OA11196A - Hiv protease inhibitors - Google Patents

Abstract

HIV protease inhibitors, obtainable by chemical synthesis, inhibit or block the biological activity of the HIV protease enzyme, causing the replication of the HIV virus to terminate. These compounds, as well as pharmaceutical compositions that contain these compounds and optionally other anti-viral agents as active ingredients, are suitable for treating patients or hosts infected with the HIV virus, which is known to cause AIDS.

Description

-ι- ΟΙ 1 Ηό

HIV PROTEASE INHIBITORS

BACKGROUND AND SUMMARY OF THE INVENTION

This invention relates to a novel sériés of Chemicalcompounds useful as HIV protease inhibitors and to the useof such compounds as antiviral agents.

Acquired Immune Deficiency Syndrome (AIDS) is a rela-tively newly recognized disease or condition. AIDS causes agraduai breakdown of the body's immune System as ’well asprogressive détérioration of the central and peripheralnervous Systems. Since its initial récognition in the early1980's, AIDS has spread rapidly and has now reached épidémieproportions within a relatively limited segment of thepopulation. Intensive research has led to the discovery ofthe responsible agent, human T-lymphotropic retrovirus III(HTLV-III), now more commonly referred to as the humanimmunodeficiency virus or HIV. HIV is a member of the class of viruses known asretroviruses. The retroviral genome is composed of RNAwhich is converted to DNA by reverse transcription. Thisretroviral DNA is then stably integrated into a host cell'schromosome and, employing the réplicative processes of thehost cells, produces new retroviral particles and advancesthe infection to other cells. HIV appears to hâve aparticular affinity for the human T-4 lymphocyte cell whichplays a vital rôle in the body's immune System. HIVinfection of these white blood cells depletes this whitecell population. Eventually, the immune System is renderedinoperative and ineffective against various opportunisticdiseases such as, among others, pneumocystic carinipneumonia, Kaposi's sarcoma, and cancer of the lymph System.

Although the exact mechanism of the formation and work-ing of the HIV virus is not understood, identification ofthe virus has led to some progress in controlling thedisease. For example, the drug azidothymidine (AZT) hasbeen found effective for inhibiting the reverse transcription of the retroviral genome of the HIV virus, -Σ- ΟΙ 1 1 96 thus giving a measure of control, though not a cure, forpatients afflicted with AIDS. The search continues fordrugs that can cure or at least provide an improved measureof control of the deadly HIV virus.

Retroviral réplication routinely features post-translational processing of polyproteins. This processingis accomplished by virally encoded HIV protease enzyme.

This yields mature polypeptides that will subsequently aidin the formation and function of infectious virus. If thismolecular processing is stifled, then the normal productionof HIV is terminated. Therefore, inhibitors of HIV proteasemay function as anti-HIV viral agents. HIV protease is one of the translated products from theHIV structural protein pol gene. This retroviral protease specifically cleaves. other structural polypeptides atdiscrète sites to release these newly activated structuralproteins and enzymes, thereby rendering the virionreplication-competent. As such, inhibition of the HIVprotease by potent compounds may prevent proviralintégration of infected T-lymphocytes during the early phaseof the HIV-1 life cycle, as well as inhibit viralproteolytic processing during its late stage. Additionally,the protease inhibitors may hâve the advantages of beingmore readily available, longer lived in virus, and lesstoxic than currently available drugs, possibly due to theirspecificity for the retroviral protease.

In accordance with this invention, there is provided anovel class of Chemical compounds that can inhibit and/orblock the activity of the HIV protease, which halts theprolifération of HIV virus, pharmaceutical compositionscontaining these compounds, and the use of the compounds asinhibitors of the HIV protease.

The présent invention relates to compounds fallingwithin formula (9) below, and pharmaceutically acceptablesalts, prodrugs, and solvatés thereof, that inhibit theprotease encoded by human immunodeficiency virus (HIV) type1 (HIV-1) or type 2 (HIV-2). These compounds are useful in the treatment of infection by HIV and the treatment of the 011196 -3- accuired immune deficiency syndrome (AIDS). The compounds,their pharmaceutically acceptable salts, and thepharmaceutical compositions of the présent invention can beused alone or in combination with other antivirals,immunomodulators, antibiotics or vaccines. Compounds of theprésent invention can also be used as prodrugs. Methods oftreating AIDS, methods of treating HIV infection and methodsof inhibiting HIV protease are disclosed.

The compounds of the présent invention are of theformula ( 9) :

wherein: R and R' are independently selected from H, asubstituted or unsubstituted alkyl-ORj. group, a cycloalkylgroup substituted with a (Cj-CJ alkyl group or a (Cj-C6)alkyl-OH group, a heterocycle group substituted with a(C^CJ alkyl group or a (Cj-C6) alkyl-OH group, an alkyl-NR2R3group, or an alkyl-S(X)(Y)R„ group, wherein

Rr is H, a substituted or unsubstituted alkyl group, oran acyl group; R2 and R3 are each independently selected from H,substituted or unsubstituted alkyl, cycloalkyl,heterocycle, and aryl groups, and acyl and sulfonylgroups; R4 is H, a substituted or unsubstituted alkyl,cycloalkyl, heterocycle, or aryl group; andX and Y are each independently selected from =0 and nothing; or a pharmaceutically acceptable prodrug, sait or solvatéthereof. 011196 -Δ -

Preferabiy in the compounds of formula 9, R is H. Morepreferably, R is H and R' is a cycloalkyl group selectedf rom:

1U

Preferably in the compounds of formula 9 when at least oneof R and R' is an alkyl-OR3 group, Rj is H. Particularlywhen at least one of R and R' is an alkyl-ORj group, thealkyl-ORj is selected from -C (CH3) 2CH2OH, -CH (CH3j CH2OH,-CH2CH2OH, -C (CH3) (CH2OH) 2, -C (CH3) 2-O-CH2-O-CH3, -C(CH3)2CH2-O-CH2-O-CH3, and -C (CH3) 2CH2-O-acyl, or a pharmaceuticallyacceptable prodrug, sait or solvaté thereof.

Preferably when at least one of R and R' is acycloalkyl group substituted with a (C1-C6)alkyl group or a(Cj-Cg)alkyl-OH group, the cycloalkyl group is selected from:

15

Preferably when at least one of R and R' is a heterocyclegroup substituted with a (Cj-Cg) alkyl group or a (C3-C€)alkyl-OH group, the heterocycle group is selected from:

wherein R3 is H, a substituted or unsubstituted alkyl,cycloalkyl, heterocycle, or aryl group, or an acyl orsulfonyl group. A preferred species of the formula (9) is [3S- [2(2S*,3S*),3 alpha,4a beta,8a beta]]-N-(l,l-dimethyl-2- hydroxyethyl)decahydro-2- [2-hydroxy-3-[(3-hydroxy-2- -Ο- 011196 methylbenzoyl)amino]-4-(phenylthio)butyl]-3-isoquinolinecarboxamide

Me O

HO

N

H Y1nX^oh

H i SPh

CL N

21 : Me Me 15 and its pharmaceutically acceptable salts, and its prodruganalogs. Preferred prodrugs can be obtained by replacingthe hydrogen in one of the alcohol groups with an acylgroup, and more preferably an amino acid acyl group. .

The présent invention further provides pharmaceuticalformulations comprising an effective amount of a compound offormula (9) or a pharmaceutically acceptable sait thereof,in combination with a pharmaceutically acceptable carrier,such as a diluent or excipient.

The présent invention further provides a method oftreating AIDS comprising administering to a host or patient,such as a primate, an effective amount of a compound of theprésent invention.

The présent invention further provides a method ofinhibiting HIV réplication comprising administering to anHIV infected cell, a cell susceptible to HIV infection or ahost or patient, such as a primate, an effective amount of acompound of the présent invention.

Detailed Description of the Invention

The présent invention provides new compounds fallingwithin formula (9), as described above, that are useful fortreating HIV infection and/or AIDS.

Applicants incorporate by reference U.S. Patent No. 5,484,926, U.S. Patent Application Nos. 08/708,411 and 08/708,607, and Japanese Patent Application Nos. JP 95- 248183 and JP 95-248184, with the caveat that the définitions of preferences, terms, variables, labels and the -6- 011196 like used in each application are applicable only to thecorresponding disclosure from that application.

In particular, since each of the above-identifiedapplications incorporated by reference was preparedseparately, the original applications may use in sortieinstances the same term, label or variable to mean somethingdifferent. For example, the variable "X" is used in eachapplication, but each application has its own distinctdéfinition of the substituent or moiety represented by thisvariable. It will be apparent to those skilled in the artthat the terms, labels and variables in each applicationincorporated by reference are limited solely to thedisclosure from that application, and may be replaced byother suitable terms, labels and variables or the likerepresenting the particular substituents and moieties. Ofcourse, those skilled in the art will realize that anysuitable set of terms, labels and variables may be used togenerically or more specifically represent the subjectmatter disclosed in the présent application, includingterms, labels, variables, and the like universallyapplicable to the incorporated disclosures of theabove-identified applications and the following disclosure.

Compounds of the formula (9) may be prodrugs, which canserve to improve the pharmaceutical properties of thecompounds, such as pharmacokinetic properties, for example,improved bioavailability or solubility. .The préparation ofprodrugs may be carried out by standard methods known tothose skilled in the art. A preferred prodrug can beobtained by acylation or alkylation of the starting alcoholwhen R or R' is CH (CH3) 2CK2OH.

Ail températures stated herein are in degrees Celsius(°C) . Ail units of measurement employed herein are inweight units except for liquids which are in volume units.

The term "alkyl" as used herein refers to straight orbranched chain groups, preferably, having one to eight, morepreferably having one to six, and most preferably havingfrom one to four carbon atoms. The term "Cj-C6 alkyl"represents a straight or branched'alkyl chain having from -Ί - 011196 one to six carbon atoms. Exemplary C^-Cg alkyl groupsinclude methyl, ethyl, n- propyl, isopropyl, butyl,isobutyl, sec-butyl, t-butyl, pentyl, neo-pentyl, hexyl,isohexyl, and the like. The terra "Cj-Cg alkyl" includeswithin its définition the term "Cl-C,, alkyl".

The term "cycloalkyl" represents a saturated orpartially saturated, mono- or poly-carbocylic ring,preferably having 5-14 ring carbon atoms. Exemplarycycloalkyls include monocyclic rings having from 3-7,preferably 3-6, carbon atoms, such as cyclopropÿl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and thelike. An exemplary cycloalkyl is a C5-C7 cycloalkyl, whichis a saturated hydrocarbon ring structure containing fromfive to seven carbon atoms.

The term "alkoxyl" represents -O-alkyl. An example ofan alkoxyl is a C^-Cg alkoxyl, which represents a straight obranched alkyl chain having from one to six carbon atomsattached to an oxygen atom. Exemplary Cj-Cg alkoxyl groupsinclude methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl,sec-butoxyl, t-butoxyl, pentoxyl, hexoxyl, and the like.C^-Cg alkoxyl includes within its définition a Cj-C4 alkoxyl.

The term "aryl" as used herein refers to a carbocyclicor heterocyclic, aromatic, 5-14 membered monocyclic orpolycyclic ring. Exemplary aryls include phenyl, naphthyl,anthryl, phenanthryl, thienyl, pyrrolyl, imidazolyl,pyrazolyl, furyl, isothiazolyl, furazanyl, isoxazolyl,thiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,triazinyl, benzo [b]thienyl, naphtho[2,3-b]thianthrenyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxathienyl,indolizinyl, isoindolyl, indolyl, indazolyl, purinyl,isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl,quinoxyalinyl, quinzolinyl, benzothiazolyl, benzimidazolyl,tetrahydroquinolinyl, cinnolinyl, pteridinyl, carbazolyl,beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,and phenoxazinyl.

The term "aryioxyl" represents -O-aryl. 011196

The term "hydrolyzable group" is a group, which whenbonded to an oxygen, forms an ester, which can be hydrolyzedin vivo to a hydroxyl group. Exemplary hydrolyzable groups,which are optionally substituted, include acyl function,sulfonate function and phosphate function. For example,such hydrolyzable groups include blocked or unblocked aminoacid residue, a hemisuccinate residue, and a nicotinateresidue.

The term "halogen" represents chlorine, fluorine,bromine or iodine. The term "halo" represents chloro,fluoro, bromo or iodo.

The term "carbocycle" represents an aromatic or asaturated or a partially saturated 5-14 membered monocyclicor polycyclic ring, such as a 5- to 7-membered monocyclic or 7- to 10-membered bicyclic ring, wherein ail the ringmembers are carbon atoms.

The term "heterocycle" represents an aromatic or asaturated or a partially saturated, 5-14 membered, monocylicor polycyclic ring, such as a 5- to 7-membered monocyclic or7- to 10-membered bicyclic ring, having from one to threeheteroatoms selected from nitrogen, oxygen and sulfur, andwherein any nitrogen and sulfur heteroatoms may optionallybe oxidized, and any nitrogen heteroatom may optionally bequaternized. The heterocyclic ring may be attached at anysuitable heteroatom or carbon atom. Examples of suchheterocycles include decahydroisoquinolinyl, octahydro-thieno[3,2-c]pyridinyl, piperidinyl, piperazinyl,azepinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl,imidazolyl, isobenzofuranyl, furazanyl, imidazolinyl,imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, thianthrenyl, triazinyl, isoxazolidinyl, morpholinyl,thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, iso-thiazolidinyl, indolyl, quinolinyl, chromenyl, xanthenyl,isoquinolinyl, benzimidazolyl, thiadiazolyl, benzopyranyl,benzothiazolyl, benzoazolyl, furyl, tetrahydrofuryl,tetrahydropyranyl, thienyl, benzothienyl, benzo[b]thienyl,naphtho[2,3-bj thienyl, thiamorpholinyl, -9- Z * ' •w » ' ‘ 011196 thiamorpholmyl suif oxide, thiamorphoiinylsulfone, oxadiazolyl, triazolyl, tetrahydroquinolinyl, tetrahydriso-quinolinyl, phenoxathienyl, indolizinyl, isoindolyl,indazolyl, purinyl, isoguinolyl, quinolyl, phthalazinyl,naphthyridinyl, quinoxyalinyl, quinzolinyl,tetrahydroquinolinyl, cinnolinyl, pteridinyl, carbazolyl,beta-carbolinyl, phenanthridinyl, acridinyl, perimidinyl,phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl,and phenoxazinyl.

The terra "thioether" includes S-aryl, such’asphenylthio and naphthylthio; S-heterocycle where theheterocycle is saturated or partially saturated; S-(C5-CJ-cycloalkyl ; and S-alkyl, such as Cj-Cg alkylthio.

In the thioether, the -aryl, the -heterocycle, the-cycloalkyl and the -alkyl can optionally be substituted.

An example of a thioether is "Cj-Cg alkylthio", whichrepresents a straight or branched alkyl chain having fromone to six carbon atoms attached to a sulfur atom.

Exemplary C^-Cg alkylthio groups include methylthio,ethylthio, propylthio, isopropylthio, butylthio, sec-butylthio, t-butylthio, pentylthio, hexylthio, and the like.

The term "mercapto" represents -SH.

The term "amino" represents -NL^, wherein L3 and L2are preferably independently selected from oxygen,carbocycle, heterocycle, alkyl, sulfonyl and hydrogen; orNC(O)L3, wherein L3 is preferably alkyl, alkoxyl, hydrogen or-NL,L2. The aryl, alkyl and alkoxyl groups can optionally besubstituted. An example of an amino is Cj-C, alkylamino,which represents a straight or branched alkyl chain havingfrom one to four carbon atoms attached to an amino group.Exemplary Cj-C, alkylamino groups include methylamino,ethylamino, propylamino, isopropylamino, butylamino, sec-butylamino, and the like. Another example of an amino isdi (Cj-CJ alkylamino, which represents two straight orbranched alkyl chains, each having from one to four carbonatoms attached to a common amino group. Exemplary di(Cj-C<)alkylamino groups include dimethylamino, ethylmezhylamino, methylpropylamino, ethylisopropylamino, -10- 1li 15

2iJ 25 011196 butylmethylamino, sec-butylethylamino, and the like. Anexample of an amino is Ci-C4 alkylsuifonylamino, which has astraight or branched alkyl chain having from one te fourcarbon atoms attached to a sulfonylamino moiety. ExemplaryCj-C4 alkylsulfonylamino groups include methylsulfonylamino,ethylsulfonylamino, propylsulfonylamino, isopropylsulfonylamino, butylsulfonylamino, sec- butylsulfonylamino, t-butylsulfonylamino, and the like.

The term "acyl" represents L6C(O)L4, wherein L6 is a single bond, -O or -N, and further wherein L4 is preferablyalkyl, amino, hydroxyl, alkoxyl or hydrogen. The alkyl andalkoxyl groups can optionally be substituted. An exemplaryacyl is a Cj-C4 alkoxycarbonyl, which is a straight orbranched alkoxyl chain having from one to four carbon atomsattached to-a carbonyl moiety. Exemplary Cj-C^ alkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, and the like. Another exemplary acyl is acarboxy wherein L>6 is a single bond and L4 is alkoxyl,hydrogen, or hydroxyl. A further exemplary acyl is N-(Cj-C4 ) alkylcarbamoyl (L6 is a single bond and L„ is an amino),which is a straight or branched alkyl chain having from oneto four carbon atoms attached to the nitrogen atom of acarbamoyl moiety. Exemplary N-(C^-CJ alkylcarbamoyl groupsinclude N-methylcarbamoyl, N-ethylcarbamoyl, N-propylcarbamoyl, N-isopropylcarbamoyl, N-butylcarbamoyl,and N-t-butylcarbamoyl, and the like. Yet another exemplary acyl is N, N-di (C,-C4) alkylcarbamoyl, which has two straightor branched alkyl chains, each having from one to fourcarbon atoms attached to the nitrogen atom of a carbamoylmoiety. Exemplary N,N-di (Ci-C4) alkylcarbamoyl groups includeN,N-dimethylcarbamoyl, N,N-ethylmethylcarbamoyl, N,N-methylpropylcarbamoyl, N,N-ethylisopropylcarbamoyl, N,N-butylmethylcarbamoyl, N,N-sec-butyiethylcarbamoyl, and thelike.

The term "sulfinyl" represents -SO-Ls, wherein L5 is preferably alkyl, amino, aryl, cycloalkyl or heterocycle. 35 -11- 011196

The alkyl, aryl, cycloalkyl and heterocycle can ail optionally be substituted.

The terra "sulfonyl" represents -SO2-L5, wherein L5 ispreferably alkyl, aryl, cycloalkyl, heterocycle or amino.

The alkyl, aryl, cycloalkyl and heterocycle can ailoptionally be substituted. An example of a sulfonyl is aCj-C4 alkylsulfonyl, which is a straight or branched alkylChain having from one to four carbon atoms attached to asulfonyl moiety. Exemplary Cj-C^ alkylsulfonyl groupsinclude methylsulfonyl, ethylsulfonyl, propylsulfonyl,isopropylsulfonyl, butylsulfonyl, sec-butylsulfonyl, t- butylsulfonyl and the like.

As indicated above, many of the groups are optionally substituted. In fact, unless specifically noted, ail of thegroups defined by the terms defined in this application maybe substituted or unsubstituted. For instance, when theterra "alkyl" is used, it should be understood to encompassboth substituted and unsubstituted alkyl unless spécifieexclusion of one or the other is positively stated.

Examples of substituents for alkyl and aryl includemercapto, thioether, nitro (NO2) , amino, aryloxyl, halogen,hydroxyl, alkoxyl, and acyl, as well as aryl, cycloalkyl andsaturated and partially saturated heterocycles. Examples ofsubstituents for heterocycle and cycloalkyl include thoselisted above for alkyl and aryl, as well as aryl and alkyl.

Exemplary substituted aryls include a phenyl ornaphthyl ring substituted with one or more substituents,preferably one to three substituents, independently selectedfrom halo, hydroxy, morpholino(Cj-C„)alkoxy carbonyl, pyridyl alkoxycarbonyl, halo (Cx-C4) alkyl, Ci-C4 alkyl, Cj-C4alkoxy, carboxy, Cj-C4 alkoxycarbonyl, carbamoyl, N- (C^-CJ alkylcarbamoyl, amino, Cj-C4 alkylamino,di(C.-CJ alkylamino or a group of the formula-(CH2)a-R' where a is 1, 2, 3 or 4 ; and R7 is hydroxy, Cj-C,alkoxy, carboxy, Cj-C, alkoxycarbonyl, amino, carbamoyl,

Ci-C4 alkylamino or di(C,-CJ alkylamino.

Anotner substituted alkyl is halo (C^-CJ alkyl, whichrepresents a straight or branched alkyl chain having from- -12- 011196 one to four carbon atoras with 1-3 halogen atoms attachée} toit. Exemplary halo (Ci-C4) alkyl groups include chloromethyl, 2- bromoethyl, 1-chloroisopropyl, 3-fluoropropyl, 2,3-dibromobutyl, 3-chloroisobutyl, iodo-t-butyl, trifluoromethyl and the like.

Another substituted alkyl is hydroxy (Ci-C„) alkyl, which represents a straight or branched alkyl chain having fromone to four carbon atoms with a hydroxy group attached toit. Exemplary hydroxy(Cj-CJ alkyl groups includehydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, ‘2-hydroxy-isopropyl, 4-hydroxybutyl and the like.

Yet another substituted alkyl is Cj-C4 alkylthio(C:-C,,) alkyl, which is a straight or branched C}-C4 alkyl groupwith a Ch-Ch alkylthio group attached to it. Exemplary Cj-C^alkylthio (C,-C4) alkyl groups include methylthiomethyl,ethylthiomethyl, propylthiopropyl, sec-butylthiomethyl, andthe like.

Yet another exemplary substituted alkyl isheterocycle(Ci- C4) alkyl, which is a straight or branchedalkyl chain having from one to four carbon atoms with aheterocycle attached to it. Exemplary heterocycle(Cj-CJ alkyls include pyrrolylmethyl, quinolinyl-methyl, 1-indolylethyl, 2-furylethyl, 3-thien-2-ylpropyl, 1-imidazolylisopropyl, 4-thiazolylbutyl and the like.

Yet another substituted alkyl is aryl (Cj-C,,) alkyl, whichis a straight or branched alkyl chain having from one tofour carbon atoms with an aryl group attached to it.Exemplary aryl(Ci-C4) alkyl groups include phenylmethyl,2-phenylethyl, 3-naphthyl-propyl, 1-naphthylisopropyl, 4-phenylbutyl and the like.

The heterocycle can, for example, be substituted with1, 2 or 3 substituents independently selected from halo,halo (Ci- CJ alkyl, Ct-C4 alkyl, Cj-C^ alkoxy, carboxy, C,-C,alkoxycarbonyl, carbamoyl, N-(Cj-C4 ) alkylcarbamoyl, amino,

Ci-C4alkylamino, di (Ci-C4) alkylamino or a group having the structure -(CH2)a-R1 where a is 1, 2, 3 or 4 and R' is hydroxy, C,-C< alkoxy, carboxy, Cj-C4 alkoxycarbonyl, amino, carbamoyl, Ct-C4 alkylamino or di (C,-C4) alkylamino. -13- 011196

Examples of substituted heterocycles include 3-N-t-butyl carboxamide decahydroisoquinolinyl, 6-N-t-butylcarboxamide octahydro-thieno [3,2-c]pyridinyl, 3- methylimidazolyl, 3-methoxypyridyl, 4-chloroquinolinyl, 4- aminothiazolyl, 8-methylquinolinyl, 6-chloroquinoxalinyl, 3- ethylpyridyl, 6-methoxybenzimidazolyl, 4-hydroxyfuryl, 4- methylisoquinolinyl, 6,8-dibromoquinolinyl, 2-methyl- 1,2,3,4-tetrahydroisoquinolinyl, N-methyl-quinolin-2-yl, 2-1-butoxycarbonyl-l·,2,3,4-isoquinolin-7-yl and the like.

Exemplary heterocyclic ring Systems represénted by A orB include (1) 5-membered monocyclic ring groups such asthienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl,isothiazolyl, furazanyl, isoxazolyl, thiazolyl and the like;(2) 6-membered monocyclic groups such as pyridyl, pyrazinyl,pyrimidinyl, pyridazinly, triazinyl and the like; and (3)polycyclic heterocyclic rings groups, such as decahydroisoquinolinyl, octahydro-thieno [3,2-c] pyridinyl,benzo[b]thienyl, naphtho[2,3-b]thianthrenyl,isobenzofuranyl, chromenyl, xanthenyl, and fully orpartially saturated analogs thereof. A cycloalkyl may be optionally substituted with'l, 2 or3 substituents independently selected from halo,halo (Cj-CJ alkyl, Cj-C4 alkyl, Cj-C4 alkoxy, carboxy, Cj-C4alkoxycarbonyl, carbamoyl, N-(Cj-C,) alkylcarbamoyl, amino, alkylamino, di (Cj-Ch) alkylamino or a group having thestructure - (CH2) a-R7 where a is 1, 2, 3 or 4 and R1 ishydroxy, Ci-C4 alkoxy, carboxy, C!-C4 alkoxycarbonyl, amino,carbamoyl, Ci~C4 alkylamino or di(C,-C4)alkylamino.

Exemplary substituted cycloalkyl groups include 3-methylcyclopentyl, 4-ethoxycyclohexyl, 5-carboxycyclo-heptyl, 6-chlorocyclohexyl and the like.

Exemplary substituted hydrolyzable groups includeN-benzyl glycyl, N-Cbz-L-valyl, and N-methyl nicotinate.

The compounds of the présent invention hâve at leastfive asymmetric centers denoted by an asterisk in theformula (9) below. -14- 011196

As a conséquence of these asymmetric centers, thecompounds of the présent invention can occur in any of thepossible stereoisomeric forms, and can be used in mixturesof stereoisomers, which can be optically active or racemic,or can be used alone as essentially pure stereisomers, i.e.,at least 95% pure. Ail asymmetric forms, individualstereoisomers and combinations thereof, are within the scopeof the présent invention.

The individual stereoisomers may be prepared from cheirrespective precursors by the procedures described above, byresolving the racemic mixtures, or by separating thediastereomers. The resolution can be carried out in theprésence of a resolving agent, by chromatography or byrepeated crystallization or by some combination of thesetechniques which are known in the art. Further detailsregarding resolutions can be found in Jacques et al.,Enantiomers, Racemates, and Resolutions, John Wiley &amp; Sons1981.

Preferably, the compounds of the présent invention aresubstantially pure, i.e, over 50% pure. More preferably,the compounds are at least 75% pure. Even more preferably,the compounds are more than 90% pure. Even more preferably,the compounds are at least 95% pure, more preferably, atleast 97% pure, and most preferably at least 99% pure.

As mentioned above, the invention includes thepharmaceutically acceptable salts of the compounds definedby formula (9). A compound of this invention may possess asufficiently acidic, a sufficiently basic, or bothfunctional groups, and accordingly react with any of anumber of inorganic or organic bases, and inorganic andorganic acids, to form a pharmaceutically acceptable sait. -15- 1ü 2Ü 25 .Xi 35 011196

The term "pharmaceutically acceptable sait", as usedherein, refers to salts of the compounds of the abcveformula which are substantially non-toxic to livingorganisms. Exemplary pharmaceutically acceptable saltsinclude those salts prepared by reaction of the compounds ofthe présent invention with a minerai or organic acid or aninorganic base. The reactants are generally combined in amutual solvent such as diethylether or benzene, for acidaddition salts, or water or alcohols for base additionsalts. The salts normally precipitate out of solutionwithin about one hour to about ten days and can be isolatedby filtration or other conventional methods. Such salts areknown as acid addition and base addition salts.

Acids that may be employed to form acid addition saltsare inorganic acids such as hydrochloric acid, hydrobromicacid, hydroiodic acid, sulfuric acid, phosphoric acid, andthe like, and organic acids such as p-toluenesulfonic,methanesulfonic acid, oxalic acid, p-bromophenylsulfonicacid, carbonic acid, succinic acid, citric acid, benzoicacid, acetic acid, and the like.

Examples of pharmaceutically acceptable salts are thesulfate, pyrosulfate, bisulfate, sulfite, bisulfite,phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide,acetate, propionate, decanoate, caprylate, acrylate,formate, isobutyrate, caproate, heptanoate, propiolate,oxalate, malonate, succinate, suberate, sebacate, fumarate,maleate, butyne-1,4-dioate, hexyne-1,S-dioate, benzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate,phenylbutyrate, citrate, lactate, g-hydroxybutyrate,glycollate, tartrate, methane-sulfonate, propanesulfonate,naphthalene-l-sulfonate, napththalene-2-sulfonate, mandelateand the like.

Preferred pharmaceutically acceptable acid addition salts are those formed with minerai acids such as hydrochloric acid and hydrobromic acid, and those formed 011196 -16- with organic acids such as maleic acid and methanesulfonicacid.

Base addition salts include those derived frominorganic and organic bases, such as ammonium or alkali oralkaline eartn métal hydroxides, carbonates, bicarbonates,and the like. Such bases useful in preparing the salts ofthis invention thus include sodium hydroxide, potassiumhydroxide, ammonium hydroxide, potassium carbonate, sodiumcarbonate, sodium bicarbonate, potassium bicarbonate,calcium hydroxide, calcium carbonate and the like. Thepotassium and sodium sait forms are particularly preferred. A "pharmaceutically acceptable prodrug" is intended tomean a compound that may be converted under physiologicalconditions or by solvolysis to a compound of the formula 9. A "pharmaceutically acceptable solvaté" is intended tomean a solvaté that retains the biological effectiveness andproperties of the biologically active components of compounds of formula 9.

Examples of pharmaceutically acceptable solvatésinclude, but are not limited to, compounds of formula 9 incombination with water, isopropanol, éthanol, methanol, DMSO, ethyl acetate, acetic acid, or ethanolamine.

It should be recognized that the particular counterionforming a part of any sait of this invention is not of acritical nature, so long as the sait as a whole ispharmacologically acceptable and as long as the counteriondoes not contribute undesired qualities to the sait as awhole. -17- 011196 A preferred compound is compound 21 i SPh

Ch N

HO

: Me Me 21 [3S-[2(2S*,3S*),3 alpha,4a beta,8a beta]]-N-(1,1-dimethyl-2-hydroxyethyl)decahydro-2-[2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4 -(phenylthio)butyl]-3-isoquinolinecarboxamide. A process for making compound 21 is provided below.

Compound 21 has also been obtained as a métabolite from theplasma of patients administered [3S- (3R,4aR*,8aR*,2'S*,3'S*)]-2-[2'-hydroxy-31-phenylthiomethyl- 4'-aza-5'-oxo-5'-(2 1 1 -methyl-31 '-hydroxyphenyl)pentyl]decahydroisoquinoline - 3-N-t-butylcarboxamide methanesulfonicacid sait, which is disclosed in U.S. Patent No. 5,484,926.

The compounds of formula 9 can be prepared according tothe following Reaction Scheme I. -18- 011196

REACTION SCHEME I

Scheroc L General Synthctlc P>th*av for the Production oi 9b and Dérivatives CONH-t-Βυ

A

R

Sttp4

Compound la, perhydroisoquinoline, which is commercially available from NSC Technologies (Chicago, IL)or Procos SpA (Milan, Italy) is subjected to prolonged acidhydrolysis in step la to obtain compound 2a. A variety of inorganic acids may be used in either an aqueous/organicsolvent mixture or in water alone at températures above 50°C. An example of such an inorganic acid is 6N aqueous HCl.Substitutes for compound la include the corresponding esters lb, thioesters le or other amides ld: çooz

lb

where Z, Z, cycloalkyl, and Z2 may eachheterocycle, or independently bearyl. alkyl, -19- 011196

Compound 2a is then protected at the amine nitrogen toobtain compound 2b in step lb. The protecting group Rp is defined as a suitably conjugating group to avoid unwanteddécomposition of activated carboxylate dérivatives ofcompound 2b in Step 2. Such protecting groups typically canbe carbamate in origin, having a general structure offormula 11: R-c 11

The identity of R" in formula 11 can be any alkyl, cycloalkyl, aryl, or heterocycle which can be removed easilyin a deprotection step after Step 2. Examples of R" include, but are notlimited to methyl, ethyl, propyl,isopropyl, n-butyl, isobutyl, t-butyl or higher branched orunbranched alkyl, 2,2,2-trichloroethyl, 2- trimethylsilylethyl, allyl, phenyl, substituted phenyl,benzyl, substituted benzyl, 9-fluorenylmethyl, 9-anthrylmethyl and higher polycyclic aromatic ring System.

The following materials, as defined below, can be obtainedfrom the Aldrich Chemical Co. (Sigma Aldrich Fluka): σ

2.2.2-lrichbroethyl - —O-^-Ç-QO H CHj 2-irimettiylsÎtytethyl = — 0+2- Ç- S<— CH3H CH3 allyl - — CHj-CHsC^

9-anthry!rnethyl = -2°- 011196

Such procecting groups typically can be installée! by anacylation reaction of the corresponding haloformate ester12a or a dicarbonate 12b:

O

X R"O X12a X = halogen

O

X

R-cr^Y 12b Y=OCOR" 1ΰ 1,8- in the presence of a suitable base in typical organicsolvents for these types of reactions such as hal’ogenatedsolvents, ethers and hydrocarbons. Such bases are typicallyinorganic, such as métal hydroxides, bicarbonates andcarbonates or organic bases such as amines like triethylamine, diethylamine, diethyl isopropylamine,diazabicyclo[2.2.2]octane (DABCO) or related di- ortrialkyl-amines, as well as amidine bases like 1,8-diazabicyclo[5.4,0] undec-7-ene (DBU) and 1,8-aiazabicyclo [4.3.0]non-5-ene (DBN) . The followingmaterials, as defined below, can be obtained from theAldrich Chemical Co. (Sigma Aldrich Fluka): CABCO= CÇ) DBN =

These reactions are typically run anywhere from belowroom température to approximately 100 °C.

The amide coupling Step 2 can be accomplished in any number of fashions depending on how the carboxyl group is activated. A group J is installed in Step 2 by reaction of the carboxylic acid 2b to produce the activated dérivative 2c . -21- 011196

Step 2

activation of thecarboxyi group 2b Rp = amine proteccng group

The group J can be any of a variety of leaving groupssuch as alkoxy, hydroxy, halogen, pseudohalogen (includingazide, cyanide, isocyanate and isothiocyanate), alkyl orarenesulfonate, aromatic heterocycle(bonded through aheteroatom) and N-hydroxyheterocycle, including hydroxysuceinimide or hydroxybenzotriazole ester. Thefollowing définitions apply to the terms above: azide — N—hfeN cyanide — Cs N ts ocyanate — c®= o is othioc yanate _N=O=S 0 II alkylsuilonate — O—S-alkyl Ô 0 II arenesutf onate — O- S- aryl N -hyd roxyhet e rocyclic N.hydroxysuccir.inu'Oe

e nitrogen heterocycle hydroxybenzotriazole

N 'n

N i

OH -22- 011196

The acyl halides (2c, J = halogen) may be prepared using inorganic halogenating agents such as thionyl chlorideor bromide, phosphorous trichloride or bromide, ohosphorouspentachloride or bromide or organic agents such as oxalylchloride or trichlorisocyanuric acid. Esters (2c, J = OR") (R" is defined above) may be prepared in a variety of waysstarting from the acid chloride 2c where J is Cl by combination with the desired alcohol in the presence of anorganic or inorganic base stated previously for theacylation of compound 12a or compound 12b. Alternatively, the ester may be produced by acid-promoted estérification inthe presence of the desired alcohol. The sulfonates (2c, J = OSO2Wj, where Wj is alkyl or aryl) are typically made byreaction of the carboxylic acid 2b with alkyl or arvlsulfonyl chlorides in the presence of an organic aminebase such as triethylamine in a non-polar solvent attempératures below 0 ’C. Alkyl and arylsulfonyl are definedas follows: o alkyfajlfonylchloride = Q—S—3tWy!

(I

O

O

U arenesulfonyl chloride - Q — S—aryl ii

O

The pseudohalogen dérivatives of 2c (J = pseudohal.ogen)are typically made from the acid halides 2c (J = halogen) by reaction with inorganic pseudohalide in the presence of abase. Such bases include, but are not limited to métalhydroxides, bicarbonates and carbonates or organic basessuch as amines like triethylamine, diethylamine, diethylisopropylamine, 1,8-diazabicyclo[2.2.2]octane (DABCO) orrelated di- or trialkylamines, as well as' amidine bases like1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1,8-diazabicyclo[4.3.0}non-5-ene (DBN). A particularlypreferred base is triethylamine. The heteroaromatic -23- 011196

dérivatives of 2c are also made from the acid halides 2c (J = halogen), utilizing the spécifie heteroaromatic compoundin the presence of an amine base in a non-polar solvent.

The N-hydroxyheterocyclic dérivatives of 2c can be made from the acid halides as above and may also be generated usingalkyl carbodiimides (alkyl-N=C=N-alkyl, where the alkylgroups can be the same or different) or aryl carbodiimides(aryl-N-C=N-aryl, where the aryl groups can be the same ordifferent) and an amine base as condensing agents.

The primary or secondary amine (shown above the arrowin Step 2 of Scheme I) used in the coupling process mayincorporate suitable protecting groups, depending on thefunctionality présent in the amine and the mode of couplingused. The mode of coupling of 2c with a primary or secondary amine can be carried out in a variety of waysdepending on the iaentity of J. When a free acid is used(2c, J = OH) the coupling can be performed using carbodiimide-based methods utilizing any of the commonreagents of this class, including dicyclohexylcarbodiimideor related dialkylcarbodiimides, EDC (salts of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) or related water-soluble reagents along with an organic amine base in polarorganic solvents such as dioxane, DMF, NMP and acetonitrilein the presence of an N-hydroxyheterocyclic compound such asN-hydroxysuccinimide or 3-hydroxybenzotriazole.

Alternatively, haloformate esters, such as 12d, may be used to temporarily activate the acid to give mixed anhydrides ofgeneral formula 2d.

12d X = halogen2d -24- 011196

Such halofcrmate esters are typically as shown in 12d above and include methyl-, ethyl-, isopropyl-, isobutyl-, n-butyl,phenyl- and related alkyl and aryl chloroformâtes, definedbelow. o

alkyl chloroformate = II

alkyl-O^Q

1iJ 15 25

O aryl chloroformate « , aryl-0 0

Formula 2d is a possible intermediate in the step fromformula 2b to formula 3. Formula 2d is an intermediate, butthe process described here results in formula 3, withoutisolation of Formula 2d.

These reactions are typically performed in a variety ofnon-polar organic solvents like halocarbons and ethers suchas diethvl ether, methyl t-butylether, diisopropyl ether,dioxane and THF at températures below 0 °C accompanied by anorganic amine base such as triethylamine, diethylamine,diethyl isopropylamine, DABCO or related di- or trialkylamines, as well as amidine bases like DBU and DBN.

When J in compound 2c is an alkyl or arenesulfonate (J = OSO2R or OSO2Ar) , the coupling can be performed in avariety of non-polar organic solvents like halocarbons andethers, such as diethyl ether, methyl t-butylether,diisopropyl ether, dioxane and THF at températures below 0’C, accompanied by an organic amine base such as triethylamine, diethylamine, diethyl isopropylamine, DABCOor related di- or trialkylamines, as well as amidine baseslike DBU and D3N.

When J in compound 2c is a halogen or pseudohalogen, the coupling may be performed in most common organic solvents such as THF, diethyl ether, dioxane, methyl t-butyl ether or other ethers; acetone, cyclohexanone, methyl isobutylketone and other ketones; esters such as ethyl, -25- 011196 methyl and isopropyl acetate; halogenated solvents such ashalogenated méthanes and ethanes, chlorobenzene and otherhalogenated benzenes; nitriles such acetonitrile andpropionitrile; lower alcohols such as éthanol, isopropanol,t-butanol and related alcohols; and polar organic solventssuch as dimethylformamide, dimethylsulfoxide, N-methyl-2-pyrrollidone and related amide-containing solvents. A baseis frequently used and may be any of a number of inorganicbases such as métal hydroxides, bicarbonates and carbonatesor organic bases such as amines like triethylamine,diethylamine, diethyl isopropylamine, DABCO or related di-or trialkylamines, as well as amidine bases like DBU andDBN.

One skilled in the art will be able to perform theamide coupling Step 2 with other possible J groups.

In Step 3 protecting group removal can be accomplished using any of the standard methods for deprotecting aparticular class of protecting group. Simple alkyl- andsubstituted alkyl carbamates can be removed with aqueoussolutions of base at températures up to about 100 °C,employing any of the common inorganic métal hydroxides suchas sodium-, lithium-, potassium- or barium hydroxide orhydroxides of other metals in at least stoichiometricamounts. Carbamate protecting groups that contain benzylgroups bonded to oxygen may be removed by hydrogenolysiswith a palladium or platinum catalyst. Alternatively,aqueous base hydrolysis may be used at températures up. toabout 100 ’C, employing any of the common inorganic métalhydroxides such as sodium-, lithium-, potassium- or bariumhydroxide or hydroxides of other metals in at leaststoichiometric amounts. A variety of anhydrous acids mayalso be used for deprotection of benzvl-based carbamates,including HCl, H3r and HI. Lewis acids of boron andaluminum such as A1C13, BBr3, BC13 in non-polar solvents arealso effective. Certain substituted benzyl, aryl or alkylgroups in which the spécifie substitution pattern is chosen for its ability to be removed under spécifie conditions may also be used. 'For example, the 2- -26- 011196 trimethylsilylethylcarbonyl group (Teoc) is a protectingcroup designed to take advantage of the spécifie reactivityof the 2-trimethylsilylethyl group in the deprotectionprocess. 2-Trimethylsilylethylcarbonyl chloride may be usedto protect the amine nitrogen and may later be removed usingsources of fluoride ion such as Hr or tetraalkylammoniumfluoride salts.

In Step 4, the perhydroisoquinoline piece of formula 4is connected to the Chloroalcohol (compound 5, Sçheme I) viaan epoxide intermediate (13) generated via the base-inducedclosure of the vicinal chlorohydrin functionality.

Compound 5 is produced by Kaneka Industries, Japan. Severalclose-open procedures in proceeding from compound 5 -compound 13 - compound 6 may be used. The epoxide 13 may beisolated or it may be reacted with 4 added either subséquentto formation of 13 or 4 may be présent from the beginning ofthe sequence. The epoxide 13 can be generated using inorganic bases such as métal hydroxides, carbonates andbicarbonates in solvents such as alcohols like methano.léthanol or isopropyl alcohol, ethers such as THF and dioxaneor mixtures of the two. The epoxide can also be generated ina 2-phase solvent System consisting of water and ahalocarbon solvent such as dichloromethane along with thebase. A phase-transfer catalyst such as a tetraalkylammonium sait may be used to facilitate theprocess. The process of opening the epoxide 13 withcompound 4 is accomplished in alcohol solvents or mixtures of an alcohol and another solvent which may be an ether or a dipolar aprotic solvent such as dimethylformamide or dimethylsulfoxide. The opening of the epoxide 13 with -27- 011196 compound 4 to give compound 6 is optimally performed over aperiod of 2-7 hours at 50 - 60 °C.

In Step 5 the carbobenzyloxy group can be removed togive the free amine 7. This can be done using HBr in acetic acid using cosolvents such as halocarbons. It can also beperformed using halides of boron such as BBr3 and BC13 oralkyl substituted boron halides such as dimethylboronbromide in halocarbon solvents like chloroform anddichloromethane at températures ranging from 0 °C up toambient température. Alternatively, the carbobenzyloxygroup can be removed by hydrolysis using aqueous/alcoholicsolutions of métal hydroxides like barium, sodium, lithiumor potassium hydroxide at températures above ambient forperiods of hours.

Step 6a is the coupling of benzoic acid dérivatives offormula 8 to give 9a. In Formula 8, Q can be a leaving group. Q can be any of the leaving groups discussed abovefor Group J. The compounds of formula 8 where Q = OH or Cl are commercially available from EMS Dottikon, Lenzburg,Switzerland and Sugai Chemical Industries, Ltd. in Japan.

The coupling can be carried out in a variety of ways,depending on the identity of Q. When a free acid is used (Q= OH) , the coupling can be performed using carbodiimidebased methods utilizing any of the common reagents of thisclass including dicyclohexylcarbodiimide or relateddialkylcarbodiimides, EDC (salts of l-(3- dimethylaminopropyl)-3-ethylcarbodiimide) or related watersoluble reagents along with an organic amine base in polarorganic solvents such as dioxane> DMF, NMP and acetonitrilein the presence of an N-hydroxyheterocyclic including N-hydroxysuccinimide or 3-hydroxybenzotriazole. When Q = a halogen or pseudohalogen, the coupling may be performed inmost common organic solvents such as THF, diethyl ether,dioxane, methyl t-butyl ether or other ethers; acetone,cyclohexanone, methyl isobutylketone and other ketones;esters such as ethyl, methyl and isopropyl acetate; -28- 011196 halogenated solvents such as halogenated méthanes and ethanes, chlorobenzene and other halogenated benzenes; nitriles such acetonitrile and propionitrile; lower alcohols such as éthanol, isopropanol, t-butanol and related alcohols, and polar organic solvents such as5 dimethylformamide, dimethylsulfoxide, N-methyl-2-pyrrollidone and related amide-containing solvents. A base is frequently used and may be any of a number of inorganic bases such as métal hydroxides, bicarbonates and carbonates or organic bases such as amines like triethylamine, 1L1 diethylamine, diethyl isopropylamine, DABCO or related di-or trialkylamines, as well as amidine bases like DBU andDBN.

Acetate removal is accomplished in step 6b with aqueous or alcoholic solutions of inorganic bases such as métal 15 hydroxides, carbonates and bicarbonates at ambienttempératures up to 100 °C. If there is a protectedfunctionality on the carboxamide group bonded to theperhydroisoquinoline ring System, it is best removed at thispoint (during or after step 6b). The nature of this step is dépendent on the exact identity of the protecting group. A preferred method for accomplishing the entire process shown in Scheme I is shown in Scheme II.

The Cbz-protected amino acid 15 was coupled with the amine22 to give the amide 16. The Cbz group was removed byhydrogénation to -29- 011196 S ch e me II. Svnlhesis of Amidc 2 ! CW U· Bu 1

Qe

I. 6N*q. HCl X

COOH

H M? Me

Me. Mc>/ OTMS

Cta 1MX Me"N tH 2. Ctod

STEPA EDC, ΒΟΒρΗ,Ο

STEPB *2

S’/.Pd'C

EiOH νχ%, H Mc

"N

STEPC

Fvrb vd roi soq ai noii n e SPh

CbzHhK'Y''^ Gai

ChloroilCDhol

Niai 1PA

STE PD

Kl

Mc 50%x|. NOH

IPA

STEPE

Me

AcO 1 COCI

Yy 20

EljN, THF, &amp;Oltlien 50% uq. hiCH

STEPF

H SPh Ck f< xx

? f XJO 21 give the amine 17. This was coupled with the chloroalcoholvia the epoxide using the in situ procedure to give theadduct 18. Conventional deprotection with base and couplingof the free primary amine with the acid chloride 20 gaverise to amide 21. Details of this process are provided below in Examples 1 A to F. The lettering A to F in SchemeII corresponds to Examples 1 A to F below.

The fcllowing Examples illustrate aspects of theinvention. These examples are for illustrative purposes andare not intended to limit the scope of the invention.

Abbreviations for the terms melting point, nuclearmagnetic résonance spectra, électron impact mass spectra,field desorption mass spectra, fast atom bombardment massspectra, infrared spectra, ultraviolet spectra, elementalanalysis, high performance liquid chromatography, and thinlayer chromatography are, respectively, m.p., NMR, EIMS,MS(FD), MS(FA3), IR, UV, Analysis, HPLC, and TLC. Inaddition, the absorption maxima listed for the IR spectraare those of interest, not ail maxima observed. -30- 011196

In conjunction with the NMR spectra, the followingabbreviations are used: singlet (s), doublet (d), doubletof doublets (dd), triplet (t), quartet (q), multiplet (m),doublet of multiplets (dm), broad singlet (br.s), broaddoublet (br.d), broad triplet (br.t), and broad multiplet(br.m). J indicates the coupling constant in Hertz (Hz).Unless otherwise noted, NMR data refer to the free base ofthe subject compound. NMR spectra were obtained on a General Electric QE-300300MHz instrument. Chemical shifts are expressed in δvalues in ppm. Mass spectra were obtained on a VG ZAB-3Spectrometer at the Scripps Research Institute, La Jolla, CA. Infra-red spectra were recorded on a Midac Corporationspectrometer. UV spectra were obtained on a Varian.Cary 3Einstrument. Thin layer chromatography was carried out usingsilica plates available from E. Merck. Melting points weremeasured on a Mettler FP62 instrument and are uncorrected.

Example 1

Procedures for the Synthesis of Ainide of Formula 21 [3S- [2(2S*,3S*) ,3 alpha,4a beta,8a beta]]-N-(1,1-dimethyl-2 -hydroxyethyl)decahydro-2- [2-hydroxy-3- [ (3-hydroxy-2-methylbenzoyl)amino]- 4 -(phenylthio)butyl] - 3 -isoquinolinecarboxamide

H SPh Cl Λ HO,

A. Perhydroisoquinoline (26.4 g, 111 mmol) (commericallyavailable from NSC Technologies (Chicago, IL) or Procos SpA (Milan, Italy) ) was suspended in water (200 m.L) and concentrated aaueous HCl (200 mL) . This mixture v;as heated -31- 011196 to reflux and stirred for 3 days, during which time it wentinto solution. The solvents were removed under reducedpressure to give a light yellow solid. The solid wasslurried in 2-propanol (200 mL) and filtered. The filtratewas evaporated under reduced pressure to an oil. EtOAc (100mL) and water (100 mL) were added and the pH of the solutionwas brought to 8.0 by the addition of 2 N aqueous KOH.

Benzyl chloroformate (15.8 mL, 111 mmol) was added dropwiseover 30 minutes and the pH was kept between 7 and 8 by theaddition of 2 N aqueous KOH. The mixture was stirred atroom température for 18 hours. EtOAc (200 mL) was added andthe organic layer was washed with 1 N aqueous HCl (100 mL),and brine (100 mL) . The organic layer was dried (MgSO4) ,filtered, and evaporated under reduced pressure to an oil.The product was purified by silica gel chromatography,eluting with 1:1 40-60 petroleum ether/EtOAc followed by100% EtOAc. The fractions containing product were collectedand evaporated under reduced pressure to give the compound 15 (11.3 g, 32% ) as a colorless oil: ]H NMR (300 MHz, CDC13) δ 7 .43-7 .28 (m, 5 H), 5.17 (br s, 2 H) , 4.76 (m, 1 H) , 3.79 (m, 1 H) , 3. .33 (m, 1 H) , 2.19 (m, 1 H) , 1.96 (m, 1 H) , 1.88- 1.15 (m, 10 H). B. 1-Hydroxybenzotriazole (4.2 g, 31.4 mmol) and EDC (6.0g, 31.4 mmol) were added to a solution of acid 15 (8.3 g,26.2 mmol) in DMF (128 mL) at ambient température. Themixture was heated at 80° C for 10 minutes. 1,l-Dimethyl-2-trimethylsilyloxyethylamine (5.1 g, 31.4 mmol, prepared from1,l-dimethyl-2-hydroxyethylamine (Aldrich Chemical Co.) andhexamethyldisilazane (Aldrich Chemical Co.)) by heating themixture neat under reflux for several hours followed byévaporation of the volatile components was added and thesolution was heated at 80° C for 17 hours. The yellowsolution was poured into EtOAc (250 mL) and 2 N aqueous HCl (250 mL) . After stirring for 10 minutes· EtOAc (750 mL) was added and the mixture was washed with H?O (3 x 500 mL) and brine (1 x 250 mL) . The combined aqueous layers were extracted with EtOAc (1 x 250 mL). The combined organic layers were dried (Na;SO.,) and purified by flash -32- 011196 chromatography (50/50 EtOAc/hexanes) to give the compound 16 as a colorless oil (7.9 g, 78%): : ’H NMR (3 00 MHz, CD3OD) δ 7.36 (m, 5 H) , 5.20 (d, J = 8.1 Hz, 1 H), 5.10 (m, 1 H), 4.53 (m, 1 H) , 3.78 (dd, J = 13 .2, 4.4 Hz, 1 H), 3.60 (m, 2 H) , 3.48 (d, J = 10.7 Hz, 1 H), 2.15-1.25 (m, 12 H), 1.31 (s, 3 H) , 1.29 (S, 3 H) .

1L C. A mixture of carbamate 16 (7.9 g, 20.4 mmol) and 5% palladium on carbon (Pd/C)(1.6 g) was hydrogenated at 50 psiH, in absolute EtOH (110 mL) at ambient température for 18hours. The mixture was filtered through Celite and evaporated in vacuo to give amine 17 as a white, crystalline solid: ]H NMR (300 MHz, CD3OD) δ 3.63 (q, J = 7.0 Hz, 2 H) , 3.34 (m, 1 H) , 3.27 (dd, J = 11.8 , 3.3 Hz, 1 H), 2.91 (m, 1 H), 2.02-1 . 15 (m, 12 H) , 1.32 (s, 3 H) , 1. 31 (s, 3 H). <» , D. Aqueous 10.2 N'NaOH (2.4 mL, 24.5 mmol) was added to awarm (27 °C) suspension of chloroalcohol (obtained fromKaneka Industries in Japan)(10.4 g, 28.6 mmol) inisopropanol (IPA)(104 mL) with mechanical stirring. After 1hour 1 N aqueous HCl in IPA (prepared by addition of 1 mL ofconcentrated aqueous HCl to 12 mL of IPA) approximately(ca.) 1 mL) was added to neutralise (pH = 7). Amine 17 (5.2

g, 20.4 mmol) was added as a solution in IPA (50 mL) and thethin suspension was heated at 60° C for 10 hours. The IPAwas removed in vacuo. The residue was diluted with EtOAc(150 mL) and washed with H2O (2 x 50 mL), saturated aqueousNaHCO3 (1 x 50 mL), and brine (1 x 50 mL). The combinedaqueous layers were extracted with EtOAc (1 x 25 mL). Thecombined organic layers were dried (Na2SOj and purified byflash chromatography (75/25 EtOAc/hexanes, then EtOAc) togive the compound 18 as a white solid (8.98 g, 76%): :H NMR

(300 MHz, CD3OD) δ 7.33 (m, 10 H) , 5.08 (AB, JAS = 12.2 Hz, = 12.1 Hz, 2 H), 3.96, (m, 2 H ) , 3 . 56 (q, J = 7.3 Hz, H) , 3.50, (m, 1 H), 3.20 (dd, J = 13.6, 9.2 Hz, 1 H), 3 . 03 (m, 1 K) , 2.64. (m, 2 H), 2.20- 1.20 (m, 14 H), 1.28 (S, 6 H -33- 011196 E. 50% aqueous NaOH (2.7 9, 1.8 mL, 33.6 mmol) vas addedto a suspension of carbamate 18 (6.75 g, 11.6 mmol) in IPA(34 mL) at ambient température. The mixture was heatedunder reflux for 12 hours. After cooling to ambienttempérature, the mixture was diluted with methyl t-butylether (MTBE) (600 mL) and washed with H2O (2 x 250 mL) andbrine (1 x 125 mL). The combined aqueous layers wereextracted with MTBE (1 x 150 mL). The combined organiclayers were dried (Na2SO4) and evaporated in vacuo to give amixture of compound 19 and benzyl alcohol as an oily white solid: '1 1 NMR (300 MHz, H) , 3.81 (m, 1 H) , 3.58 (m, 1 H) , 2.05 (m, 1 H) 1.28 (s, 3 H) . F. Trie thylamine (3.2 solution of the mixture

2U from 18) and benzyl alcohol in EtOH (23 mL) at ambienttempérature. A solution of 3-acetoxy-2-methylbenzoylchloride (20)(obtained according to procedures set forth inU.S. Patent Application Serial No. 08/708,411, filedSeptember 5, 1996, which is specifically incorporated byreference herein) (2.4 g, 11.5 mmol) in THF (4 mL) wasadded. After 2 hours 50% aqueous NaOH (4.1g, 2.8 mL, 52.2mmol) was added and the mixture was heated under reflux for1 hour. After cooling to ambient température, the mixturewas neutralized to pH = 7 with 2 N aqueous HCl (26 mL).

This mixture was diluted with EtOAc (500 mL) and washed withH2O (1 x 250 mL) , saturated aqueous NaHCO3 (2 x 250 mL) , H,0 (1 x 250 mL), and brine (1 x 125 mL). The organic layer was dried (Na,SOj and purified by flash chromatography (75/25

EtOAc/hexanes) to give amide 21 as a white foam (1173-57A, 1.39 g, 23%). The ‘H NMR indicated the presence of 11 wt %

EtOAc which could not be removed in vacuo. -34- 011196 10

Analysis : :Η NMR (300 MHz, CD3OD) δ 7.53 (d, J = 7.3 Hz, 2 H), 7.32 (t, J = 7.0 Hz, 2 H), 7.20 (t, J = 7.3 Hz, 1 H), 7.06 (t, j'= 8.1 Hz, 1 H), 6.92 (d, J = 8.1 Hz, 1 H), 6.83 (d, J = 8.1Hz, 1 H), 4.42 (m, 1 H), 4.08 (m, 1 H), 3.61 (dd, J = 13.6,4.0 Hz, 1 H), 3.45 (AB, JAB =11.0 Hz, ΔυΑΒ = 18.0 Hz, 2 H) ,3.29 (dd, J = 13.6, 10.3 Hz, 1 H), 3.10 (m, 1 H), 2.66 (m, 2 H), 2.28 (s, 3 H), 2.22 (m, 2 H), 2.04 (m, 1 H), 1.86-1.20 (m, 11 H), 1.19 (s, 3 H), 1.18 (s, 3 H). nC NMR (75.5 MHz, CD3OD) δ 175.7, 172.5, 155.9, 138.8, 136.7, 129 . 8, 128 .9, 126.3, 126.0, 122.4 , 118 • 4, 70.3, 69.9, 68 2, 59.3, 58.8, 54.9, 53.0, 36 . 5 , 31.1, 30.7, 26 a • - Z 26.0, 23.1, 23.0, 20.8, 12 . 1 . 1θ du

Example 2 HIV Protease inhibition activity and anti HIV activity incell culture of compound 21

Tight binding kinetics analysis was used to déterminethe magnitude of the Kj values of compound 21. The K£= 5.6 ±0.91nM.

Methods

Expression of HIV-1 protease HIV-1 protease gene was isolated from the viral strainIIIB (Ratner, L. et al., Nature, 316, 227-284 (1985)). In order to increase the stability of purified protease (Rose,J.R. et al., J. Biol. Chem., 268, 11939-11945 (1993)), the glutamine residue at position 7 (Q7) was mutated to serine(S) bv replacing the 33 base pairs segment between the Ndeland BstEII sites of the protease gene sequence withsynthetic oligonucleotides encoding the Q7S mutation. Themodified gene sequence was inserted in.to the plasmid vectorpGZ (Menge, K.L. et la., Biochemistry, 34:15934-15942 (1995' under the control of phage T7 promoter. The resulting -35- 0 ι 1126 construct, pGZ/HP-19Q7S#9, was transformed into E. coiistrain BL21(DE3) purchased from Novagen, Inc.

Expression of HIV-1 PR: Cultures were grown in 2YTmedia (1.6% Trypticase Pepton, 1% Yeast extract, 0.5 % NaClat an initial pH 7.5) containing 200 gg/L ampicillin in 100L fermentor (Biolafitte SA) at 37°C for 5 hours and theninduced by addition of 1 mM IPTG (Isopropyl-3-D- thiogalactopyranoside). The température of the cultureduring induction was raised to 42°C to increaseaccumulations of the recombinant HIV-1 protease as insolubleinclusion bodies. After 2 hours at 42°C, cells wereharvested by crossflow filtration using Pellicon 0.1 μπιWPP000C5 cassette #10 (Millipore) and the cell paste wasstored frozen at -70°C.

Purification of Recombinant HIV-1 Protease: Ail stepsunless otherwise indicated were carried out at 4°C. Proteinconcentrations were determined using BioRad protein assaysolution with bovine sérum albumin (BioRad, Richmond, CA) asa standard. Chromatographie steps and the purity of HIV PRwas analyzed by sodium dodecylsulfate polyacrylamide gelelectrophoresis (SDS-PAGE). Final purity of HIV-PR was> 98%. Typical final yield from each 100 L culture was ~120mg.

Cell paste from 100L culture was resuspended in 300 mLof lysis buffer (50 mM Tris-Cl pH 8.0, 25 mM NaCl, 20 mM 2-mercaptoethanol) and microfluidized in MicrofluidicsCorporation fluidizer at 22,000 psi. The crude cell lysatewas clarified by centrifugation at 14,000 rpm for 20minutes. KIV PR was round predominantly in the pellet in theform of inclusion bodies. The inclusion bodies weresubsequently washed multiple times in the lysis buffercontaining in addition 0.1% Trition-X100 and 1 M urea, andafter each washing procedure, the inclusion bodies were -36- 011196 pelleted by centrifugation at 5,000 rpm for 20 minutes.Purified inclusion bodies were solubilized in buffercontaining 50 mM Tris-Cl, pH 8.0, 25 mM NaCl, 20 mM 2-mercaptoethanol, and 8 M urea. Solution was clarified bycentrifugation at 14,000 rpm and applied at room températureto a 300 mL Fast Flow Q-Sepharose column (Pharmacia,Piscataway, NJ) equilibrated with the same buffer. Underthese conditions HIV PR did not bind to the column andessentially pure enzyme was found in the flow-throughfractions. To renature the protein, the fractions from FastFlow Q-Sepharose column were dialyzed against three changesof buffer containing 25 mM NaH2PO4 pH 7.0, 25 mM NaCl, 10 mMDTT and 10% glycerol. After refolding, small quantifies ofprecipitated material were removed by centrifugation andrésultant enzyme préparation were concentrated, dialyzedagainst 0.5 M NaCl, 50 mM MES pH 5.6, 10 mM DTT, frozen insmall aliquots at' ~2 mg/mL and stored at -70°,

Tight-Binding Kinetics Assay and Analysis

Proteolytic activity of purified HIV-1 protease wasmeasured using a modified chromogenic assay developed byRichards at al. (Richards, A.D. et al. J. Biol. Chem., 256,773-7736 (1990)). The synthetic peptide His-Lys-Ala-Arg-Val-Leu-Phe(paraNO2)-Glu-Ala-Nle-Ser-NH2 (American PeptideCompany) (Nie is norleucine) was used as a substrate. Theassay was carried out in 0.5 M NaCl, 50 mM MES pH 5.6, 5 mMDDT, and 2% DMSO at 37°C. Cleavage of the scissile bondbetween leuci.ne and paranitro-phenylalanine (Phe para-NO2)was assayed by spectrophotometric monitoring of the decreaseon absorbance at 305 nm. Initial velocity was determined asthe rate of décliné of absorbance during the first 100seconds of the enzymatic réaction. Under these conditions,and using Q7S HIV-1 protease, the Michaelis constant (Km)for this substrate is 59 ± 17 μΜ. -37- 011I96

For détermination of the inhibition of compound.21, asaturating concentration of substrate of 200 uM was used.Between 13 and 20 concentrations of inhibitors wereevaluated and the velocity of reaction was measured at eachconcentration as described above. The apparent Ki (Ki app),set forth above, was determined by computer assisted non-linear least square fitting of the data to the tight bindingéquation of Morrison (Morrison, J.F., Biochem. Biophys.

Acta, 185, 269-286 (1963)).

Example 3

Antiviral activity of compound 21 against HIV-1 in cellculture

Cells and virus strains:

The CEM-SS and MT-2 human T cell lines and HIV-1strains RF and IIIB were obtained from the AIDS Research andReference Program, Division of AIDS, NIAID, and NIH.

Cell protection assays:

The inhibitory effects of each agent on HIV-1 réplication were measured by the MTT dye réduction method(Alley, M.C. et al., Cancer Res. 48: 589-601 (1988)).Compounds were dissolved in DMSO at a concentration of40 mg/ml then diluted 1:200 in culture medium (RPMI,supplemented with 10% fêtai bovine sérum). From eachdiluted stock, 100 μΐ was added to a 96-well plate andserial half-log dilutions were prepared. In separate tubes,MT-2 cells and CEM-SS cells were infected with HIV-1 IIIB orHIV-l RF at a multiplicity of infection (m.o.i.) of 0.01 and0.03, respectively. Following a 4-hour absorption period,100 μΐ of infected or uninfected cells were added to thewells of the drug containing plate to give a finalconcentration of 1 x 104 cells/well. Six days (CEM-SScells) or 7 days (MT-2 cells) later, MTT (5 mg/ml) was addedto test plates and the amount of formazan produced was -38- 011196 quantified spectrophotometrically at 570 nm. Data wereexpressed as the percentage of formazan produced in drug-treated cells compared to formazan produced in wells ofuninfected, drug-free cells. The ED50 was calculated as theconcentration of drug that increased the percentage offormazan production in infected, drug-treated cells to 50%of that produced by uninfected, drug-free cells.

Cytotoxicity (TC50) was calculated as the concentration ofdrug that decreased the percentage of formazan produced inuninfected, drug-treated cells to 50% of that. produced inuninfected, drug-free cells. The therapeutic index (TI) wascalculated by dividing the cytotoxicity (TC50) by theantiviral efficacy (ED50) .

Table 1

Antiviral Activity and Cytotoxicity Evaluations of Compound 21

in an Acute Infection of CEM-SS cells with HIV-1 RF

Compound ed50 (nM) ed95 (nM) TC50 (μΜ) Therapeuticindex a 21 34.2 154.1 96.6 2825 azidophymidine (AZT) 52.3 543.1 >374.5 >7161 dideoxycytidine (ddC) 94.70 142.0 37.69 398 â Therapeutic index = Cytotoxicity (TC50) + Antiviralactivity (ED5C) . -39- 011196

Table 2

Antiviral Activity and Cytotoxicity Evaluations of Compound21

in an Acute Infection of MT-2 cells with HIV-i IIIB

Compound ed50 (nM) ed95 (nM) TC50 (μΜ) Therapeu tic 21 85.6 ND 92.6 1082 AZT 430.7 ND 109.4 254 ddC 5924 ND 176.3 30 1t 15 aTherapeutic index = Cytotoxicity (TC50) Antiviral activity (ED50) .

As noted above, the compounds of the présent inventionare useful for inhibiting HIV protease, which is an enzymeassociated with viral component production and assembly. Anembodiment of the présent invention is a method of treatingHIV infection comprising administering to a host or patient,such as a primate, an effective amount of a compound offormula (9) or a pharmaceutically acceptable sait thereof.Another embodiment of the présent invention is a method oftreating AIDS comprising administering to a host or patientan effective amount of a compound of formula (9) or apharmaceutically acceptable sait thereof. A furtherembodiment of the présent invention is a method ofinhibiting HIV protease comprising administering to an HIV infected cell or a host or patient, such as a primate, infected with HIV, an effective amount of a compound of formula (1) or a pharmaceutically acceptable sait thereof.

The term "effective amount" means an amount of a compound of formula (9) or its pharmaceutically acceptable' 2>j -40- 011156 sait that is effective to inhibit the HIV protease meaiatedviral component production and assembly. The spécifie doseof compound administered according to this invention toobtain therapeutic or inhibitory effects will, of course, bedetermined by the particular circumstances surrounding thecase, including, for example, the compound administered, theroute of administration, the condition being treated andthe individual host or patient being treated. An exemplarydaily dose (administered in single or divided doses)contains a dosage level of from about 0.01 mg/kg to about 50mg/kg of body weight of a compound of this invention.Preferred daily doses generally are from about 0.05 mg/kg toabout 40 mg/kg and, more preferably, from about 1.0 mg/kg toabout 30 mg/kg.

The compounds of the invention may be administered by avariety of routes, including oral, rectal, transdermal,subeutaneous, intravenous, intramuscular and intranasalroutes. The compounds of the présent invention arepreferably formulated prior to administration. Therefore,another embodiment of the présent invention is apharmaceutical composition or formulation comprising aneffective amount of a compound of formula (9) or apharmaceutically acceptable sait thereof and a pharmaceutically acceptable carrier, such as a diluent orexcipient therefor.

The active ingrédient preferably comprises from 0.1% to99.9% by weight of the formulation. By "pharmaceuticallyacceptable" it is meant that the carrier, such as thediluent or excipient, is compatible with the otheringrédients of the formulation and not deleterious to thehost or patient.

Pharmaceutical formulations may be prepared from thecompounds of the invention by known procedures using known -41- 011196 and readily available ingrédients. In making the compositions of the présent invention, the active ingrédientwill usually be admixed with a carrier, or diluted by acarrier, or enclosed within a carrier, which may be in theform of a capsule, sachet, paper or other suitablecontainer. When the carrier serves as a diluent, it may bea solid, semi-solid or liquid material which acts as avehicle, excipient or medium for the active ingrédient.

Thus, the compositions can be in the form of tablets, pills,powders, lozenges, sachets, cachets, élixirs, suspensions,émulsions, solutions, syrups, aérosols (as a solid or in aliquid medium), ointments (containing, for example, up to10% by weight of the active compound), soft and hard gelatincapsules, suppositories, stérile injectable solutions,stérile packaged powders and the like.

The following formulation examples are illustrativeonly and are not intended to limit the scope of theinvention. The term "active ingrédient" represents acompound of formula (9) or a pharmaceutically acceptablesait thereof. -42- 011196

Formulation 1

Hard gelatin capsules are prepared using the followingingrédients :

Quantity (ma/caüsule) Active ingrédient 250 Starch, dried 200 Magnésium stéarate 10 Total 4 60 mg Formulation 2 A tablet is prepared using the ingrédients below:

Quantity (mg/tablet )

Active ingrédient 250

Cellulose, microcrystalline 400

Silicon dioxide, fumed 10

Stearic acid 5

Total 665 mg

The components are blended and compressed to form tablets each weighing 665 mg. 011156 -4 3-

Formulation 3

An aérosol solution is prepared containing the following components:

Weight

Active ingrédient 0.25Methanol 25.75Propellant 22 (Chlorodifluoromethane) 74.00Tota 100.00

The active compound is mixed with éthanol and the mixtureadded to a portion of the propellant 22, cooled to -30°C andtransferred to a filling device. The required amount is thenfed to a stainless Steel container and diluted with theremainder of the propellant. The valve units are then fittedto the container.

Formulation 4

Tablets, each containing 60 mg of active ingrédient, aremade as follows:

Quantity(mg/tablet)

Active ingrédient

Starch

Microcrystalline cellulosePoiyvinylpyrrolidone (as 10% solution in water)Sodium carboxymethyl starchMagnésium stéarateTalc 60 45 35

Total 150 -44- 011196

The active ingrédient, starch and cellulose are passed througha No. 45 mesh U.S. sieve and mixed thoroughly. The agueoussolution containing polyvinylpyrrolidone is mixed with therésultant powder, and the mixture then is passed through a Mo.14 mesh U.S. sieve. The granules so produced are dried at 50°Cand passed through a No. 18 mesh U.S. sieve. The sodiumcarboxymethyl starch, magnésium stéarate and talc, previouslypassed through a No. 60 mesh U.S. sieve, are then added to thegranules which, after mixing, are compressed on a tabletmachine to yield tablets each weighing 150 mg.

Formulation 5

Capsules, each containing 80 mg of active ingrédient, aremade as follows:

Quantity (ma/capsule)

Active ingrédient 80 mg Starch 59 mg Microcrystalline cellulose 59 mg Magnésium stéarate 2 mg Total 200 mg

The active ingrédient, cellulose, starch and magnésium stéarate are blended, passed through a No. 45 mesh U.S. sieve,and filled into hard gelatin capsules in 200 mg quantities.Formulation 6

Suppositories, each containing 225 mg of activeingrédient, are made as follows:

Active ingrédient 225 mg Saturated fatty acid glycerides 2 , 000 mg Total 2,225 mg The active ingrédient is passed through a No sieve and suspended in the saturated fatty acid glycerides previously melted usina the minimum heat necessary. The

ïLi 2ΓΙ -as- 011196 mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7

Suspensions, each containing 50 mg of active ingrédientper 5 ml dose, are made as follows:

Active ingrédient 50 mg

Sodium carboxymethyl cellulose 50 mg

Syrup 1.25 ml

Benzoic acid solution 0.10 ml

Flavor q.v.

Color q.v.

Purified water to total 5 ml

The active ingrédient is passed through a No. 45 mesh U.S.sieve and mixed with the sodium carboxymethylcellulose andsyrup to form a smooth paste. The benzoic acid solution,flavor and color are diluted with a portion of the water andadded, with stirring. Sufficient water is then added toproduce the required volume.

Formulation 8

An intravenous formulation is prepared as follows:

Active ingrédient 100 mg

Isotonie saline 1,000 mL

The solution of the above ingrédients generally is administered intravenously to a subject at a rate of 1 ml perminute. -46- 011I9b 01119b

Formulation 9 A tablet is prepared using the ingrédients below: * Quantity (mcr/tablet ) V 5 Active ingrédient 2 92 mg calcium silicate 14 6 mg crospovidone 14 6 mg Magnésium stéarate 5 mq Total 58 9 mg

Claims (12)

1. -47- 0111 5 6 aiti: 01119 6

   1. A compound of the formula (9) :

   wherein: R and R' are independently selecced from H, a substicutedor unsubscituted alkyl-OR, group, a cycioalkyl groupsubsticuted with a (C,-Cr) alkyl group or a (C -CJ alkyl-OHgroup, a heterocycle group substituted with a (C:-CJ alkylgroup or a (0,-C,-) alkyl-OH group, an alkyl-NRR, group, or analkyl - S (X) (Y) R,. group, wherein R: is H, a substicuced or unsubsicuced alkyl group, or anacyl group; R and R, are each independently selecced from K,substicuted cr unsubstituted alkyl, cycioalkyl,heterocycle, and aryl groups, and acyl and sulfonylgroupe ; R. îs H, a substicuted or unsubstituted alkyl, cycioalkyl,heterocycle, cr aryl group; and X and Y are each independently selected from =0 andnothing; or a pharmaceuticallv acceptable prodrug, sait or solvatéC b. "S L' S C Z
2. 2. Λ compound according to claim 1, wherein R is H, or a pnarmaceutically acceptable prodrug, sait, or solvaté -49- 011196 group, or a pharmaceutically acceptable prodrug, sale orsolvaté thereof.
3. 7. A compound according to claim 1, wherein saidcompound has trie formula 21:

   or a pharmaceutically acceptable prodrug, sait or solvaté thereof. S. A compound according to claim 2, wherein w'nen R' isa cycloalkyl grcup substituted with a (C,-CJalkyl group or a(C;-CJ alkyl-OH group, said cycloalkyl group is selected from:

   10 or a pharmaceutically acceptable prodrug, sait or solvatéthereof. 9 . (9b) : A sait according to claim 1, having the formula

   IG. A pharmaceutical composition comprising·. (a) an effective amount of compound of claim 1; and (b) a pharmaceutically acceptable carrier therefor.11·. A pharmaceutical composition comprising: (a) an effective amount of compound of claim 7 ; and (b) a onarmaceuticallv acceDtable carrier therefor. 113 011196 -50-
4. 12. A compound according to claim 1, which has a purity of more than 90%. 13. A compound according to claim 1, which has a purity of at least 95%. 14 . A compound according to claim 1, which has a purity of at least 97%. 15. A compound according to claim b which has a purity of at least 99%. 16. A compound according to claim 7, which has a purity of more than 90%. 17. A compound according to claim 7, which has a purity of at least 95%. 18. A compound according to claim 7, which has a purity of at least 97%. 19 . A compound according to claim 7, which has a 2'* 3d purity of at least 99%.
5. 20. A pharmaceutical composition according to claim10, wherein the compound has a purity of more than 90%.
6. 21. A pharmaceutical composition according to claim10, wherein the compound has a purity of at least 95%.
7. 22. A pharmaceutical composition according to claim10, wherein the compound has a purity of at least 97%.
8. 23. A pharmaceutical composition according. to claim 10, wherein the compound has a purity of at least than 99%.
9. 24. A pharmaceutical composition according to claim 11, wherein the compound has a purity of more than 90%.
10. 25. A pharmaceutical composition according to claim11, wherein the compound has a purity of at least 95%.
11. 26. A pharmaceutical composition according to claim11, wherein the compound has a purity of at least 97%.
12. 27. A pharmaceutical composition according to claim11, wherein the compound has a purity of at least 99%.