Note: Descriptions are shown in the official language in which they were submitted.
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WO 97138684 PCT/US97/06351
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
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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.
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WO 97/38684 PCT/US97/06351
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
infection, Ann Intem Med, 121(10): 769-785.
Lipton, SA (1994) Neuronal injury associated with HIV-l and potential
treatment with calcium-channel and NMDA antagonists, Dev Neurosci, 16(3-4):
145-151
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
WO 97/38684 PCT/US97/06351
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
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
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.
Lafon-Cazal, M, Pietri, S, Culcasi, M, Bockaert, J (1993)
NMDA-dependent superoxide production and neurotoxicity, Nature, 364:
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.
Cao, X. and Phillis, J.W. (1994) a-Phenyl-N-tert-butyl-nitrone Reduces
Cortical Infarct and Edema in Rats Subjected to Focal Ischemia. Brain ~es.
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
WO 97/38684 PCT/US97/06351
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
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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.
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-~, AIDS, 9: 137-143.
Pulliarn L., Berens, ME, Rosenblum, ML 1988. A normal human brain
cell agg~egale model for neurobiological studies, J Neurosci Res 21 :521-530.
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.
Robinson, C (1995) N-acetylcysteine, Drugs of the Future, 20(6):
559-563.
Sandstrom, PA, Roberts, B, Folks, TM, Buttke,TM (1993) HIV gene
expression enhances T-cell susceptibility to hydrogen peroxide induced
apoptosis, 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 Hum
Retroviruses, 9: 299-306.
Floyd, RA, Watson,JJ, Wong, PK (1984) Sensitive assay of hydroxyl
free radical formation utilizing high pressure liquid chromatography and
,
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WO 97/38684 PCTJUS97/06351
electrochemical detection of phenol and salicylate hydroxylation products, J
Biochem Biophys Methods, 10: 221-235.
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-
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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
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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.
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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.
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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
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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
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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
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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-
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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~
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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
17
,,
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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)
18
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WO 97138684 PCT/US97/06351
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).
19
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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
WO 97/38684 PCT/US97/06351
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
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WO 97/38684 PCT/US97/06351
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.
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WO 97/38684 PCT/US97/06351
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 .
.
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WO 97138684 PCT/US97/06351
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.
24
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WO 97/38684 PCT/US97/06351
~ 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.
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WO 97/38684 PCT/US97/06351
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
26
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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.
, ,
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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
28
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WO 97138684 PCT/US97/06351
( > 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
29
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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.
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Experimental De~ n
Experiment# System TNF-~ (pg/ml) Colllpound' (~M)
Neural Cell 0 0
Bilayers
o 100
100 ~
100 100
2 Neural Cell 0 0
Bilayers
100 0
100 100
3 Brain 0 0
Aggregate
0 100
100 0
100 100
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.
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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.
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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.
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Further Brain Aggre~ation Studies
Further studies were conducted as follows:
Experiment # gpl20(ng/ml)TNF-(x(ng/ml) Compound' (~M)
4 - 0 0
- 1 0.1
- 1 0.3
- 1 3.0
S O - O
- 3
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.
34
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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.
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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.
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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.
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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.
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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
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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.
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The data suvgests N-tert-butyl-4-acetamidobenzamide had a protective
effect.
41
