaminobenzamide:
Acetylation of the aminoben7~mide yields N-tert-butyl-2,3-diacetamidobenzamide .

S Example lS
Preparation of N-tert-amyl-2~4-diacetamidobenzamide The method of Example 3 is repeated using 2,4-dinitrobenzoyl chloride and tert-amyl amine in the amidation step. This yields N-tert-amyl-2,4-dinitrobenzamide .
Reduction of the nitrobenzamide with hydrazine yields N-tert-amyl-2,4-diaminobenzamide .
Acetylation of the aminobenzamide yields N-tert-amyl-2,4-cet~midobenzamide.

Example 16 Preparation of N-tert-butyl-2~5-di~cet~nlidobenzamide The method of Example 3 is repeated using 2,5-dinitrobenzoyl chloride in the amidation step. This yields N-tert-butyl-2,5-dinitrobenzamide.
Reduction of the nitrobenzamide with hydrazine yields N-tert-butyl-2,5-diaminobçn7~mide.
Acetylation of the aminob~.n7~mid~ yields N-tert-butyl-2,5-~i~cet~midobenzamide.

Example 17 Preparation of N-tert-butyl-2~6-diacetamidobenzamide The method of Example 3 is repeated using 2,6-dinitrobenzoyl chloride 25 in the amidation step. This yields N-tert-butyl-2,6-dinitrobenzamide.

CA 022~2403 1998-10-16 Reduction Gf the nitrobenzamide with hydrazine yields N-ter~-butyl-2,6-diaminobenzamide .
Acetylation of the aminobenzamide yields N-tert-butyl-2,6-ret~midobenzamide.

Example 18 Preparation of N-tert-butyl-3~4-diacetamidobenzamide The method of Example 3 is repeated using 3,4-dinitrobenzoyl chloride in the amidation step. This yields N-tert-butyl-3,4-dinillube,l~mide.
Reduction of the nitrobenzamide with hydrazine yields N-tert-butyl-3,4-10 diaminobe~7~mi~e.
Acetylation of the aminoben_amide yields N-tert-butyl-3,4-et~midoben7amide.

Example 19 Preparation of N-tert-butyl-3.5-diacetamidobenzamide The method of Example 3 is repeated using 3,5-dinitrobenzoyl chloride in the amidation step. This yields N-tert-butyl-3,5-dinitrobenzamide.
Reduction of the nitrobenzamide with hydrazine yields N-tert-butyl-3,5-minoben7~mi-1e.
Acetylation of the aminoben_amide yields N-tert-butyl-3,5-20 di~et~midobenzamide.

Preparation 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 of 25 magnesium stearatc 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 CA 022~2403 1998-10-16 WO 97t38684 PCT/US97106351 tablets were ~minic~Pred to a patient suffering from HIV dementia on a daily, twice daily or thrice daily regimen they would slow the progress of the patient's disease.

Example 2 1 The compound of Example 2 is admixed as a dry powder with a starch diluent in an approximate 1:1 weight ratio. The mixture is filled into 250 mg capsules (125 mg of active benzamide). If these capsules were ~(lmini~tçred to a patient susceptible to coming down with HIV dementia on a daily, twice daily or thrice daily regimen they would slow or prevent the onset of the HIV
dementia.

Example 22 The compound of Example 3 is suspended in a sweetened flavored aqueous medium to a concentration of approximately 50 mg/mL. If S mLs of this liquid material was ~-lmini~tered to a patient suffering from HIV dementia on a daily, twice daily or thrice daily regimen they would slow the progress of the patient's disease.

Example 23 The compound of Example 4 is admixed as a dry powder with a dry gelatin binder in an approximate 1:2 weight ratio. A minor amount of m~gnesium stearate is added as a lubricant. The mixture is formed into 450-900 mg tablets (150-300 mg of active ben7~mirle) in a tablet press. If these 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's disease.

, , WO 97/38684 PCT/US97/063Sl Example 24 The compound of Example 14 is dissolved in a buffered sterile saline injectable aqueous medium to a concentration of approximately S mg/ml. If 50 mLs of this liquid material was ~-lmini~t~red to a patient suffering from HIV
S deTnPnti~ on a daily, twice daily or thrice daily regimen this dose would slow the plu~r~ ss of the patient~s .li~
It will be appreciated that any of the compounds of Formula I could be employed in any of these 1~l~ se-~lalive formulations, and that any of these formulations could be ~limini~tered in any of these manners so as to treat any of 10 the HIV dementia con~liti- ns described in this specification.

Biological Testing These tests utilized two neural cell culture systems for de~el~ ing the efficacy of N-~ert-butyl-4-~et~mido~çn7~mide (nCo,-,pound I") in reversing ne.lloto~icity which mimic that observed with HIV dementia. In both assays, 15 human neural cell cultures were used either as a bilayer (neurons on an astrocyte layer) or a three ~lim~on~ional model (brain cell aggregates). TNF-oc (100 pg/ml) was used as the neurotoxin and the length of incubation was 72 hours. A considerable body of evidence supports the notion that TNF-~ is one of the neurotoxins responsible for HIV dementia. Brain concentrations of TNF-20 ~ are elevated in deep grey matter from AIDS patients with mild HIVdementia. Achim, C, Heyes, MP, Wiley, CA (1993) Quantitation of human immuno~lefficiency virus, immune activation facyors, and quinolinic acid in AIDS brains, J Clin Invest 91: 2769-2775. The distribution of mes~Pnger RNA
t;A~.,essing TNF-~ in the brain follows a similar pattern. Wes~l-lingh, SL, 25 Power, C, Glass, JD, Tyor, WR et al. (1993) Intracereberal cytokine me~nger RNA expression in aquired immuniodeficiency syndrome dementia, Annals of Neurology, 33: 576-582. Gelbard et al. have shown that HIV-l infected monocytes in culture with astroglial cells produce concentrations ( > 200 pg/ml) of TNF- ~ sufficient to cause neurotoxicity. Gelbard, HA, Dzenko, KA, DiLoreto, D, delCero, C, delCerro, M, Epstein, LG (1994) N~urolu~-ic effects of tumor necrosis factor alpha in primary human neuronal cultures are m~iqted by activation of the glutq-m~q-te AMPA receptor subtype:
S Tmplirqtions for AIDS neuropathogenesis, Dev Neurosci, 15: 417-422. TNF-~
is reported to cause its neurotoxicity by inducing apoptosis. Selmaj, K, Raine, CS, Farocq, M, Norton, WT, Brosnan, CF (1991) Cytokine cytotoxicity against oligodendrocytes. Apoptosis in~uce~d by Lymphotoxins, J Imrnunol, 147:
1522-1529. Recently, it was shown that gpl20 exerts toxic effects through i~duction of IL-6 and TNF-~. Yeung, MC, Pulliam, L., Lau, AS (1995) The HIV envelope protein gpl20 is toxic to human brain-cell cultures through the induction of interleukin-6 and tumor necrosis factor-~Y, A~DS, 9:137-143.

Brain A.~r~ qtp P1~IUI~, Brain cell aggregates were p~pa,ed from second trimester abortion tissue as previously described. Pulliam L., Berens, ME, Rosenblum, ML
1988. A normal human brain cell aggregate model for neurobiological studies, J Neurosci Res 21 :521-530. Briefly, human brain tissue between 16 and 18 weeks gestation are gently di~soci~ted through nylon screens to obtain single cells. A~ro~i.,.ately 4 X 107 cells within 4 ml DME supplement~d with 0.6%
dextrose, 50 mg/ml gentq-micin and 10% FCS are distributed into 25 ml DeLong flasks. Aggregates are constantly rotated and incubqt~d at 37~C in an atmosphere of 10% C02. After 2-3 days, aggregates are transferred to 50 ml flasks and 5 ml of DME supplemented with 15% FCS (exchange medium) added. Each flask contains several thousand aggregates that c. n be sampled over time. Five ml of medium is exchanged every other day in culture. After 10-12 days in culture samples are taken for histology and trypan blue exclusion is p~lÇo...-ed to determine viability. Samples are screened for HIV, Hepatitis A, B, C and mycoplasma. Aggregates remain viable for approximately 40 days CA 02252403 l998- l0- l6 in culture. Brain cell aggregates are differenti~t~d at the time of sampling in that they express neural cell l-~a~ for identifi~tion. Brain cell aggregates contain all the cells of the CNS- approximately 40% neurons, 40% astrocytes, 10% oligodendrocytes with myelin and 10% microglia. Neural cell S apoptosis/death was measured by DNA fr~gml nt~tion Elisa technique according to manufactures directions (Boehringher ~f~nnheim).

Neural Cell Bilayer ~ocelu.k Brain aggregates were plcpalcd as described above. Several aggregates are placed in each well of a multi-well chamber slide (Nunc) coated with Cell 10 TAK (Collaborative Research) at a concentration of 20 ug/ml. Cells migrate from the brain aggregates within 3 days. Astrocytes form a monolayer with neurons on top and rare microglia ( < 1 %)/oligodendrocytes ( ~ 1%). These cultures are confluent within 1 week. Monolayers can be ,~inl;1in~d for up to three weeks. Char~ t;on of cell types is d~le~ ined by using 15 immlmohi~t~rhPmistry and the antibodies neuron specific enolase (NSE, Dako) for neurons and glial fibrillary acidic protein (GFAP, Dako) for the identific~tion of astrocytes. Confocal microscopy was used to visuali~ and identify neurons and astrocytes by size and shape. Neuronal viability was detel."ined by exposing chambers with and without different treatments to AO
20 and ethidium bromide (EtBr). Neurons and total cell counts were de~er~ ed by AO st~ining with visual confirmation by phase microsc~y. Enumeration of cell viability by co---pule-ized software was pelro~.ned at the time of microscopy; in addition, a visual printout of the fields observed always accomp~nied the data.

CA 02252403 l998- l0- l6 WO 97/38684 PCT/lJS97/06351 Experimental De~ n Experiment# System TNF-~ (pg/ml) Colllpound' (~M) Neural Cell 0 0 Bilayers o 100 100 ~

2 Neural Cell 0 0 Bilayers 3 Brain 0 0 Aggregate Test compound is N-tert-butyl-4-~cet~mi~oben7~mide.

Results EA~.il,lent 1 (Figure 1): This was a human neural cell bilayer e~pç~imPnt N-tert-butyl~-acet~midQben7~mide ("Compound I") showed some toxicity relative to the control. The TNF-~ tre~tment produced a high degree of cell death, over 61 ~o. N-tert-butyl-4-~rPt~midobPn7~mitle tre~tment produced subst~ntial pluteclion.
Experiment 2 (Figure 2): This experiment was a repeat of experiment 1 using a different brain pr~ation. Results çccpnt~ y duplicated those from the first experiment, except the TNF-~ tre~tmPnt gave less neuronal toxicity.

CA 02252403 l998- l0- l6 4 PCTIUS97/063~i1 Experiment 3 (Figure 3): This experiment utilized human brain aggregates. In this experiment, apoptosis/cell death was measured by an immunoassay for quantitation of cytoplasmic histone-associated DNA
fragments. In this experiment, N-tert-butyl-4-acetamidoben_amide trP~tments S gave substantial protection both with and without the TNF- ~ treatments. The bars in Figure 4 replesent the mean of duplicate experiments. The error bars in this figure express the individual values.

Physical/Chemical Parameters N-te~-butyl-4-acetamidobenzamide was studied to determine 10 physical/chemical p"~p~"ies which suggest its suitability for this application.
The following results were obtained:

N-tert-butyl-4-~- et~midobçn7~mide tl,2(min) in Aqueous 3000 HCI Solution (p~Il) Octanol-Water Partition 31 This shows that N-ten-butyl-4-acetamidobenzamide is lipophilic and slowly cleared from the body. N-tert-butyl-4-~et~midoben7~mide is a compound of particular interest for HIV dementia because, at least in the rat, it 20 shows excellent brain distribution, bioavailability and pharmacokinetic profile.
N-tert-butyl-4-~cet~midobenzamide is also significantly stable at a pH
commonly observed in the stomach.

CA 022F,2403 1998- lO- 16 Brain penetration Gf N-tert-butyl-4-acetamidoben_amide Following a 30 mg/kg oral dose, blood and brain samples from the same ~nim~ were analyzed for N-tert-butyl-4-~cet~midoben_amide at 4 and 8 hours post-dose with the following results:

Time Post-Dose Mean Brain Concentration Mean Blood Concentration (hours) (~Lg/g) +/- SEM (llg/ml) +l- SEM
4 8.9 +l- 3.2 43 +/- 7.9 8 9.1 +/- 1.7 39 +/- 4.2 Absolute Bioavailability of N-terl-butyl-4-acetamidobenzamide Oral Suspension The absolute bioavailability of N-tert-butyl-4-acetamidobçn7~mide in rats was determined by comparing the area under the curve following a 20 mg/kg dose of the ben7~mide dissolved in 1 % methyl cellulose. Blood concentrations were 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 AUCs determined. Four ~nim~ls were dosed orally and 4 animals were dosed IV.
Route Mean AUC +/- SEM Absolute (~g hr ml~') Bioavailability IV 252 +/- 73 -Oral 130 +/- 33 52%

The pharmacokinetic profile of a 30 mg/kg dose to Sprague Dawley rats can be found in Figure 4. The apparent tl,2 for N-tert-butyl-4-~cet~midoben7~mi(1e in this experiment was 8 hours, a very long t"2 for a drug in rat- a good predictor of once-a-day dosing if N-tert-butyl-4-25 acetamidoben_amide would ever be dosed in man. Such a dosing regimenwould be a significant therapeutic advantage in the clinic.

Further Brain Aggre~ation Studies Further studies were conducted as follows:
Experiment # gpl20(ng/ml)TNF-(x(ng/ml) Compound' (~M) - 1 0.1 - 1 0.3 - 1 3.0 S O - O

Test compound is N-tert-butyl-4-acetamidoben_amide.

As shown in the following Table, Experiment 4 showed that at unexpectedly low concentrations, N-ten-butyl-4-acet~midoben7~mi-1e provided complete protection in human brain aggregates from DNA fragmentation, a 15 measure of apoptosis, induced by 1 ng TNF-~. Some degree of dose proportionality was found. The results at all test compound concentrations are statistically 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 ~M test compound group is statistically significant from the other two treatment groups.

CA 022~2403 1998-10-16 Table Fxperiment 4 Results Experiment DNA Fragmentation % Protection (Absorbance + SF.n=3) Control 0.663 + 0.048 --TNF Only 1.592 + 0.156 --TNF + 0.1 ~M Compoundl 0.955 + 0.101 78 TNF + 0.3 ~LM Compound' 0.835 + 0.051 87 TNF + 3.0 ~M Compound' 0.801 + 0.123 90 Test compound is N-tert-butyl-4-acetamidoben7~mide.

The data above suggests that protection from apoptosis can be achieved at concentrations of approximately 1 ~M and below.

A l~M concentration of N-tert-butyl-4-acetamidobenzamide is in the 15 order of 0.2 ~g/ml. To achieve this concentration in rat brain would require a blood concentration of only 1 ~g/ml based on the brain/blood ratio data presented previously. If some degree of dose proportionality is found with lower doses of N-tert-butyl-4-a~et~midobenzamide, a 6 mg/kg dose to rats should achieve this concentration even at 24 hours post-dose (trough value).
20 Using liver blood flow differences to scale the clearance of drug in rats to that in man as described in Pulliam, L, Herndier, B, McGrath, MS (1991) Purified trichosanthin (GLQ223~)) exacerbation of indirect HlV-associated neurotoxicity in vitro, AIDS, 5: 1237-1242, a dose of 1.5 mg/kg to man would be predicted to achieve at 24 hours post-dose the 1 ~M target concentration in the brain for 25 protection from apoptosis.

Consistent with the results above, N-tert-butyl-4-acet~midQbenzamide also provided complete protection in human brain aggregates from toxicity in~luced by 1 ng TNF-~, although the concentration of the ben7~mide needed was considerably higher than that found to prevent DNA fragmentation. These S data are as follows:

Experiment LDH Release % Protection Absorbance i SD (n=) Control 0. 875 + 0.022 --T~nF 1.071 _ 0.036 --T~nF + 0.1 ~ M Co m poundl 1.114 + 0.023 0 TNF + 0.3 ~ M Co m poundl 1.103 + 0.034 0 TNF + 3.G ~M Compoundl 0.864 0.028 100 Test compound is N-tert-butyl-4-~et~midobenzamide.

15 Experiment 5:

In this experiment, N-tert-butyl-4-acetamidobenzamide provided significant protection in human brain ag~egates from cell toxicity induced by 1 ng gpl20. The difference in absorbance was statistically signifi~nt for all groups at p < 0.003.

Experiment LDH Release %
Absorbance i SD(n=) Protection Control 0.328 + 0.011 --gpl20 0.575 + 0.008 --gpl20 + 3.0 ~M Compoundl 0.427 _ 0.034 60%

Test compound is N-tert-butyl-4-acetamidoben_amide.

There was no evidence in this experiment for DNA fragmentation induced at this concentration of gpl20.
~0 Experiment 6:
Using procedures essentially the same as those described above for determining LDH release induced by TNF, programmed cell death (PCD) analysis was performed by ELISA using standardized kits (Boehriger M~nnheim). The results were as follows:

Experiment 6A PCD
Control 0 _ 0 359 TNF-~ 1.18+0.759 TNF-~Y + 10.0 ~M Compound' 1.15 + 0.125 TNF-(x + lO.O,uM Compound2 1.021 _ 0.099 TNF-lx + 10.0 ~M Compound3 0.34 _ 0.029 Test compound is N-tert-butyl-4-acetamidobenzamide.
2 Test compound is N-tert-butyl-4-aminobenzamide.
3 Test compound is N-tert-amyl-4-acetamidobçn7~mide.

Experiment 6B PCD
Control 0 + 0.69 TNF-~ 1.16 _ 0.088 TNF-~ + 10.0 ~M Compound' 1.05 + 0.043 TNF-a + 10.0 ~M Compound2 0.567 + 0.026 TNF-~ + 10.0 ~M Compound3 0.671 + 0.043 Test compound is N-tert-butyl-4-~ce~midobenzamide.
2 Test compound is N-ter~-butyl-4-aminobenzamide.
10 3 Test compound is N-tert-amyl-4-~cet~mi(lobenzamide.

Experiment 6C PCD
Control 0 + 0.032 TNF-cY 0.674 + 0.058 TNF-a + 10.0 ~lM Compound4 0.565 + 0.042 4 Test compound is N-isopropyl-4-acetamidobenzamide.

Experiment 6D PCD
Control 0 + 0.018 TNF-~ 0.531 + 0.034 TNF-a + lO.O,uM Compound4 0.016 + 0.03 4 Test compound is N-isopropyl-4-acetamidobenzamide.

The data from Experiments 6A-D demonstrate that various benzamides of this invention provided protection in human brain aggregates from toxicity 25 induced by 1 ng TNF-c~ as measured by PCD analysis.

CA 022~2403 1998-10-16 In vivo Tests In order to determine the effectiveness of this approach for treating ADC, a series of in vivo biological tests were carried out.

In vivo Test A
S 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. gpl20 was obtained from Intracel Corporation, Cambridge, Massachusetts. These materials were used in the following preliminary test:
Treatment of Animals: Sprague-Dawley neonatal rats (sixteen siblings) were divided into four groups. Starting at day one after birth until day six, the neonates received 60 ~1 subcutaneous injections of the following treatments.
Group 1: phosphate buffer-saline (PBS), Group 2: 5 ng gpl20 in PBS, Group 3: 5 ng gpl20 plus PBN (50 mg/kg) in PBS, and Group 4: PBN (50 mg/kg) in 15 PBS. Rats were weighed daily and the amount of PBN injected was adjusted accordingly.
Behavioral Assessments: Time required to perform two developmental milestones were measured to determine the adverse effects of gpl20 ~1mini.ctration on behavioral development as reported by Hill et al. and to 20 determine the possible protective action of PBN on these parameters.
Behavioral parameters studied were surface righting (animal placed head down on 45~ inclined screen will turn around and climb up.) These two tests have been shown to be the most sensitive tests for ~cce~.cment of the neurological disorder caused by gpl20 treatment. Furthermore, they can be examined early 25 enough in the life of the animal (day 3 for surface righting and day 6 for negative geotaxis) that their determination will not interfere with NO trapping in the brain which we performed at the end of the first week of the life of the animal. Animals were tested for the time required for surface righting on day 3 CA 022~2403 1998-10-16 W O 97/38684 PCTrUS97/063Sl and day 4 after bir;h, immediately prior to receiving the injections on those days, and on day 6 (2 hrs after the last injection that the ~nim~l~ received) aswell as day 7 (20 hrs following the last injections) for the time required to perform negative geotaxis. The angle chosen for the setting used for negative 5 geotaxis was decreased from 45~ (the angle used by Hill et al) to 35~ since under the experimental setting employed, animals were not able to stay on the screen set at 45~ and would slide down before being able to make an attempt to turn upward.

In vivo Test B
10 Protection by N-tert-butyl-4-acetamidobenzamide from gpl20-induced behavioral changes The striking results obtained with PBN prompted preliminary experiments with N-tert-butyl-4-acetamidobenzamide in the same model. The results are suggestive that N-tert-butyl-4-acetamidobenzamide is effective as 15 demonstrated by the data shown below obtained on neonates that had been adminictered gpl20 at 10 ng per dose starting on 3 day old animals. N-tert-butyl-4-~ce~midobenzamide was given at an oral dose of 35 mg/kg 2 hours prior to administering the gpl20. Treatment with N-tert-butyl-4-acet~midobenzamide and gpl20 continued daily. The negative geotaxis test was conducted on day ~.
Negative Geotaxis (sec) Tre ~tment 3 h Post-Last Dose gpl20 (Day 6) Vehicle 8.89 _ 3.74 gpl20 18.0_ 13.8 gpl20 + Compound' 8.39 3.94 Compound' 8.56 + 5.11 Test compound is N-tert-butyl-4-acetamidobenzamide.

The data suvgests N-tert-butyl-4-acetamidobenzamide had a protective effect.

Claims (20)

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for treating HIV dementia comprising a benzamide compound of the formula:

wherein R' is a saturated alkyl of from 3 to 5 carbon atoms, each R is 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 not tert-butyl, iso-butyl, or propyl; 2) when n is 1 and R is -NO2 at the 2 positionof the ring, R' is not iso-butyl or propyl; and 3) when n is 2 and R' is tert-butyl and both Rs are -NO2, the R groups are not at the 3 and 5 positions of the ring, in a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of Claim 1 wherein the benzamide compound is an acetamidobenzamide of the formula:

where R' is a saturated alkyl of from 3 to 5 carbon atoms and n is 1 or 2.

3. The pharmaceutical composition of Claim 2 wherein n is 1.

4. The pharmaceutical composition of Claim 3 wherein R' is tert-butyl.

5. The pharmaceutical composition of Claim 3 wherein R' is tert-amyl.

6. The pharmaceutical composition of Claim 3 wherein the benzamide compound is N-tert-butyl-4-acetamidobenzamide.

7. The pharmaceutical composition of Claim 1 wherein the carrier is an oral carrier.

8. The pharmaceutical composition of Claim 1 wherein the carrier is an injectable carrier.

9. A method for treating HIV dementia comprising administering to a patient in need of such treating an effective HIV dementia complex-treating amount of a composition of Claims 1-8.

10. The method of Claim 9 wherein the administering is oral.

11. The method of Claim 9 wherein the administering is parenteral.

12. The method of Claim 11 wherein the administering is by injection.

13. The method of Claim 9 wherein the treating is therapeutic.

14. The method of Claim 9 wherein the treating is prophylactic.

15. The use of a benzamide compound of the formula:

wherein R' is a saturated alkyl of from 3 to 5 carbon atoms, each R is 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 not tert-butyl, iso-butyl, or propyl; 2) when n is 1 and R is -NO2 at the 2 position of the ring, R' is not iso-butyl or propyl; and 3) when n is 2 and R' is tert-butyl and both Rs are -NO2, the R groups are not at the 3 and 5 positions of the ring, in the manufacture of a pharmaceutical composition for the treatment of HIV dementia.

16. The use of Claim 15 wherein the benzamide compound is an acetamidobenzamide of the formula:

where R' is a saturated alkyl of from 3 to 5 carbon atoms and n is 1 or 2.

17. The use of Claim 16 wherein n is 1.

18. The use of Claim 17 wherein R' is tert-butyl.

19. The use of Claim 17 wherein R' is tert-amyl.

20. The use of Claim 17 wherein the benzamide compound is N-tert-butyl-4-acetamidobenzamide.