Indazoles of cyclic ureas useful as HIV protease inhibitors

FIELD OF THE INVENTION
This invention relates generally to indazoles of cyclic ureas which are useful as inhibitors of HIV protease, and pharmaceutical compositions and diagnostic kits comprising the same. The present invention also relates to the use of such compounds in processes for the identification of HIV protease inhibitors and for the inhibition or detection of HIV in a bodily fluid sample.
BACKGROUND OF THE INVENTION
Two distinct retroviruses, human immunodeficiency virus (HIV) type-1 (HIV-1) or type-2 (HIV-2), have been etiologically linked to the immunosuppressive disease, acquired immunodeficiency syndrome (AIDS). HIV seropositive individuals are initially asymptomatic but typically develop AIDS related complex (ARC) followed by AIDS. Affected individuals exhibit severe immunosuppression which predisposes them to debilitating and ultimately fatal opportunistic infections.
The disease AIDS is the end result of an HIV-1 or HIV-2 virus following its own complex life cycle. The virion life cycle begins with the virion attaching itself to the host human T-4 lymphocyte immune cell through the bonding of a glycoprotein on the surface of the virion's protective coat with the CD4 glycoprotein on the lymphocyte cell. Once attached, the virion sheds its glycoprotein coat, penetrates into the. membrane of the host cell, and uncoats its RNA. The virion enzyme, reverse transcriptase, directs the process of transcribing the RNA into single-stranded DNA. The viral RNA is degraded and a second DNA strand is created. The now double-stranded DNA is integrated into the human cell's genes and those genes are used for virus reproduction.
At this point, RNA polymerase transcribes the integrated DNA into viral RNA. The viral RNA is translated into the precursor gag-pol fusion polyprotein. The polyprotein is then cleaved by the HIV protease enzyme to yield the mature viral proteins. Thus, HIV protease is responsible for regulating a cascade of cleavage events that lead to the virus particle's maturing into a virus that is capable of full infectivity.
The typical human immune system response, killing the invading virion, is taxed because the virus infects and kills the immune system's T cells. In addition, viral reverse transcriptase, the enzyme used in making a new virion particle, is not very specific, and causes transcription mistakes that result in continually changed glycoproteins on the surface of the viral protective coat. This lack of specificity decreases the immune system's effectiveness because antibodies specifically produced against one glycoprotein may be useless against another, hence reducing the number of antibodies available to fight the virus. The virus continues to reproduce while the immune response system continues to weaken. Eventually, the HIV largely holds free reign over the body's immune system, allowing opportunistic infections to set in and without the administration of antiviral agents, immunomodulators, or both, death may result.
There are at least three critical points in the virus's life cycle which have been identified as possible targets for antiviral drugs: (1) the initial attachment of the virion to the T-4 lymphocyte or macrophage site, (2) the transcription of viral RNA to viral DNA (reverse transcriptase, RT), and (3) the processing of gag-pol protein by HIV protease.
The genomes of retroviruses encode a protease that is responsible for the proteolytic processing of one or more polyprotein precursors such as the pol and gag gene products. See Wellink, Arch. Virol. 98 1 (1988). Retroviral proteases most commonly process the gag precursor into the core proteins, and also process the pol precursor into reverse transcriptase and retroviral protease.
The correct processing of the precursor polyproteins by the retroviral protease is necessary for the assembly of the infectious virions. It has been shown that in vitro mutagenesis that produces protease-defective virus leads to the production of immature core forms which lack infectivity. See Crawford et al., J. Virol. 53 899 (1985); Katoh et al., Virology 145 280 (1985). Therefore, retroviral protease inhibition provides an attractive target for antiviral therapy. See Mitsuya, Nature 325 775 (1987).
The ability to inhibit a viral protease provides a method for blocking viral replication and therefore a treatment for viral diseases, such as AIDS, that may have fewer side effects, be more efficacious, and be less prone to drug resistance when compared to current treatments. As a result, three HIV protease inhibitors, Roche's saquinavir, Abbott's ritonavir, and Merck's indinavir, are currently being marketed and a number of potential protease inhibitors are in clinical trials, e.g., Vertex's VX-478, Agouron's nelfinavir, Japan Energy's KNI-272, and Ciba-Geigy's CGP 61755.
As evidenced by the protease inhibitors presently marketed and in clinical trials, a wide variety of compounds have been studied as potential HIV protease inhibitors. One core, cyclic ureas, has received significant attention. For example, in PCT Application Number WO94/19329, Lam et al generically describe cyclic ureas of the formula: ##STR2## and methods of preparing these ureas. However, Lam et al does not specifically describe the indazolyl cyclic ureas of the present invention.
In another PCT publication, WO96/29329, Jahdav et al describe cyclic ureas of the formula: ##STR3## wherein R.sup.3 and R.sup.4 are preferably benzyl and R.sup.1 preferably is a benzyl group having a heterocycle linked via a heteratom attached thereto. Jahdav et al does not, though, specifically describe the indazolyl cyclic ureas of the present invention.
Even with the current success of protease inhibitors, it has been found that HIV patients can become resistant to a single protease inhibitor. Thus, it is desirable to develop additional protease inhibitors to further combat HIV infection.
SUMMARY OF THE INVENTION
Accordingly, one object of the present invention is to provide novel protease inhibitors.
It is another object of the present invention to provide pharmaceutical compositions with protease inhibiting activity comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one of the compounds of the present invention or a pharmaceutically acceptable salt or prodrug form thereof.
It is another object of the present invention to provide a novel method for treating HIV infection which comprises administering to a host in need of such treatment a therapeutically effective amount of at least one of the compounds of the present invention or a pharmaceutically acceptable salt or prodrug form thereof.
It is another object of the present invention to provide a novel method for treating HIV infection which comprises administering to a host in need thereof a therapeutically effective combination of (a) one of the compounds of the present invention and (b) one or more compounds selected form the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors.
It is another object of the present invention to provide a method of inhibiting HIV present in a body fluid sample which comprises treating the body fluid sample with an effective amount of a compound of the present invention.
It is another object of the present invention to provide a kit or container containing at least one of the compounds of the present invention in an amount effective for use as a standard or reagent in a test or assay for determining the ability of a potential pharmaceutical to inhibit HIV protease, HIV growth, or both.
These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors' discovery that compounds of formula I: ##STR4## or pharmaceutically acceptable salts or prodrug forms thereof, are effective protease inhibitors.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[1] Thus, in a first embodiment, the present invention provides a novel compound of formula I: ##STR5## or a pharmaceutically acceptable salt form thereof, wherein: one of D and D' is NH.sub.2 and the other selected from H, halogen, C.sub.1-6 alkoxy, and C.sub.1-6 alkyl;
X is selected from a bond, CH.dbd.CHCH.sub.2, CH.sub.2 CH.sub.2 CH.sub.2, CH.sub.2 CH.sub.2, CH(OH), and CH.sub.2 ;
Y is selected from OR.sup.1, SR.sup.1, NR.sup.1 R.sup.6, --CN, --OC(O)NR.sup.1 R.sup.2, --NR.sup.3 C(O)OR.sup.1, --NR.sup.3 C(O)NR.sup.1 R.sup.2, --C(O)NR.sup.1 R.sup.2, --NR.sup.3a C(O)R.sup.1, --C(O)R.sup.1, --C(O)OR.sup.1, --C(OH) (R.sup.2)R.sup.1, --SO.sub.2 R.sup.5, --SO.sub.2 NR.sup.1 R.sup.2, and --(CH.sub.2).sub.m R.sup.4 ;
R.sup.1 is selected from H, (CH.sub.2).sub.n R.sup.5, C.sub.1-6 alkyl substituted with 0, 1, 2, or 3 R.sup.1a, and C.sub.2-6 alkenyl substituted with 0, 1, or 2 R.sup.1a ;
R.sup.1a is selected from C.sub.1-6 alkoxy, OH, .dbd.O, halogen, C.sub.3-6 cycloalkyl, and NR.sup.6 R.sup.6a ;
R.sup.2 is selected from H, C.sub.1-4 alkyl substituted with 0, 1, or 2 R.sup.2b, phenyl substituted with 0, 1, or 2 R.sup.7, and a 5-10 membered heterocyclic system containing from 1-3 heteroatoms selected from the group consisting of N, O, and S substituted with 0, 1, or 2 R.sup.7 ;
R.sup.2b is selected from C.sub.1-6 alkoxy, OH, halogen, and NR.sup.6 R.sup.6a ;
R.sup.3 is selected from H and CH.sub.3 ;
R.sup.3a is selected from H and C.sub.1-3 alkyl;
R.sup.4 is selected from a 5-10 membered heterocyclic system containing from 1-3 heteroatoms selected from the group consisting of N, O, and S substituted with 0, 1, or 2 R.sup.7 ;
R.sup.5 is selected from a C.sub.3-10 carbocyclic residue substituted with 0, 1, or 2 R.sup.7, and a 5-10 membered heterocyclic system containing from 1-3 heteroatoms selected from the group consisting of N, O, and S substituted with 0, 1, or 2 R.sup.7 ;
R.sup.6 and R.sup.6a are independently selected from H and C.sub.1-4 alkyl;
R.sup.7 is selected from halogen, OH, .dbd.O, C.sub.1-6 alkoxy, --CN, --NO.sub.2, NR.sup.6 R.sup.6a, COR.sup.8, C.sub.1-6 alkyl, CF.sub.3, and S(O).sub.p (C.sub.1-6 alkyl);
R.sup.8 is selected from H, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, and NR.sup.6 R.sup.6a ;
m is 0, 1, 2, or 3;
n is 0, 1, or 2; and,
p is 0, 1, or 2.
[2] In a preferred embodiment, the present invention provides a novel compound of formula I, wherein:
D is NH.sub.2 ;
D' is H;
X is CH.sub.2 ; and,
Y is selected from OR.sup.1, SR.sup.1, NR.sup.1 R.sup.6, --CN, --OC(O)NR.sup.1 R.sup.2, --NR.sup.3 C(O)OR.sup.1, --NR.sup.3 C(O)NR.sup.1 R.sup.2, --C(O)NR.sup.1 R.sup.2, --NR.sup.3a C(O)R.sup.1, --C(O)R.sup.1, --SO.sub.2 R.sup.5, --SO.sub.2 NR.sup.1 R.sup.2, and --(CH.sub.2).sub.m R.sup.4.
[3] In a more preferred embodiment, the present invention provides a novel compound of formula I, wherein:
Y is selected from OR.sup.1, SR.sup.1, --OC(O)NR.sup.1 R.sup.2, --NR.sup.3 C(O)OR.sup.1, --NR.sup.3 C(O)NR.sup.1 R.sup.2, --C(O)NR.sup.1 R.sup.2, --NR.sup.3a C(O)R.sup.1, --C(O)R.sup.1, --C(O)OR.sup.1, --SO.sub.2 R.sup.5, --SO.sub.2 NR.sup.1 R.sup.2, and --(CH.sub.2).sub.m R.sup.4.
R.sup.1 is selected from H, (CH.sub.2).sub.n R.sup.5 and C.sub.1-6 alkyl substituted with 0, 1, 2, or 3 R.sup.1a ;
R.sup.2 is selected from H, C.sub.1-4 alkyl substituted with 0, 1, or 2 R.sup.2b, phenyl substituted with 0, 1, or 2 R.sup.7, and a 5-10 membered heterocyclic system containing from 1-3 heteroatoms selected from the group consisting of N, O, and S substituted with 0, 1, or 2 R.sup.7 ; and,
R.sup.5 is selected from a C.sub.6-10 aromatic carbocyclic residue substituted with 0, 1, or 2 R.sup.7, and a 5-10 membered aromatic heterocyclic system containing from 1-3 heteroatoms selected from the group consisting of N, O, and S substituted with 0, 1, or 2 R.sup.7.
[4] In an even more preferred embodiment, the present invention provides a novel compound of formula I, wherein:
Y is selected from OR.sup.1, SR.sup.1, --OC(O)NR.sup.1 R.sup.2, --C(O)NR.sup.1 R.sup.2, --NR.sup.3 C(O)OR.sup.1, --NR.sup.3a C(O)R.sup.1, --C(O)R.sup.1, and --(CH.sub.2).sub.m R.sup.4.
R.sup.1 is selected from (CH.sub.2).sub.n R.sup.5 and C.sub.1-6 alkyl;
R.sup.4 is selected from a 5-10 membered aromatic heterocyclic system containing from 1-3 heteroatoms selected from the group consisting of N, O, and S substituted with 0, 1, or 2 R.sup.7 ;
R.sup.5 is selected from a C.sub.6-10 aromatic carbocyclic residue substituted with 0, 1, or 2 R.sup.7, and a 5-10 membered aromatic heterocyclic system containing from 1-2 heteroatoms selected from the group consisting of N, O, and S substituted with 0, 1, or 2 R.sup.7 ;
R.sup.7 is selected from halogen, OH, C.sub.1-6 alkoxy, --CN, --NO.sub.2, NR.sup.6 R.sup.6a, COR.sup.8, C.sub.1-6 alkyl, CF.sub.3, and S(O).sub.2 (C.sub.1-4 alkyl); and,
m is 0, 1, or 2.
[5] In a further preferred embodiment, the present invention provides a novel compound of formula I, wherein:
R.sup.2 is selected from H, C.sub.1-4 alkyl substituted with 0, 1, or 2 R.sup.2b, phenyl substituted with 0, 1, or 2 R.sup.7, and a 5-10 membered heterocyclic system containing from 1-2 heteroatoms selected from the group consisting of N, O, and S substituted with 0, 1, or 2 R.sup.7 ; and,
R.sup.7 is selected from Cl, F, I, OH, C.sub.1-6 alkoxy, --CN, --NO.sub.2, NR.sup.6 R.sup.6a, COR.sup.8, C.sub.1-6 alkyl, CF.sub.3, and S(O).sub.2 (C.sub.1-4 alkyl); and,
n is 0 or 1.
[6] In another preferred embodiment, the present invention provides a novel compound of formula I, wherein:
D is NH.sub.2 ;
D' is H;
X is CH.sub.2 ; and,
Y is selected from:
phenylmethoxy; phenylthio; 2-benzimidazolylthio; 2-quinolinylmethoxy; 4-trifluoromethylphenoxy; 6-quinolinoxy; 5-quinolinoxy; 4-quinolinoxy; 2-methylphenoxy; 3-fluorophenylmethoxy; 2-fluorophenylmethoxy; ethoxy; 7-quinolinoxy; 4-methylphenylmethoxy; 4-trifluoromethylphenylmethoxy; 3-methylphenoxy; 3-aminophenoxy; 2-aminophenoxy; 3-methylphenylmethoxy; 3-trifluoromethylphenylmethoxy; 5-(2-methyl)benzthiazoloxy; 5-benzthiazoloxy; n-butoxy; 4-cinnolinoxy; 4-fluoro-2-aminophenoxy; methyl carbamoyl; 4-cyano-2-methoxyphenoxy; i-butyl carbamoyl; methoxy; 1-(5-methyl)indazole; ethyl carbamoyl; N-phenyl carbamoyl; 1-pyrazole; n-propyl carbamoyl; 3,4-dimethoxyphenoxy; n-butyl carbamoyl; i-propyl carbamoyl; t-butyl carbamoyl; trifluoroacetamido; 2,2,2-trifluoroethyl carbamoyl; 3-tetrahydrofuranyl carbamoyl; 1-(4-methyl)pyrazole; 1-(4-chloro)pyrazole; 1-(4-bromo)pyrazole; 2-pyridinyl carboxamido; phenyl carboxamido; 1-(4-amino)pyrazole; 1-(3-trifluoromethyl)pyrazole; 1-(3,5-dimethoxy)pyrazole; 1-(3-amino)pyrazole; 2-furanyl carboxamido; 2-thiophenyl carboxamido; 1-(4-iodo)pyrazole; 1-(4-nitro)pyrazole; 1-(3-nitro)pyrazole; t-butyl carboxamido; 3-trifluoromethylphenyl carboxamido; 3-methoxyphenyl carboxamido; 4-methoxyphenyl carboxamido; 2-quinolinyl carboxamido; 2-pyrazinyl carboxamido; 1-i-quinolinyl carboxamido; 2-methoxyphenyl carboxamido; 1-(4-carboethoxy)pyrazole; 2-fluorophenyl carboxamido; 3-fluoro-5-trifluoromethylphenoxy; 4-fluorophenyl carboxamido; 8-(1,2,3,4-tretrahydro)quinolinyl carboxamido; 2-(6-methyl)pyridinyl carboxamido; 2-aminophenyl carboxamido; 3-aminophenyl carboxamido; 4-aminophenyl carboxamido; 2,6-difluorophenyl carboxamido; 1-(4-acetyl)pyazole; 1-(4-carbomethoxy)pyrazole; 2-pyridylmethoxy; 2-methyl-4-thiazolemethoxy; propoxy; N,N-dimethylcarbamoyl; 6-methyl-2-pyridylmethoxy; phenoxy; 2-isopropyl-4-thiazolemethoxy; 4-methylphenoxy; 4-methoxyphenoxy; 4-cyanophenoxy; 3-pyridoxy; 2-methyl-6-benzothiazoloxy; 5-trifluoromethyl-2-pyridylmethoxy; N-methylcarbamoyl; N-isopropylcarbamoyl; 2-methyl-5-pyridoxy; 1,2,4-triazole; benzoyl; N-t-butylcarboxamido; N,N-diethylcarboxamido; N-phenylcarboxamido; pyrrolidinecarboxamido; 4-fluorophenoxy; 3-fluorophenoxy; 4-fluorophenylmethoxy; 2-naphthylmethoxy; 2-naphthoxy; 2-fluorophenoxy; 4-chlorophenoxy; 2,4-di-fluorophenoxy; 4-(methylsulfonyl)phenoxy; 2-fluoro-3-pyridoxy; 2-benzothiazolemethoxy; 3-cyanophenoxy; 3,4-di-fluorophenoxy; 2-fluoro-4-pyridylmethoxy; 6-fluoro-2-pyridylmethoxy; 5-chloro-3-pyridoxy; 3-chloro-6-pyridazoxy; 8-quinolinoxy; 6-chloro-2-pyridylmethoxy; 2-chloro-3-pyridoxy; 3-methoxy-phenoxy; 2-methoxy-phenoxy; 3-furanoxy; 3-acetylphenoxy; 2-thiazole; 2-benzimidazole; N-methyl-2-imidazole; 4-acetyl phenoxy; 6-cyano-2-pyridyl; 2-pyrizole; 2-benzothiazole; phenymethyl; 2-pyridyl; 2-isoxazole; and, 2-furan;
In a second embodiment, the present invention provides a novel pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt form thereof.
In a third embodiment, the present invention provides a novel method for treating HIV infection which comprises administering to a host in need of such treatment a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt form thereof.
In a fourth embodiment, the present invention provides a novel method of treating HIV infection which comprises administering, in combination, to a host in need thereof a therapeutically effective amount of:
(a) a compound of formula I; and,
(b) at least one compound selected from the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors.
In another preferred embodiment, the reverse transcriptase inhibitor is a nucleoside reverse transcriptase inhibitor.
In another more preferred embodiment, the nucleoside reverse transcriptase inhibitor is selected from AZT, 3TC, ddI, ddC, and d4T and the protease inhibitor is selected from saquinavir, ritonavir, indinavir, VX-478, nelfinavir, KNI-272, CGP-61755, and U-103017.
In an even more preferred embodiment, the nucleoside reverse transcriptase inhibitor is selected from AZT and 3TC and the protease inhibitor is selected from saquinavir, ritonavir, and indinavir.
In a still further preferred embodiment, the nucleoside reverse transcriptase inhibitor is AZT.
In another still further preferred embodiment, the protease inhibitor is indinavir.
In a fifth embodiment, the present invention provides a pharmaceutical kit useful for the treatment of HIV infection, which comprises a therapeutically effective amount of:
(a) a compound of formula I; and,
(b) at least one compound selected from the group consisting of HIV reverse transcriptase inhibitors and HIV protease inhibitors, in one or more sterile containers.
In a sixth embodiment, the present invention provides a novel method of inhibiting HIV present in a body fluid sample which comprises treating the body fluid sample with an effective amount of a compound of formula I.
In a seventh embodiment, the present invention to provides a novel a kit or container comprising a compound of formula I in an amount effective for use as a standard or reagent in a test or assay for determining the ability of a potential pharmaceutical to inhibit HIV protease, HIV growth, or both.
DEFINITIONS
As used herein, the following terms and expressions have the indicated meanings. The compounds herein described may have asymmetric centers. Compounds of the present invention containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms or by synthesis from optically active starting materials. Many geometric isomers of olefins, C.dbd.N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated.
When any variable (e.g., R.sup.6) occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-2 R.sup.6, then said group may optionally be substituted with up to two R.sup.6 groups and R.sup.6 at each occurrence is selected independently from the definition of R.sup.6. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
When a bond to a substituent is shown to cross a bond connecting two atoms in a ring, then such substituent may be bonded to any atom on the ring. When a substituent is listed without indicating the atom via which such substituent is bonded to the rest of the compound of a given formula, then such substituent may be bonded via any atom in such substituent. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
As used herein, "C.sub.1-6 alkyl" is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, examples of which include, but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, pentyl, and hexyl; "Alkenyl" is intended to include hydrocarbon chains of either a straight or branched configuration and one or more unsaturated carbon-carbon bonds which may occur in any stable point along the chain, such as ethenyl, propenyl, and the like.
"Halo" or "halogen" as used herein refers to fluoro, chloro, bromo, and iodo; and "counterion" is used to represent a small, negatively charged species such as chloride, bromide, hydroxide, acetate, sulfate, and the like.
As used herein, "carbocycle" or "carbocyclic residue" is intended to mean any stable 3- to 7-membered monocyclic or bicyclic or 7- to 13-membered bicyclic or tricyclic, any of which may be saturated, partially unsaturated, or aromatic. Examples of such carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl,; [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin), [2.2.2]bicyclooctane, fluorenyl, phenyl, naphthyl, indanyl, adamantyl, or tetrahydronaphthyl (tetralin).
As used herein, the term "heterocycle" or "heterocyclic system" is intended to mean a stable 5- to 7-membered monocyclic or bicyclic or 7- to 10-membered bicyclic heterocyclic ring which is saturated partially unsaturated or unsaturated (aromatic), and which consists of carbon atoms and from 1 to 4 heteroatoms independently selected from the group consisting of N, O and S and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The nitrogen and sulfur heteroatoms may optionally be oxidized. The heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom which results in a stable structure. The heterocyclic rings described herein may be substituted on carbon or on a nitrogen atom if the resulting compound is stable. If specifically noted, a nitrogen in the heterocycle may optionally be quaternized. It is preferred that when the total number of S and O atoms in the heterocycle exceeds 1, then these heteroatoms are not adjacent to one another. As used herein, the term "aromatic heterocyclic system" is intended to mean a stable 5- to 7-membered monocyclic or bicyclic or 7- to 10-membered bicyclic heterocyclic aromatic ring which consists of carbon atoms and from 1 to 4 heterotams independently selected from the group consisting of N, O and S. It is preferred that the total number of S and O atoms in the heterocycle is not more than 1.
Examples of heterocycles include, but are not limited to, 1H-indazole, 2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl, 4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl, 6H-1,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazalonyl, carbazolyl, 4aH-carbazolyl, .beta.-carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl (benzimidazolyl), isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl., oxazolyl, oxazolidinylperimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, carbolinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, xanthenyl. Preferred heterocycles include, but are not limited to, pyridinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, indolyl, benzimidazolyl, lH-indazolyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, or isatinoyl. Also included are fused ring and spiro compounds containing, for example, the above heterocycles.
As used herein, "HIV reverse transcriptase inhibitor" is intended to refer to both nucleoside and non-nucleoside inhibitors of HIV reverse transcriptase (RT). Examples of nucleoside RT inhibitors include, but are not limited to, AZT, ddC, ddI, d4T, and 3TC. Examples of non-nucleoside RT inhibitors include, but are no limited to, viviradine (Pharmacia and Upjohn U90152S), TIBO derivatives, BI-RG-587, nevirapine, L-697,661, LY 73497, and Ro 18,893 (Roche).
As used herein, "HIV protease inhibitor" is intended to refer to compounds which inhibit HIV protease. Examples include, but are not limited, saquinavir (Roche, Ro31-8959), ritonavir (Abbott, ABT-538), indinavir (Merck, MK-639), Vx-478 (Vertex/Glaxo Wellcome), nelfinavir (Agouron, AG-1343), KNI-272 (Japan Energy), CGP-61755 (Ciba-Geigy), and U-103017 (Pharmacia and Upjohn). Additional examples include the protease inhibitors disclosed in WO93/07128, WO 94/19329, WO 94/22840, PCT Application Number US96/03426, WO96/28464, WO95/33464, and WO95/06030.
As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.
The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosure of which is hereby incorporated by reference.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
"Prodrugs" are intended to include any covalently bonded carriers which release the active parent drug according to formula I or other formulae or compounds of the present invention in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound of the present invention, for example formula (I), are prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Prodrugs include compounds of the present invention wherein the hydroxy or amino group is bonded to any group that, when the prodrug is administered to a mammalian subject, cleaves to form a free hydroxyl or free amino, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, or benzoate derivatives of alcohol and amine functional groups in the compounds of formula I; phosphate esters, dimethylglycine esters, aminoalkylbenzyl esters, aminoalkyl esters and carboxyalkyl esters of alcohol functional groups in the compounds of formula I; and the like. Additional examples include compounds wherein the two hydroxy groups of formula I join to form an epoxide; --OCH.sub.2 SCH.sub.2 O--; --OC(.dbd.O)O--; --OCH.sub.2 O--; --OC(.dbd.S)O--; --OC(.dbd.O)C(.dbd.O)O--; --OC(CH.sub.3).sub.2 O--; --OC((CH.sub.2).sub.3 NH.sub.2)(CH.sub.3)O--; --OC(OCH.sub.3)(CH.sub.2 CH.sub.2 CH.sub.3)O--; or --OS(.dbd.O)O--.
"Stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contempleted by the present invention.
"Substituted" is intended to indicate that one or more hydrogens on the atom indicated in the expression using "substituted" is replaced with a selection from the indicated group(s), provided that the indicated atom's normal valency is not exceeded, and that the substitution results in a stable compound. When a substituent is keto (i.e., .dbd.O) group, then 2 hydrogens on the atom are replaced.
"Therapeutically effective amount" is intended to include an amount of a compound of the present invention or an amount of the combination of compounds claimed effective to inhibit HIV infection or treat the symptoms of HIV infection in a host. The combination of compounds is preferably a synergistic combination. Synergy, as described for example by Chou and Talalay, Adv. Enzyme Regul. 22:27-55 (1984), occurs when the effect (in this case, inhibition of HIV replication) of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent. In general, a synergistic effect is most clearly demonstrated at suboptimal concentrations of the compounds. Synergy can be in terms of lower cytotoxicity, increased antiviral effect, or some other beneficial effect of the combination compared with the individual components.
SYNTHESIS
The compounds of the present invention can be prepared in a number of ways known to one skilled in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or by variations thereon as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reactions are performed in a solvent appropriate to the reagents and materials employed and suitable for the transformations being effected. It will be understood by those skilled in the art of organic synthesis that the functionality present on the molecule should be consistent with the transformations proposed. This will sometimes require a judgment to modify the order of the synthetic steps or to select one particular process scheme over another in order to obtain a desired compound of the invention. It will also be recognized that another major consideration in the planning of any synthetic route in this field is the judicious choice of the protecting group used for protection of the reactive functional groups present in the compounds described in this invention. An authoritative account describing the many alternatives to the trained practitioner is Greene and Wuts (Protective Groups In Organic Synthesis, Wiley and Sons, 1991). All references cited herein are hereby incorporated in their entirety herein by reference.
Two of the common starting materials useful for making compounds of the present invention are shown below. ##STR6## The cyclic urea can be prepared as shown in Example 6 of U.S. Pat. No. 5,530,124, the contents of which are hereby incorporated by reference. The vinyl ether can be prepared as shown in Example 3 of U.S. Pat. No. 5,532,357, the contents of which are hereby incorporated by reference.
One can alkylate these starting materials with an appropriately substituted indazolylmethyl group as shown below. ##STR7## Descriptions of reactions of this type will be provided in the Example section. The remaining urea nitrogen can then be alkylated with an appropriate benzyl group, e.g., m-nitrobenzylchloride. The ester group of the indazole can then be further modified to obtain the presently claimed compounds. These modifications will be described in detail in the following section.
It may be advantageous to first alkylate the cyclic urea with a benzyl group. ##STR8## The aminobenzyl-cyclic urea can be obtained from reduction of the reaction product of a cyclic urea and m-nitrobenzylchloride. By alkylating with the benzyl group first, one can modify the benzyl substituent so that it will not be effected by subsequent modifications of the indazole ester.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.
EXAMPLES
Abbreviations used in the Examples are defined as follows: ".degree. C." for degrees Celsius, "d" for doublet, "dd" for doublet of doublets, "eq" for equivalent or equivalents, "g" for gram or grams, "mg" for milligram or milligrams, "mL" for milliliter or milliliters, "H" for hydrogen or hydrogens, "hr" for hour or hours, "m" for multiplet, "M" for molar, "min" for minute or minutes, "MHz" for megahertz, "MS" for mass spectroscopy, "nmr" or "NMR" for nuclear magnetic resonance spectroscopy, "t" for triplet, and "TLC" for thin layer chromatography.
General. All reactions were run under a nitrogen atmosphere at room temperature and most were unoptimized. The reactions were followed by TLC. Reactions that had been run over night were done so out of convenience. Reagents were used as received. DMF, THF and acetonitrile were dried over molecular sieves. All other solvents were reagent grade. Ethanol and methanol were absolute and water was deionized. Melting points were determined in open capillary tubes on a Mel-Temp apparatus and are uncorrected. Column chromatographies were done on flash silica gel. Exceptions to any of the conditions above are note in the text. ##STR9## Method A: The 5-methylisatin (5.08 g, 31.6 mmol) was dissolved with NaOH (1.30 g, 32.5 mmol) in water (10 mL). The solution was chilled to 0.degree. C.) and added to a chilled (0.degree. C.) solution of NaNO.sub.2 (2.19 g, 31.7 mmol) in water (10 mL). The combined solution was added (5-10 min.) with stirring to a chilled (0.degree. C.) solution of concentrated H.sub.2 SO.sub.4 (6.33 g, 64.6 mmole) in water (60 mL) using a dropping funnel with the tip below the surface of the acid. During the addition, ice was added to the reaction as needed to maintain a 0.degree. C.) operating temperature and ether was added to control foaming. The diazonium salt solution was added (15-20 min.) with stirring to a chilled (0.degree. C.) mixture of tin chloride dihydrate (17.4 g, 77.0 mmol) in concentrated H.sub.2 SO.sub.4 (30 mL) by way of a dropping funnel with the tip below the surface of the acid. During the addition, ice was added to the reaction as needed to maintain a 0.degree. C.) operating temperature and ether was added to control foaming. After stirring for one hour, the reaction was filtered giving a golden yellow powder. The powder was dissolved in water (50 mL) by adding solid KOH until a constant pH of 8.5 was achieved. The mixture was filtered through celite with water and washed with ethyl acetate (3.times.30 mL). The aqueous fraction was acidified (pH 2) with concentrated HCl and the precipitate was filtered which gave 4.52 g of 2 as a yellow powder (81%): mp >350.degree. C.; mass spec. 177 (M+H). ##STR10## Method B: A solution of 2 (59.0 g, 335 mmol) and concentrated H.sub.2 SO.sub.4 (4 mL) in methanol (500 mL) was refluxed over night. The reaction was poured into water (2500 mL) and the precipitate was filtered which gave 53.7 g of 3 as a yellow powder (84%): mp 178-9.degree. C.; mass spec. 191 (M+H).
Method C: To a chilled (0.degree. C.) solution of 3 (32.0 g, 168 mmole) and tritylchloride (56.3 g, 202 mmole) in THF (110 mL) was added NaH (8.76 g, 219 mmole). The reaction was stirred over night while allowed to slowly warm to room temperature. It was diluted with dichloromethane (200 mL) and washed with water (2.times.100 mL) and saturated NaCl (30 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a brown oil that crystallized on standing. Recrystallization from methanol/dichloromethane gave 62.9 g of 4 as yellow prisms (87%): mp 192-3.degree. C.; mass spec. 433.1 (M+H).
Method D: A mixture of 4 (11.2 g, 25.9 mmole), NBS (4.58 g, 25.9 mmol) and benzoyl peroxide (301 mg, 1.24 mmole) in degassed carbon tetrachloride (100 mL) was refluxed for one hour. Refluxing was stopped when a TLC (dichloromethane) of the reaction indicated the starting material (R.sub.f 0.37) was almost gone and dibrominated indazole (R.sub.f 0.48) was starting to form. After cooling to room temperature, the reaction mixture was filtered through celite with carbon tetrachloride and the filtrate evaporated at reduced pressure which gave a yellow oil that crystallized on standing. Recrystallization from ethyl acetate gave 8.99 g of 5 as creamy white prisms (76%): mp 214-5.degree. C.; mass spec. 511.2 (M+H). ##STR11## Method E: To a chilled (0.degree. C.) solution of 6 (4.22 g, 11.1 mmole) and 5 (10.0 g, 16.6 mmole) in DMF (40 mL) was added NaH (0.489 g, 12.2 mmole). The reaction was stirred over night while allowed to slowly warm to room temperature. It was diluted with dichloromethane (100 mL), washed with saturated NH.sub.4 Cl (1.times.80 mL), water (2.times.80 mL) and saturated NaCl (30 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a creamy white foam. Flash chromatography (hexane/ethyl acetate, 20%) gave 8.08 g of 7 as a white foam (90%): mp 113-4.degree. C.; mass spec. 811.6 (M+H).
Method F: A mixture of 7 (4.95 g, 4.95 mmole), m-nitrobenzyl chloride (2.55 g, 14.9 mmole) and potassium iodide (0.852 g, 5.16 mmol) in acetonitrile (70 mL) was refluxed over night. The reaction was diluted with ether (60 mL), washed with water (3.times.40 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a yellow foam. Flash chromatography (dichloromethane/methanol, 0%-5%) gave 4.60 g of 8 as a white foam (99%): mp 126-8.degree. C.; mass spec. 932.8 (M+H). ##STR12## Method G: To a chilled (-78.degree. C.) solution of 8 (4.60 g, 4.93 mmole) in dichloromethane (45 mL) was added DIBAL-H (lN in dichloromethane, 11.8 mL, 11.8 mmole) and the reaction was stirred for 2 hours. After diluting with ether (60 mL), the reaction was washed with 1N HCl (3.times.40 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a light yellow foam. Flash chromatography (hexane/ethyl acetate, 10%) gave 4.18 of 9 as a white foam (94%): mp 123-5.degree. C.; mass spec. 904.7 (M+H).
Method H: To a chilled (0.degree. C.) solution of 9 (9.84 g, 10.9 mmole) and diisopropylethylamine (3.01 mL, 17.4 mmol) in DMF (50 mL) was added methanesulfonyl chloride (1.26 mL, 16.3 mmol). After stirring over night, the reaction diluted with ether (100 mL), washed with water (3.times.50 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a light yellow foam. Flash chromatography (dichloromethane) gave 9.88 g of 10 as a creamy white powder (98%): mp 167-8.degree. C.; mass spec. 923 (M+H). ##STR13## Method I: To a chilled (5.degree. C.) solution of 11 (2.41 g, 5.59 mmole) in acetic acid (25 mL), was added NaNO.sub.2 (405 mg, 5.87 mmole). After stirring 15 min., NaN.sub.3 was added (400 mg, 6.15 mmol) and the reaction was stirred 30 min. After pouring into water (200 mL), the precipitate was filtered and washed with water (3.times.30 mL) which gave 2.50 g of 12 as a creamy white powder (98%): mp 173-4.degree. C.; mass spec. 458.3 (M+H).
Method J: A solution of 12 (2.50 g, 5.477 mmole) was stirred in 2,2-dimethoxypropane (25 mL) with p-toluenesulfonic acid monohydrate (31 mg) for 6 hours. The reaction was diluted with ether (60 mL), washed with water (3.times.30 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave 2.53 g of 13 as a light yellow foam (99%): mp 52-4.degree. C.; mass spec. 498.4 (M+H). ##STR14## Method K: A solution of 10 (2.10 g, 2.28 mmole) and sodium azide (178 mg, 2.74 mmol) in DMF (20 mL) was stirred for 2 hours. The reaction was diluted with ether (50 mL), washed with water (3.times.20 mL) then saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave 2.08 g of 14 as a white powder (98%): mp 110-1.degree. C.; mass spec. 930.5 (M+H).
Method L: A solution of 14 (1.60 g, 1.72 mmole), triphenylposphene (540 mg, 2.06 mmole) and 30% NH.sub.4 OH (5 mL) in THF (20 mL) was stirred over night. The reaction was diluted with ether (75 mL), washed with water (3.times.30 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure giving a yellow foam. The foam was dissolved in dichloromethane (6 mL), petroleum ether (100 mL) was added with stirring and the turbid ether was decanted. The remaining pasty thick oil was evaporated at reduced pressure which gave 1.38 g of 15 as a light creamy white powder (89%): mp 98-100.degree. C.; mass spec. 903.5 (M+H). ##STR15## Method M: A mixture of 16 (1.03 g, 1.21 mmol) and MnO.sub.2 (1.12 g, 12.9 mmol) in dichloromethane was stirred over night. The reaction was filtered though celite with dichloromethane and evaporated at reduced pressure which gave an orange foam. Flash chromatographed (hexane/ethyl acetate 30%) gave 847 mg of 17 as a white foam (83%): mass spec. 920.8 (M+H). ##STR16## Method N: To a chilled (-78.degree. C.) solution of 18 (90 mg, 0.098 mmol) in THF (3 mL) was added MeMgBr (150 mL of 1.4 N in THF, 0.22 mmol). After stirring for 30 min., the reaction was poured into 1N HCl/ethyl acetate (20 mL/30 mL), washed with water (2x20 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a white film (18A) that was carried on to the next reaction without purification. ##STR17## Method O: To a chilled (-78.degree. C.) solution of 17 (106 mg, 0.125 mmol) in THF (3 mL) was added MeMgBr (108 mL of 1.4 N in THF, 0.151 mmol). After stirring for 60 min., the reaction was poured into 1N HCl/ethyl acetate (20 mL/30 mL), washed with water (2.times.20 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a white powder (17A) that was carried on to the next reaction without purification.
Method P: To a chilled (0.degree. C.) solution of 17 (221 mg, 0.250 mmol) and TMS-trifluoromethane (32 mL, 0.300 mmol) in THF (3 mL) was added TBAF (15 mL of 1 N in THF, 0.015 mmol). After stirring for 30 min., 1 N HCl was added (5 mL). After stirring for an additional 15 min., the reaction was diluted with ethyl acetate (30 mL), washed with water (3.times.20 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a yellow oil (17B) that was carried on to the next reaction without purification. ##STR18## Method Q: To a solution of 10 (2.95 g, 3.20 mmole) and ethanol (0.220 mL, 4.80 mmole) in DMF (15 mL) was added sodium hydride (192 mg, 4.80 mmole). After stirring for two h, the reaction was diluted with ether (80 mL), washed with water (3.times.50 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a light brown foam (19) that was carried on to the next reaction without purification.
Method R: To a solution of 10 (2.61 g, 2.86 mmole) and 6-hydroxyquinoline (1.25 g, 8.59 mmole) in DMF (25 mL) was added sodium hydride (172 mg, 4.29 mmole). After stirring over night, the reaction was diluted with ether (100 mL), washed with water (3.times.50 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a creamy white foam (20) that was carried on to the next reaction without purification. ##STR19## Method S: To a solution of 9 (163 mg, 0.183 mmole) and benzyl bromide (43 mL, 0.365 mmole) in DMF (3 mL) was added sodium hydride (11 mg, 0.275 mmole). After stirring 3 h, the reaction was diluted with ether (30 mL), washed with water (3.times.20 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a white foam (21) that was carried on to the next reaction without purification. ##STR20## Method T: To a solution of 10 (181 mg, 0.199 mmole) and thiophenol (41 mL, 0.397 mmole) in DMF (3 mL) was added sodium hydride (12 mg, 0.299 mmole). After stirring 2 h, the reaction was diluted with ether (30 mL), washed with water (3.times.20 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a tan foam (22) that was carried on to the next reaction without purification.
Method U: To a solution of 9 (195 mg, 0.218 mmole) and o-fluoronitobenzene (69 mL, 0.655 mmole) in DMF (3 mL) was added sodium hydride (13 mg, 0.328 mmole). After stirring 1.5 h, the reaction was diluted with ether (30 mL), washed with water (3.times.20 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a yellow film (23) that was carried on to the next reaction without purification. ##STR21## Method V: To a solution of 10 (2.99 g, 3.24 mmole) and pyrazole (265 mg, 3.89 mmole) in DMF (30 mL) was added sodium hydride (143 mg, 3.57 mmole). After stirring 1 h, the reaction was diluted with ether (100 mL), washed with water (3.times.50 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a white foam (24) that was carried on to the next reaction without purification.
Method W: A solution of 10 (152 mg, 165 mmol) and 2-methoxypyridine (35 mL, 330 mmol) in DMF (3 mL) was stirred over night at 100.degree. C.). The reaction was diluted with ether (25 mL), washed with 0.5 N HCl (3.times.10 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave an orange foam (25) that was carried on to the next reaction without purification.
Method X: A solution of the urea 10 (182 mg, 197 mmol), excess K.sub.2 CO.sub.3 and 4-hydroxypyridine (38 mL, 395 mmol) in DMF (3 mL) was stirred over night. The reaction was diluted with ether (25 mL), washed with water (3.times.10 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a yellow oil (26) that was carried on to the next reaction without purification. ##STR22## Method Y: Ammonia (.about.5 mL condensed) was bubbled with stirring into a chilled solution (-78.degree. C.) of trimethyl aluminum (5.21 mmol) in dichloromethane (15 mL). After warming to room temperature and stirring for one hour, 27 was added. After stirring at 35.degree. C. over night, the reaction was diluted with ether (70 mL), washed with 1 N HCl (3.times.30 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a white foam (28) that was carried on to the next reaction without purification. ##STR23## Method Z: To a solution of 15 (3.00 g, 2.66 mmole) and diisopropylethylamine (555 mL, 3.19 mmol) in dichloromethane (30 mL) was added benzoyl chloride (338 mL, 2.93 mmole). After stirring 1 h, the reaction was diluted with ether (80 mL), washed with 0.5 N HCl (2.times.30 mL), saturated NaHCO.sub.3 (2.times.30 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a white foam (29) that was carried on to the next reaction without purification.
Method AA: A solution of 15 (178 mg, 0.197 mmole), 2-quinoline carboxylic acid (41 mg, 0.237 mmol) and EDC (42 mL, 3.19 mmol) in dichloromethane (3 mL) was stirred over night. The reaction was diluted with ether (30 mL), washed with 0.5 N HCl (2.times.10 mL), saturated NaHCO.sub.3 (2.times.10 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a yellow powder (30) that was carried on to the next reaction without purification.
Method BB: To a solution of 15 (2.01 g, 2.23 mmol) and diisopropylethylamine (504 mL, 2.90 mmol) was added ethyl chloroformate (254 mL, 2.67 mmol). After stirring 30 min., the reaction was diluted with ether (25 mL), washed with 0.5 N HCl (2.times.10 mL), saturated NaHCO.sub.3 (2.times.10 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a creamy white powder (31) that was carried on to the next reaction without purification.
Method CC: To a solution of 2,2,2-trifluoroethanol (13. mL, 176 mmol) and diisopropylethylamine (31 mL, 176 mmol) in dichloromethane (3 mL) was added p-nitrophenyl chloroformate (35.8 mg, 176 mmole). After stirring 15 min., 15 (137 mg, 152 mmol) was added. After stirring over night, the reaction was diluted with ether (25 mL), washed with 0.5 N HCl (2.times.10 mL), saturated NaHCO.sub.3 (2.times.10 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave an orange oil (32) that was carried on to the next reaction without purification. ##STR24## Method DD: A solution of 9 (156 mg, 173 mmol), phenylisocyanate (23 mL, 207 mmol) and diisopropylethylamine (30 mL, 173 mmol) in dichloromethane (3 mL) was stirred over night. The reaction was diluted with ether (25 mL), washed with 0.5 N HCl (3.times.10 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure giving a white powder (33) which was carried on to the next reaction without purification.
Method EE: To a solution of 15 (154 mg, 171 mmol) and diisopropylethylamine (89 mL, 513 mmol) in dichloromethane (3 mL) was added N,N-dimethylcarbamoyl chloride (31 mL, 341 mmol). After stirring 1.5 h, the reaction was diluted with ether (25 mL), washed with 0.5 N HCl (3.times.10 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a white powder (34) that was carried on to the next reaction without purification. ##STR25## Method FF: A solution of 20 (2.86 mmole) in dichloromethane/TFA (20%, 20 mL) was stirred 2 hours. The reaction was diluted with ether (100 mL), washed with saturated NaHCO3 (3.times.40 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a creamy white foam (35) that was carried on to the next reaction without purification.
Method GG: A mixture of 35 (805 mg, 1.08 mmole), Pd/C (10%, 161 mg) and ammonium formate (81 mg, 1.29 mmol) in methanol (15 mL) was stirred 6 hours. The reaction was filtered though celite with methanol. The filtrate was diluted with ether (100 mL), washed with water (3.times.50 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a creamy white foam. Flash chromatography (dichloromethane chloride/methanol, 6%, with 0.1% NH.sub.4 OH) gave 626 mg of 36 as a creamy white powder (81% for two steps). ##STR26## Method HH: A mixture of 37 (3.25 mmole), Zn.degree. (7.90 g, 121 mmol) and calcium chloride (397 mg, 3.58 mmol) in ethanol (30 mL) was refluxed over night. The reaction was filtered though celite with methanol and the filtrate evaporated at reduced pressure which gave a creamy white powder. Flash chromatography (dichloromethane/methanol, 4%, with 0.1% NH.sub.4 OH) gave 1.10 g of 38 as a white powder (50% for three steps). ##STR27## Method II: A mixture of 39 (144 mmole) and tin chloride dihydrate (162 mg, 720 mmol) in ethyl acetate (20 mL) was refluxed for 4 hours. The reaction was diluted with ethyl acetate (20 mL), washed with saturated NaHCO.sub.3 (3.times.15 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a white powder. Flash chromatography (dichloromethane/methanol, 5%, with 0.1% NH.sub.4 OH) gave 92 mg of 40 as a white powder (83% for three steps). ##STR28## Method JJ: A solution of 41 (182 mmole), triphenylposphene (57 mg, 218 mmole) and 30% NH.sub.4 OH (2 mL) in THF (8 mL) was refluxed 4 hours. The reaction was diluted with ether (25 mL), washed with saturated sodium bicarbonate (3.times.10 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a yellow film. Flash chromatography (dichloromethane/methanol, 5%, with 0.1% NH.sub.4 OH) gave 95 mg of 42 as a white powder (64% for three steps). ##STR29## Method KK: A solution of 10 (4.80 g, 5.20 mmol) and KCN (0.407 g, 6.24 mmol) in DMF (50 mL) was stirred overnight. The reaction was added to EtOAc and washed with water (2.times.), sat. NaCl, dried over Na.sub.2 SO.sub.4 and evaporated leaving 4.44 g of a pale yellow foam. This material was deprotected (Method FF) and reduced (Method II) to give 43.
A mixture of 43 (43 mg, 72 mmol), hydrogen peroxide (30%, 16 mL, 143 mmol) and excess K.sub.2 CO.sub.3 in DMSO (5 mL) was stirred 1 hour. The reaction was diluted with ethyl acetate (15 mL), washed with water (3.times.7 mL) and saturated NaCl (7 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a white powder. Precipitation from dichloromethane/methanol with ether gave 29 mg of 44 as a white powder (65%). ##STR30## Method LL: To a solution of 9 (161 mg, 0.178 mmole) and diisopropylethylamine (37 mL, 0.214 mmol) in dichloromethane (3 mL) was added benzoyl chloride (23 mL, 0.196 mmole). After stirring 1 h, the reaction was diluted with ether (20 mL), washed with 0.5 N HCl (2.times.10 mL), saturated NaHCO.sub.3 (2.times.10 mL) and saturated NaCl (10 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a colorless film (45) that was carried on to the next reaction without purification. ##STR31## Method MM: The starting cyclic urea can be prepared by known methods, e.g., see Tetr. Lett. 1995, 36(28), 4967. To a chilled (0.degree. C.) solution of the starting cyclic urea (5.01 g, 14.2 mmole) and 5 (7.27 g, 14.2 mmole) in DMF (40 mL) was added NaH (0.626 g, 15.6 mmole). The reaction was stirred for 1 h while allowed to warm to room temperature. After diluting with ether (60 mL), the reaction was washed with saturated NH.sub.4 Cl (1.times.40 mL), water (2.times.40 mL) and saturated NaCl (20 mL), dried (MgSO.sub.4) and evaporated at reduced pressure which gave a creamy white foam. Flash chromatography 20 (hexane/ethyl acetate, 20-30%) gave 6.95 g of 46 as a white foam (63%). ##STR32## Method NN: To a 0.degree. C. solution of the chloride 10 (150 mg, 0.163 mmol) and 2-morpholino-2-phenylacetonitrile (39 mg, 0.195 mmol) in DMF (1 mL), was added NaH (10 mg, 0.244 mmol). After stirring for 2 h, the reaction was added to EtOAc and washed with water, sat. NaCl, dried (Na.sub.2 SO.sub.4), and evaporated. The resulting white solid was refluxed in EtOH (4 mL) and 3N HCl (1 mL) for 2 h. The reaction was added to EtOAc and washed with sat. NaHCO.sub.3, sat. NaCl, dried (NaSO.sub.4) and evaporated leaving 125 mg of a pale yellow glass (47) which was used in the next step without further purification. ##STR33## Method OO: A suspension of the nitrile 43 (2.00 g, 2.19 mmol) in EtOH (40 mL) and 25% NaOH (10 mL) was refluxed for 1.5 h. The reaction was added to EtOAc and added washed with sat. NaCl (3.times.), dried (Na.sub.2 SO.sub.4), and evaporated leaving 2.24 g of an orange foam (48) which was used without further purification.
Method PP: A solution of 48 (0.161 mmol), amine (1.2 mmol), BOP-Cl (1.2 mmol), and Et.sub.3 N (0.322 mmol) in CH.sub.2 Cl.sub.2 (2 mL) was stirred overnight. The reaction was washed with sat. NaHCO.sub.3, sat. NaCl, dried (Na.sub.2 SO.sub.4) and evaporated leaving the amide (49) which was used without further purification. ##STR34## Method a: To a -78.degree. C. solution of BuLi (190 .mu.L, 0.467 mmol) in dry ether (2 mL) under argon, was added a solution of SEM-protected imidazole (92 mg, 0467 mmol) in dry ether (1 mL). After 10 min., a solution of the aldehyde 17 (200 mg, 0.234 mmol) in dry THF (2 mL) was added dropwise (5-15 min.). The reaction was stirred at -78.degree. C. for another 10 to 60 minutes and then quenched with saturated NH.sub.4 Cl. The reaction was partitioned between EtOAc and saturated NH.sub.4 Cl, washed with brine, dried (Na.sub.2 SO.sub.4) and evaporated to give an orange oil (50) (320 mg, 100% yield).
Method b: A solution of 50 (100 mg) in TFA (1 mL) was stirred for 0.5-12 h at room temperature. The reaction was diluted with EtOAc and neutralized with 10% NaOH, washed with water and brine, dried (Na.sub.2 SO.sub.4) and evaporated to give a pale yellow solid (51) (85 mg).
Method c: A solution of 51 (1.0 g) in MeOH (10 mL) and 10% NaOH (1 mL) was stirred for 20 min. Ethylene diamine (1 mL) was added, and the reaction was stirred for 5-60 min. The reaction was diluted with EtOAc, neutralized with aqueous HCl, washed with water and brine, dried (Na.sub.2 SO.sub.4) and evaporated to give a pale yellow solid Flash chromatography on silica gel (9% MeOH/CH.sub.2 Cl.sub.2) gave a white solid (350 mg) which was triturated with ether to give a white solid (52) (330mg). ##STR35## Method d: A solution of 52 (20 mg) in TFA (1 mL) and Et.sub.3 SiH (0.1-0.2 mL) was heated at 60-80.degree. C. overnight. The reaction was diluted with EtOAc, neutralized with 10% NaOH, washed with water and brine, dried (Na.sub.2 SO.sub.4) and evaporated to give an orange oil. Flash chromatography on silica gel (8% MeOH/CH.sub.2 Cl.sub.2) gave a white semi-solid (17 mg) which was triturated with ether to give a white solid (53) (13.5 mg). ##STR36## Method e: A solution of 50 (140 mg) in EtOH (2 mL) and 3N HCl (1 mL) was refluxed overnight. The reaction was diluted with EtOAc, neutralized with 10% NaOH, washed with water and brine, dried (Na.sub.2 SO.sub.4) and evaporated to give an orange oil. Flash chromatography (8% MeOH/CH.sub.2 Cl.sub.2) gave a white glass (20 mg) which was triturated with ether to give a pale orange solid (55) (15.8 mg).
Method f: A mixture of 17 (80 mg, 0.09 mmol), aniline (8.5 .mu.L, 0.09 mmol), AcOH (5.3 .mu.L, 0.09 mmol) and NaBH.sub.3 CN (15 mg) in MeOH (2 mL) was stirred for 2 h. The reaction was diluted with EtOAc, washed with saturated sodium bicarbonate (2.times.) and brine, dried (Na.sub.2 SO.sub.4) and evaporated to give a pale yellow glass (56). ##STR37## Method g: A solution of 17 (79 mg, 0.09 mmol), 2-aminopyrimidine (7 mg, 0.09 mmol) and benzotriazole (11 mg, 0.09 mmol) in toluene (2 mL) was refluxed for 3.5 h. The solvent was evaporated, and the residue was refluxed in THF with excess of NaBH.sub.4 overnight. The reaction was diluted with EtOAc washed with 10% NaOH (2.times.), water and brine, dried (Na.sub.2 SO.sub.4) and evaporated to give pale yellow glass. Flash chromatography (25-35% EtOAc/hexane) gave a glass (57) (22 mg).
Method h: A modified Method R. A suspension of the chloride (10) (200 mg, 0.164 mmol), 4-hydroxyacetophenone (45 mg, 0.328 mmol) and potassium carbonate (50 mg) was in ACN (2 mL) refluxed for 5.5 h. The reaction was diluted with EtOAc washed with 10% NaOH (2.times.), water, brine, dried (Na.sub.2 SO.sub.4) and evaporated to give a colorless glass (220 mg, 100% yield). ##STR38## Method i: To a 0.degree. C. suspension of benzyl triphenylphosphonium chloride (86 mg, 0.22 mmol) in THF (2 mL) was added a solution of potassium t-butoxide in THF (0.24 mL, 0.24 mmol). An orange colored ylide formed immediately. After 10 min., a solution of the aldehyde (58) (200 mg, 0.22 mmol) in THF (2 mL) was added. The orange color turned slightly yellow after 1 h. TLC (30% EtOAc/hexane) indicated no more starting material (the aldehyde). The reaction was diluted with EtOAc, washed with water (2.times.), brine, dried (Na.sub.2 SO.sub.4) and evaporated to give a white solid (266 mg) which was recrystallized from EtOH to give a white solid (59) (100 mg). ##STR39## Method j: A mixture of (58) (2.5 g), nitromethane (25 mL) and ammonium acetate (0.5 g) was refluxed for 2.5 h. The reaction was diluted with EtOAc washed with water (2.times.), brine, dried (Na.sub.2 SO.sub.4) and evaporated to give a yellow solid (3.23 g).
A solution of the nitroethene (3.2 g) and NaBH.sub.4 (0.32 g) in MeOH (75 mL) was stirred for 40 minutes. The reaction was diluted with EtOAc washed with water, brine, dried (Na.sub.2 SO.sub.4) and evaporated to give a yellow solid/glass (3.3 g). Chromatography (30% EtOAc/hexane) gave 60 (1.41 g).
Method k: To a solution of the nitroethane (60) (105 mg, 0.11 mmol) in benzene (2 mL)was added PhNCO (72 .mu.L, 0.66 mmol), TMS-acetylene (63 .mu.L, 0.44 mmol) and Et.sub.3 N (3 drops). After stirring at room temperature for 3 h, the reaction was heated to 50.degree. C. for 2.5 h, and filtered through celite. The filtrate was diluted with EtOAc, washed with water, saturated NaHCO.sub.3, brine, dried (Na.sub.2 SO.sub.4) and evaporated to give a glass. Flash chromatography (1% MeOH/CH.sub.2 Cl.sub.2) gave (61). ##STR40## Method m: A solution of 5-TMS-isoxazole (100 mg) in EtOH (5 mL) and concentrated ammonia (2 mL) was refluxed for 7.5 h. The reaction was diluted with EtOAc, washed with water (2.times.), brine, dried (Na.sub.2 SO.sub.4) and evaporated to give an orange glass (62) (105 mg). ##STR41## Method n: A solution of the aniline (63) (6.3 g, 6.4 mmol), Boc-glycine (2.0 g, 11.6 mmol), BOP-Cl (2.5 g, 10.2 mmol) and Et.sub.3 N (1.8 mL, 12.7 mmol) in methylene chloride (60 mL) was stirred overnight. The reaction was diluted with methylene chloride, washed with saturated NaHCO.sub.3 (2.times.), brine, dried (Na.sub.2 SO.sub.4) and evaporated to give a white solid (64) (7.7 g), which was carried on to next step reaction without purification.
Table 1 contains representative examples which were made via the methods indicated.
TABLE 1__________________________________________________________________________1 STR42## -Ex. # R Methods Used MP (.degree. C.) MS (M + H)__________________________________________________________________________1 phenylmethoxy S,FF,GG 109-11 682.6 2 phenylthio T,FF,HH 122-3 684.6 3 hydroxy G,FF,GG 144-6 592.5 4 2-benzimidazolylthio T,FF,HH 164-.6 724.7 5 2-quinolinylmethoxy S,FF,GG 126-7 733.6 6 4-trifluoromethylphenoxy R,FF,GG 124-5 736.7 7 6-quinolinoxy R,FF,GG 144-5 719.7 8 5-quinolinoxy R,FF,GG 147-9 719.7 9 4-quinolinoxy R,FF,GG 177-8 719.7 10 2-methylphenoxy R,FF,GG 251-2 682.6 11 3-fluorophenylmethoxy S,FF,GG 188-9 700.6 12 2-fluorophenylmethoxy S,FF,GG 1O1-3 700.6 13 ethoxy Q,FF,GG 129-30 620.6 14 7-quinolinoxy R,FF,GG 143-4 719.7 15 4-methylphenylmethoxy S,FF,GG 117-8 696.7 16 4-trifluoromethylphenylmethoxy S,FF,GG 124-6 750.7 17 3-methylphenoxy R,FF,GG 128-9 682.6 18 3-aminophenoxy R,FF,GG 124-6 683.3 19 2,2,2-trifluoroethoxy Q,FF,GG 236-7 674.3 20 2-aminophenoxy U,FF,GG 131-3 683.4 21 3-methylphenylmethoxy S,FF,GG 104-6 696.2 22 3-trifluorormethylphenylmethoxy S,FF,GG 97-9 750.3 23 5-(2-methyl)benzthiazoloxy R,FF,GG 135-7 739.4 24 5-benzthiazoloxy R,FF,GG 142-4 725.2 25 n-butoxy Q,FF,GG 107-9 648.4 26 4-cinnolinoxy R,FF,GG 171-3 720.3 27 4-fluoro-2-aminophenoxy U,FF,GG 127-9 701.3 28 cyclopropylmethoxy Q,FF,GG 118-20 646.3 29 cyano K,FF,II 167-9 601.3 30 methyl carbamoyl BB,FF,GG 142-3 649.3 31 4-cyano-2-methoxyphenoxy R,FF,GG 131-2 723.4 32 carboxamido K,KK,GG 148-9 619.3 33 i-butyl carbamoyl BB,FF,GG 123-4 691.6 34 methoxy Q,FF,GG 108-9 606.4 35 1-(5-methyl)indazole V,FF,GG 147-9 706.4 36 N,N-dirmethyl urea EE,FF,GG 151-3 662.4 37 ethyl carbamoyl BB,FF,GG 134-6 663.4 38 cyclopropylmethyl carbamoyl CC,FF,GG 130-2 689.4 39 N-phenyl carbamate DD,FF,GG 136-7 711.3 40 1-pyrazole V,FF,GG 138-9 642.3 41 3-oxazolid-2-one V,FF,GG 143-4 661.3 42 n-propyl carbamoyl BB,FF,GG 123-4 677.3 43 3,4-dimethoxyphenoxy AA,FF,GG 119-20 728.3 44 N'-pyrro1idine urea EE,FF,GG 166-7 688.3 45 n-butyl carbamoyl AA,FF,GG 121-2 691.3 46 amino K,L,FF,GG 164-5 591.2 47 i-propyl carbamoyl AA,FF,GG 127-8 677.2 48 t-butyl carbamoyl CC,FF,GG 136-8 691.4 49 trifluoroacetamido Z,FF,GG 157-8 687.4 50 1-pyrid-2-one W,FF,GG 145-6 669.5 51 2,2,2-trifluoroethyl carbamoyl CC,FF,GG 127-8 747.4 52 3-tetrahydrofuranylcarbamoyl CC,FF,GG 138-9 735.5 53 3-allylyl carbamoyl CC,FF,HH 115-6 675.5 54 1-(4-methyl)pyrazole V,FF,GG 133-4 656.5 55 1-(4-chloro)pyrazole V,FF,HH 120-1 676.5 56 1-pyrid-4-one X,FF,GG 171-2 669.6 57 1-(4-bromo)pyrazole V,FF,HH 136-7 720.4 58 2-pyridiny1 carboxamido Z,FF,GG 139-40 696.5 59 phenyl carboxamido Z,FF,GG 143-4 695.5 60 1-(4-amino)pyrazole V,FF,GG 134-5 657.5 61 1-(3-trifluoromethyl)pyrazole V,FF,GG 207-8 710.5 62 2-oxetanemethoxy Q,FF,GG 127-8 662.5 63 1-(3,5-dimethoxy)pyrazole V,FF,GG 141-2 670.6 64 1-(3-amino)pyrazole V,FF,GG 138-9 657.6 65 2-furanyl carboxamido Z,FF,GG 148-9 685.5 66 2-thiophenyl carboxamido Z,FF,GG 145-6. 701.5 67 1-(4-iodo)pyrazole V,FF,II 206-7 768.3 68 1-(4-nitro)pyrazole I,J,MM,G,H, 214-5 687.5 V,FF,GG,JJ 69 1-(3-nitro)pyrazole I,J,MM,G,H, 148-9 687.5 V, FF,GG,JJ 70 t-butyl carboxamido Z,FF,GG 141-2 675.6 71 3-trifluoromethylphenyl carboxamido Z,FF,GG 137-8 763.5 72 3-methoxyphenyl carboxamido Z,FF,GG 133-4 725.4 73 4-methoxyphenyl carboxamido Z,FF,GG 148-9 725.4 74 2-quinolinyl carboxamido AA,FF,GG 153-4 746.4 75 2-pyrazinyl carboxamido AA,FF,GG 155-6 697.4 76 1-i-quinolinyl carboxamido AA,FF,GG 149-50 746.5 77 2-methoxyphenyl carboxamido Z,FF,GG 144-5 725.5 78 1-(4-carboethoxy)pyrazole V,FF,GG 135-6 714.5 79 benzoyloxy LL,FF,GG 126-7 696.5 80 2-f1uoropheny1 carboxamido Z,FF,GG 138-9 713.4 81 3-fluoro-5-trifluoromethylphenoxy R,FF;GG 228-9 754.4 82 4-fluorophenyl carboxamido Z,FF,GG 150-1 713.4 83 8-(1,2,3,4-tertrahydro)quinolinyl AA,FF,GG 152-3 750.4 carboxarnido 84 2-(6-methyl)pyridinyl carboxamido AA,FF,GG 141-2 710.4 85 2-aminophenyl carboxarnido AA,FF,GG 146-7 710.5 86 3-aminophenyl carboxarnido AA,FF,GG 159-60 710.6 87 4-aminophenyl carboxarnido AA,FF,GG 168-9 710.5 88 2,6-difluorophenyl carboxarnido Z,FF,GG 148-50 731.3 89 1-(4-acetyl)pyrazole V,FF,GG 131-2 684.4 90 1-(4-carbomethoxy)pyrazole V,FF,GG,B 138-40 700.4 91 2-pyridylmethoxy R,FF,GG 160 683.4 (dec.) 92 2-methyl-4-thiazolemethoxy S,FF,HH 112-6 703.4 93 propoxy Q,FF,HH 112-6 634.5 94 N,N-dimethylcarbamoyl S,FF,HH 134-6 663.6 95 6-methyl-2-pyridylmethoxy S,FF,GG 110-4 697.7 96 phenoxy R,FF,HH 222-4 668.6 97 2-isopropyl-4-thiazolemethoxy S,FF,HH 97-102 731.6 98 4-methyiphenoxy R,FF,GG 114-8 682.6 99 4-methoxyphenoxy R,FF,GG 113-7 698.6 100 4-cyanophenoxy R,FF,II 127-32 693.6 101 phenylsulfonyl R,FF,GG 142-47 716.7 102 2-tetrahydrofuranmethoxy R,FF,GG 109-13 676.4 103 3-pyridoxy R,FF,GG 119-23 669.3 104 2-methyl-6-benzothiazoloxy R,FF,GG 132-37 739.4 105 5-trif1uoromethyl-2-pyridylmethoxy S,FF,GG 112-6 751.3 106 N-methylcarbamoyl DD,FF,GG 125-30 649.3 107 N-isopropylcarbamoyl DD,FF,GG 134-9 677.5 108 2-methyl-5-pyridoxy R,FF,GG 128-31 683.5 109 1,2,4-triazo1e R,FF,GG 137-41 643.6 110 benzoyl NN,II 189-91 680.5 111 N-t-butylcarboxamido PP,FF,GG 142-7 675.5 112 N,N-diethylcarboxamido PP,FF,GG 130-34 675.6 113 N-phenylcarboxamido PP,FF,GG 140-4 695.6 114 pyrrolidinecarboxamido PP,FF,GG 141-5 673.6 115 4-fluorophenoxy R,FF,GG 120-5 703 (M+18) 116 3-fluorophenoxy R,FF, GG 245-8 686.6 117 4-f1uorophenylmethoxy S,FF, GG 108-l1 700.6 118 2-naphthylmethoxy S,FF,GG 110-15 732.7 119 2-naphthoxy R,FF,GG 174-7 735 (M+18) 120 2-fluorophenoxy R,FF,GG 227-30 686.6 121 4-chlorophenoxy R,FF,II 136-40 702.6 122 2,4-di-f1uorophenoxy R,FF,GG 190-3 704.6 123 phenyl sulfonarnido R,FF,GG 185-90 748 (M+18) 124 4-(methylsulfonyl)phenoxy R,FF,GG 145-50 746.3 125 2-f1uoro-3-pyridoxy S,FF,GG 110-15 701.4 126 2-benzothiazolmethoxy R,FF,GG 124-8 739.2 127 3-cyanophenoxy R,FF,GG 121-5 693.4 128 3,4-di-f1uorophenoxy R,FF,GG 217-21 704.3 129 2-piperonoxy R,FF,GG 150-5 712.4 130 2-f1uoro-4-pyridylmethoxy R,FF,GG 110-14 701.3 131 6-f1uoro-2-pyridylmethoxy S,FF,GG 108-13 701.3 132 5-ch1oro-3-pyridoxy R,FF,II 121-6 703.2 133 3-ch1oro-6-pyridazoxy S,FF,II 142-7 704.3 134 8-quinolinoxy R,FF,GG 147-52 719.4 135 6-ch1oro-2-pyridylmethoxy S,FF,II 110-14 717.3 136 2-ch1oro-3-pyridoxy R,FF,II 180-3 703.4 137 3-methoxy-phenoxy R,FF,GG 241-4 698.3 138 2-methoxy-phenoxy R,FF,GG 200-4 698.3 139 3-furanoxy R,GG,FF 115-20 662.5 140 3-acetylphenoxy R,FF,GG 113-7 710.5 141 2-thiazole a,d,GG 128-31 659.4 142 2-benzimidazole a,d,c,GG 170-5 692.5 143 1-(2-benzimidazole)-1-(OH) a,d,c,GG 708.5 144 N-methyl-2-imidazole a,d,GG 125-30 656.5 145 phenylamino f,b,GG 124-8 667.5 146 4-acety1 phenoxy h,FF,GG 125-30 710.6 147 6-cyano-2-pyridyl S,FF,II 110-15 708.6 148 3-pyrazole a,b,d,c,GG 145-50 642.5 149 2-benzothiazole a,d,GG 127-30 709.4 150 phenymethyl i,GG,b 110-15 666.5 151 1-(2-benzothiazole)-1-(OH) a,d,GG 150-5 725.4 152 2-pyridyl a,d,GG 130-3 653.5 153 2-isoxazole j,k,b,m,II 115-20 643.5 154 2-tetrahydrofuran a,d,GG 125-8 646.5 155 2-furan a,d,GG 124-7 642.3__________________________________________________________________________
Utility
The compounds of formulae I and II possess HIV protease inhibitory activity and are therefore useful as antiviral agents for the treatment of HIV infection and associated diseases. The compounds of formulae I and II possess HIV protease inhibitory activity and are effective as inhibitors of HIV growth. The ability of the compounds of the present invention to inhibit viral growth or infectivity is demonstrated in standard assay of viral growth or infectivity, for example, using the assay described below.
The compounds of formulae I and II of the present invention are also useful for the inhibition of HIV in an ex vivo sample containing HIV or expected to be exposed to HIV. Thus, the compounds of the present invention may be used to inhibit HIV present in a body fluid sample (for example, a serum or semen sample) which contains or is suspected to contain or be exposed to HIV.
The compounds provided by this invention are also useful as standard or reference compounds for use in tests or assays for determining the ability of an agent to inhibit viral clone replication and/or HIV protease, for example in a pharmaceutical research program. Thus, the compounds of the present invention may be used as a control or reference compound in such assays and as a quality control standard. The compounds of the present invention may be provided in a commercial kit or container for use as such standard or reference compound.
Since the compounds of the present invention exhibit specificity for HIV protease, the compounds of the present invention may also be useful as diagnostic reagents in diagnostic assays for the detection of HIV protease. Thus, inhibition of the protease activity in an assay (such as the assays described herein) by a compound of the present invention would be indicative of the presence of HIV protease and HIV virus.
As used herein ".mu.g" denotes microgram, "mg" denotes milligram, "g" denotes gram, ".mu.L" denotes microliter, "mL" denotes milliliter, "L" denotes liter, "riM" denotes nanomolar, ".mu.M" denotes micromolar, "mM" denotes millimolar, "M" denotes molar and "nm" denotes nanometer. "Sigma" stands for the Sigma-Aldrich Corp. of St. Louis, Mo.
HIV RNA Assay
DNA Plasmids and in vitro RNA transcripts:
Plasmid pDAB 72 containing both gag and pol sequences of BH10 (bp 113-1816) cloned into PTZ 19R was prepared according to Erickson-Viitanen et al. AIDS Research and Human Retroviruses 1989, 5, 577. The plasmid was linearized with Bam HI prior to the generation of in vitro RNA transcripts using the Riboprobe Gemini system II kit (Promega) with T7 RNA polymerase. Synthesized RNA was purified by treatment with RNase free DNAse (Promega), phenol-chloroform extraction, and ethanol precipitation. RNA transcripts were dissolved in water, and stored at -70.degree. C. The concentration of RNA was determined from the A.sub.260.
Probes:
Biotinylated capture probes were purified by HPLC after synthesis on an Applied Biosystems (Foster City, Calif.) DNA synthesizer by addition of biotin to the 5' terminal end of the oligonucleotide, using the biotin-phosphoramidite reagent of Cocuzza, Tet. Lett. 1989, 30, 6287. The gag biotinylated capture probe (5-biotin-CTAGCTCCCTGCTTGCCCATACTA 3') was complementary to nucleotides 889-912 of HXB2 and the pol biotinylated capture probe (5'-biotin-CCCTATCATTTTTGGTTTCCAT 3') was complementary to nucleotides 2374-2395 of HXB2. Alkaline phosphatase conjugated oligonucleotides used as reporter probes were prepared by Syngene (San Diego, Calif.). The pol reporter probe (5' CTGTCTTACTTTGATAAAACCTC 3') was complementary to nucleotides 2403-2425 of HXB2. The gag reporter probe (5' CCCAGTATTTGTCTACAGCCTTCT 3') was complementary to nucleotides 950-973 of HXB2. All nucleotide positions are those of the GenBank Genetic Sequence Data Bank as accessed through the Genetics Computer Group Sequence Analysis Software Package (Devereau Nucleic Acids Research 1984, 12, 387). The reporter probes were prepared as 0.5 .mu.M stocks in 2 .times. SSC (0.3 M NaCl, 0.03 M sodium citrate), 0.05 M Tris pH 8.8, 1 mg/mL BSA. The biotinylated capture probes were prepared as 100 .mu.M stocks in water.
Streptavidin coated plates:
Streptavidin coated plates were obtained from Du Pont Biotechnology Systems (Boston, Mass.).
Cells and virus stocks:
MT-2 and MT-4 cells were maintained in RPMI 1640 supplemented with 5% fetal calf serum (FCS) for MT-2 cells or 10% FCS for MT-4 cells, 2 mM L-glutamine and 50 .mu.g/mL gentamycin, all from Gibco. HIV-1 RF was propagated in MT-4 cells in the same medium. Virus stocks were prepared approximately 10 days after acute infection of MT-4 cells and stored as aliquots at -70.degree. C. Infectious titers of HIV-1(RF) stocks were 1-3.times.10.sup.7 PFU (plaque forming units)/mL as measured by plaque assay on MT-2 cells (see below). Each aliquot of virus stock used for infection was thawed only once.
For evaluation of antiviral efficacy, cells to be infected were subcultured one day prior to infection. On the day of infection, cells were resuspended at 5.times.10.sup.5 cells/mL in RPMI 1640, 5% FCS for bulk infections or at 2.times.10.sup.6 /mL in Dulbecco's modified Eagles medium with 5% FCS for infection in microtiter plates. Virus was added and culture continued for 3 days at 37.degree. C.
HIV RNA assay:
Cell lysates or purified RNA in 3 M or 5 M GED were mixed with 5 M GED and capture probe to a final guanidinium isothiocyanate concentration of 3 M and a final biotin oligonucleotide concentration of 30 nM. Hybridization was carried out in sealed U bottom 96 well tissue culture plates (Nunc or Costar) for 16-20 hours at 37.degree. C. RNA hybridization reactions were diluted three-fold with deionized water to a final guanidinium isothiocyanate concentration of 1 M and aliquots (150 .mu.L) were transferred to streptavidin coated microtiter plates wells. Binding of capture probe and capture probe-RNA hybrid to the immobilized streptavidin was allowed to proceed for 2 hours at room temperature, after which the plates were washed 6 times with DuPont ELISA plate wash buffer (phosphate buffered saline(PBS), 0.05% Tween 20.) A second hybridization of reporter probe to the immobilized complex of capture probe and hybridized target RNA was carried out in the washed streptavidin coated well by addition of 120 .mu.l of a hybridization cocktail containing 4.times. SSC, 0.66% Triton X 100, 6.66% deionized formamide, 1 mg/mL BSA and 5 nM reporter probe. After hybridization for one hour at 37.degree. C., the plate was again washed 6 times. Immobilized alkaline phosphatase activity was detected by addition of 100 .mu.L of 0.2 mM 4-methylumbelliferyl phosphate (MUBP, JBL Scientific) in buffer .delta. (2.5 M diethanolamine pH 8.9 (JBL Scientific), 10 mM MgCl.sub.2, 5 mM zinc acetate dihydrate and 5 mM N-hydroxyethyl-ethylene-diamine-triacetic acid). The plates were incubated at 37.degree. C. Fluorescence at 450 nM was measured using a microplate fluorometer (Dynateck) exciting at 365 nM.
Microplate based compound evaluation in HIV-1 infected MT-2 cells:
Compounds to be evaluated were dissolved in DMSO and diluted in culture medium to twice the highest concentration to be tested and a maximum DMSO concentration of 2%. Further three-fold serial dilutions of the compound in culture medium were performed directly in U bottom microtiter plates (Nunc). After compound dilution, MT-2 cells (50 .mu.L) were added to a final concentration of 5.times.10.sup.5 per mL (1.times.10.sup.5 per well). Cells were incubated with compounds for 30 minutes at 37.degree. C. in a CO.sub.2 incubator. For evaluation of antiviral potency, an appropriate dilution of HIV-1 (RF) virus stock (50 .mu.L) was added to culture wells containing cells and dilutions of the test compounds. The final volume in each well was 200 .mu.L. Eight wells per plate were left uninfected with 50 .mu.L of medium added in place of virus, while eight wells were infected in the absence of any antiviral compound. For evaluation of compound toxicity, parallel plates were cultured without virus infection.
After 3 days of culture at 37.degree. C. in a humidified chamber inside a CO.sub.2 incubator, all but 25 .mu.L of medium/well was removed from the HIV infected plates. Thirty seven .mu.L of 5 M GED containing biotinylated capture probe was added to the settled cells and remaining medium in each well to a final concentration of 3 M GED and 30 nM capture probe. Hybridization of the capture probe to HIV RNA in the cell lysate was carried out in the same microplate well used for virus culture by sealing the plate with a plate sealer (Costar), and incubating for 16-20 hrs in a 37.degree. C. incubator. Distilled water was then added to each well to dilute the hybridization reaction three-fold and 150 .mu.L of this diluted mixture was transferred to a streptavidin coated microtiter plate. HIV RNA was quantitated as described above. A standard curve, prepared by adding known amounts of pDAB 72 in vitro RNA transcript to wells containing lysed uninfected cells, was run on each microtiter plate in order to determine the amount of viral RNA made during the infection.
In order to standardize the virus inoculum used in the evaluation of compounds for antiviral activity, dilutions of virus were selected which resulted in an IC.sub.90 value (concentration of compound required to reduce the HIV RNA level by 90%) for dideoxycytidine (ddC) of 0.2 .mu.g/mL. IC.sub.90 values of other antiviral compounds, both more and less potent than ddC, were reproducible using several stocks of HIV-1 (RF) when this procedure was followed. This concentration of virus corresponded to .about.3.times.10.sup.5 PFU (measured by plaque assay on MT-2 cells) per assay well and typically produced approximately 75% of the maximum viral RNA level achievable at any virus inoculum. For the HIV RNA assay, IC.sub.90 values were determined from the percent reduction of net signal (signal from infected cell samples minus signal from uninfected cell samples) in the RNA assay relative to the net signal from infected, untreated cells on the same culture plate (average of eight wells). Valid performance of individual infection and RNA assay tests was judged according to three criteria. It was required that the virus infection should result in an RNA assay signal equal to or greater than the signal generated from 2 ng of pDAB 72 in vitro RNA transcript. The IC.sub.90 for ddC, determined in each assay run, should be between 0.1 and 0.3 .mu.g/mL. Finally, the plateau level of viral RNA produced by an effective protease inhibitor should be less than 10% of the level achieved in an uninhibited infection. A compound was considered active if its IC.sub.90 was found to be less than 1 .mu.M.
For antiviral potency tests, all manipulations in microtiter plates, following the initial addition of 2.times. concentrated compound solution to a single row of wells, were performed using a Perkin Elmer/Cetus ProPette.
Dosage and Formulation
The antiviral compounds of this invention can be administered as treatment for viral infections by any means that produces contact of the active agent with the agent's site of action, i.e., the viral protease, in the body of a mammal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents. They can be administered alone, but preferably are administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
The dosage administered will, of course, vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; and the effect desired. A daily dosage of active ingredient can be expected to be about 0.001 to about 1000 milligrams per kilogram of body weight, with the preferred dose being about 0.1 to about 30 mg/kg.
Dosage forms of compositions suitable for administration contain from about 1 mg to about 100 mg of active ingredient per unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition. The active ingredient can be administered orally in solid dosage forms, such as capsules, tablets and powders, or in liquid dosage forms, such as elixirs, syrups and suspensions. It can also be administered parenterally, in sterile liquid dosage forms.
Gelatin capsules contain the active ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts, and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, supra, a standard reference text in this field.
Useful pharmaceutical dosage-forms for administration of the compounds of this invention can be illustrated as follows:
Capsules
A large number of unit capsules can be prepared by filling standard two-piece hard gelatin capsules each with 100 mg of powdered active ingredient, 150 mg of lactose, 50 mg of cellulose, and 6 mg magnesium stearic.
Soft Gelatin Capsules
A mixture of active ingredient in a digestible oil such as soybean oil, cottonseed oil or olive oil can be prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 mg of the active ingredient. The capsules should then be washed and dried.
Tablets
A large number of tablets can be prepared by conventional procedures so that the dosage unit is 100 mg of active ingredient, 0.2 mg of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 mg of microcrystalline cellulose, 11 mg of starch and 98.8 mg of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.
Suspension
An aqueous suspension can be prepared for oral administration so that each 5 mL contain 25 mg of finely divided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 mg of vanillin.
Iniectable
A parenteral composition suitable for administration by injection can be prepared by stirring 1.5% by weight of active ingredient in 10% by volume propylene glycol and water. The solution is sterilized by commonly used techniques.
Combination of components (a) and (b)
Each therapeutic agent component of this invention can independently be in any dosage form, such as those described above, and can also be administered in various ways, as described above. In the following description component (b) is to be understood to represent one or more agents as described previously. Thus, if components (a) and (b) are to be treated the same or independently, each agent of component (b) may also be treated the same or independently.
Components (a) and (b) of the present invention may be formulated together, in a single dosage unit (that is, combined together in one capsule, tablet, powder, liquid, etc.) as a combination product. When component (a) and (b) are not formulated together in a single dosage unit, component (a) may be administered at the same time as component (b) or in any order; for example component (a) of this invention may be administered first, followed by administration of component (b), or they may be administered in the reverse order. If component (b) contains more that one agent, e.g., one RT inhibitor and one protease inhibitor, these agents may be administered together or in any order. When not administered at the same time, preferably the administration of component (a) and (b) occurs less than about one hour apart. Preferably, the route of administration of component (a) and (b) is oral. The terms oral agent, oral inhibitor, oral compound, or the like, as used herein, denote compounds which may be orally administered. Although it is preferable that component (a) and component (b) both be administered by the same route (that is, for example, both orally) or dosage form, if desired, they may each be administered by different routes (that is, for example, one component of the combination product may be administered orally, and another component may be administered intravenously) or dosage forms.
As is appreciated by a medical practitioner skilled in the art, the dosage of the combination therapy of the invention may vary depending upon various factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the kind of concurrent treatment, the frequency of treatment, and the effect desired, as described above.
The proper dosage of components (a) and (b) of the present invention will be readily ascertainable by a medical practitioner skilled in the art, based upon the present disclosure. By way of general guidance, typically a daily dosage may be about 100 milligrams to about 1.5 grams of each component. If component (b) represents more than one compound, then typically a daily dosage may be about 100 milligrams to about 1.5 grams of each agent of component (b). By way of general guidance, when the compounds of component (a) and component (b) are administered in combination, the dosage amount of each component may be reduced by about 70-80% relative to the usual dosage of the component when it is administered alone as a single agent for the treatment of HIV infection.
The combination products of this invention may be formulated such that, although the active ingredients are combined in a single dosage unit, the physical contact between the active ingredients is minimized. In order to minimize contact, for example, where the product is orally administered, one active ingredient may be enteric coated. By enteric coating one of the active ingredients, it is possible not only to minimize the contact between the combined active ingredients, but also, it is possible to control the release of one of these components in the gastrointestinal tract such that one of these components is not released in the stomach but rather is released in the intestines.
Another embodiment of this invention where oral administration is desired provides for a combination product wherein one of the active ingredients is coated with a sustained-release material which effects a sustained-release throughout the gastrointestinal tract and also serves to minimize physical contact between the combined active ingredients. Furthermore, the sustained-released component can be additionally enteric coated such that the release of this component occurs only in the intestine. Still another approach would involve the formulation of a combination product in which the one component is coated with a sustained and/or enteric release polymer, and the other component is also coated with a polymer such as a low viscosity grade of hydroxypropyl methylcellulose or other appropriate materials as known in the art, in order to further separate the active components. The polymer coating serves to form an additional barrier to interaction with the other component. In each formulation wherein contact is prevented between components (a) and (b) via a coating or some other material, contact may also be prevented between the individual agents of component (b).
Dosage forms of the combination products of the present invention wherein one active ingredient is enteric coated can be in the form of tablets such that the enteric coated component and the other active ingredient are blended together and then compressed into a tablet or such that the enteric coated component is compressed into one tablet layer and the other active ingredient is compressed into an additional layer. Optionally, in order to further separate the two layers, one or more placebo layers may be present such that the placebo layer is between the layers of active ingredients. In addition, dosage forms of the present invention can be in the form of capsules wherein one active ingredient is compressed into a tablet or in the form of a plurality of microtablets, particles, granules or non-perils, which are then enteric coated. These enteric coated microtablets, particles, granules or non-perils are then placed into a capsule or compressed into a capsule along with a granulation of the other active ingredient.
These as well as other ways of minimizing contact between the components of combination products of the present invention, whether administered in a single dosage form or administered in separate forms but at the same time or concurrently by the same manner, will be readily apparent to those skilled in the art, based on the present disclosure.
Pharmaceutical kits useful for the treatment of HIV infection, which comprise a therapeutically effective amount of a pharmaceutical composition comprising a compound of component (a) and one or more compounds of component (b), in one or more sterile containers, are also within the ambit of the present invention. Sterilization of the container may be carried out using conventional sterilization methodology well known to those skilled in the art. Component (a) and component (b) may be in the same sterile container or in separate sterile containers. The sterile containers of materials may comprise separate containers, or one or more multi-part containers, as desired. Component (a) and component (b), may be separate, or physically combined into a single dosage form or unit as described above. Such kits may further include, if desired, one or more of various conventional pharmaceutical kit components, such as for example, one or more pharmaceutically acceptable carriers, additional vials for mixing the components, etc., as will be readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, may also be included in the kit.
Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Number	Name	Date	Kind
5532356	Smyser et al.	Jul 1996
5532357	Rodgers et al.	Jul 1996

Number	Date	Country
9419329	Sep 1994	WOX
9629329	Sep 1996	WOX

Indazoles of cyclic ureas useful as HIV protease inhibitors

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