Compounds of Formula I are disclosed and methods of treating viral infections with compositions comprising such compounds.

FIELD OF THE INVENTION

The present invention relates to substituted isoindoline compounds, pharmaceutical compositions, and methods of use thereof for (i) inhibiting HIV replication in a subject infected with HIV, or (ii) treating a subject infected with HIV, by administering such compounds.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus type 1 (HIV-1) leads to the contraction of acquired immune deficiency disease (AIDS). The number of cases of HIV continues to rise, and currently over twenty-five million individuals worldwide suffer from the virus. Presently, long-term suppression of viral replication with antiretroviral drugs is the only option for treating HIV-1 infection. Indeed, the U.S. Food and Drug Administration has approved twenty-five drugs over six different inhibitor classes, which have been shown to greatly increase patient survival and quality of life. However, additional therapies are still required because of undesirable drug-drug interactions; drug-food interactions; non-adherence to therapy; and drug resistance due to mutation of the enzyme target.

Currently, almost all HIV positive patients are treated with therapeutic regimens of antiretroviral drug combinations termed, highly active antiretroviral therapy (“HAART”). However, HAART therapies are often complex because a combination of different drugs must be administered often daily to the patient to avoid the rapid emergence of drug-resistant HIV-1 variants. Despite the positive impact of HAART on patient survival, drug resistance can still occur. The emergence of multidrug-resistant HIV-1 isolates has serious clinical consequences and must be suppressed with a new drug regimen, known as salvage therapy.

Current guidelines recommend that salvage therapy includes at least two, and preferably three, fully active drugs. Typically, first-line therapies combine three to four drugs targeting the viral enzymes reverse transcriptase and protease. One option for salvage therapy is to administer different combinations of drugs from the same mechanistic class that remain active against the resistant isolates. However, the options for this approach are often limited, as resistant mutations frequently confer broad cross-resistance to different drugs in the same class. Alternative therapeutic strategies have recently become available with the development of fusion, entry, and integrase inhibitors. However, resistance to all three new drug classes has already been reported both in the lab and in patients. Sustained successful treatment of HIV-1-infected patients with antiretroviral drugs will therefore require the continued development of new and improved drugs with new targets and mechanisms of action.

For example, over the last decade HIV inhibitors have been reported to target the protein-protein interaction between HIV-1 integrase and Lens Epithelium Derived Growth Factor/p75 (“LEDGF”). LEDGF is a cellular transcriptional cofactor of HIV-1 integrase that promotes viral integration of reverse transcribed viral cDNA into the host cell's genome by tethering the preintegration complex to the chromatin. Because of its crucial role in the early steps of HIV replication, the interaction between LEDGF and integrase represents another attractive target for HIV drug therapy.

U.S. provisional patent application 62/027,359 discloses certain isoindoline compounds having the following formula:

SUMMARY OF THE INVENTION

Briefly, in one aspect, the present invention discloses compounds of Formula I:

X is O or CH2;

R2is H, C1-6alkyl, C5-14aryl, C3-7cycloalkyl, C3-7cycloalkenyl, C3-9heterocycle, or C5-9heteroaryl, wherein each R2group is optionally substituted by one to four substituents selected from halo, C1-6alkyl, C1-6hetereoalkyl, or C1-6alkylene or C1-6hetereoalklylene wherein said C1-6alkylene or C1-6hetereoalklylene is bonded to adjacent carbon atoms on said C5-14aryl, C3-7cycloalkyl, C3-7cycloalkenyl, C3-9heterocycle, or C5-9heteroaryl to form a fused ring;

each R6is independently H, or C1-3alkyl, C5-14aryl, C3-9heterocycle, C5-9heteroaryl, —C(O)NR47or —C(O)NHR4, or both R6may together comprise 2-4 carbon atoms and join together to form a bridged ring system;

and wherein each heterocycle, heteroaryl, heteroalkyl, and heteroalkylene comprises one to three heteroatoms selected from S, N, B, or O.

In another aspect the present invention discloses pharmaceutically acceptable salts of the compounds of Formula I.

In another aspect, the present invention discloses pharmaceutical compositions comprising a compound of Formula I or a pharmaceutically acceptable salt thereof.

In another aspect, the present invention discloses a method for treating a viral infection in a patient mediated at least in part by a virus in the retrovirus family of viruses, comprising administering to said patient a composition comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof. In some embodiments, the viral infection is mediated by the HIV virus.

In another aspect, a particular embodiment of the present invention provides a method of treating a subject infected with HIV comprising administering to the subject a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof.

In yet another aspect, a particular embodiment of the present invention provides a method of inhibiting progression of HIV infection in a subject at risk for infection with HIV comprising administering to the subject a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof. Those and other embodiments are further described in the text that follows.

In accordance with another embodiment of the present invention, there is provided a method for preventing or treating a viral infection in a mammal mediated at least in part by a virus in the retrovirus family of viruses which method comprises administering to a mammal, that has been diagnosed with said viral infection or is at risk of developing said viral infection, a compound as defined in Formula I, wherein said virus is an HIV virus and further comprising administration of a therapeutically effective amount of one or more agents active against an HIV virus, wherein said agent active against the HIV virus is selected from the group consisting of Nucleotide reverse transcriptase inhibitors; Non-nucleotide reverse transcriptase inhibitors; Protease inhibitors; Entry, attachment and fusion inhibitors; Integrase inhibitors; Maturation inhibitors; CXCR4 inhibitors; and CCR5 inhibitors.

DETAILED DESCRIPTION OF THE INVENTION

Preferably X is O.

Preferably R2is optionally substituted phenyl. Most preferably, R2is phenyl substituted by one to four substituents selected from fluorine, methyl, —CH2CH2CH2O— wherein said —CH2CH2CH2O— is bonded to adjacent carbon atoms on said phenyl to form a bicyclic ring, or —NHCH2CH2O— wherein said —NHCH2CH2O— is bonded to adjacent carbon atoms on said phenyl to form a bicyclic ring.

Preferably the stereochemistry on the carbon to which XR1is bound is as depicted below.

“Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, and oxalate. Suitable salts include those described in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002.

EXAMPLES

The compounds of this invention may be made by a variety of methods, including well-known standard synthetic methods. Illustrative general synthetic methods are set out below and then specific compounds of the invention are prepared in the working examples.

The following examples serve to more fully describe the manner of making and using the above-described invention. It is understood that these examples in no way serve to limit the true scope of the invention, but rather are presented for illustrative purposes. In the examples below and the synthetic schemes above, the following abbreviations have the following meanings. If an abbreviation is not defined, it has its generally accepted meaning.

dd=doublet of doublets

h or hr=hours

HCV=hepatitus C virus

HPLC=high performance liquid chromatography

J=coupling constant (given in Hz unless otherwise indicated)

M+H+=parent mass spectrum peak plus H+

ppm=parts per million

To an ice-cooled solution of 1-bromobut-2-yne (581 g, 2.2 eq) in DMF (3.5 L) was added NaH (60%, 199 g, 2.5 eq) carefully and the mixture was stirred at 0° C. under N2 atmosphere for 15 min. Then a solution of benzyl carbamate (300 g, 1.985 mol, 1 eq) in DMF (500mL) was added dropwise at 0° C. for 1 h and the resulting mixture was allowed to warm to ambient temperature for 2h. After being quenched cautiously with H2O, the reaction was extracted with ether (×2). The organic layer was washed with H2O (×3), brine, dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (silica gel, 0-5% EtOAc in hexane) to afford the title compound (398 g, 79%).1H NMR (400 MHz, CDCl3) δ 7.41-7.27 (m, 5H), 5.17 (s, 2H), 4.18 (s, 4H), 1.81 (t, J=2.3 Hz, 6H). LC-MS (ESI+): m/z (M+H)=256.3

To a 10 L flask was added EtOAc (7.5 L) followed by Ac2O (400 mL). After stirring at RT for 30 minutes the mixture was cooled to 0° C. and treated with another portion of Ac2O (2.1 L). After 1 hour at 0° C., the solution was allowed to warm to RT. To the solution was added ethyl 2-hydroxy-4-(trimethylsilyl)but-3-ynoate (520 g, 2.60 mol). After stirring at RT for 1 hour the solution was washed with 1N aqueous NaOH (3×, 20 L total). The solution was then washed with brine (5 L), dried over Na2SO4and concentrated to dryness at reduced pressure. The crude product was purified by flash chromatography (silica gel, 0-5% EtOAc/petroleum ether) to give the title compound (590 g, 94%) as a yellow oil.1H NMR (400 MHz, CHLOROFORM-d) δ=5.69 (s, 1 H), 4.36-4.21 (m, 2H), 2.19 (s, 3H), 1.32 (t, J=7.2 Hz, 3H), 0.25-0.15 (m, 9H).

A mixture of benzyl (S)-5-(1-(tert-butoxy)-2-ethoxy-2-oxoethyl)-4,7-dimethyl-6-(phenylethynyl)isoindoline-2-carboxylate (35 mg, 0.065 mmol) and NaOH (78 mg, 1.95 mmol) in EtOH (0.8 mL) and H2O (0.4 mL) was stirred at 100° C. After 2 h, the reaction mixture was cooled to ambient temperature acidified with 1N HCl and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound (25 mg, quant. yield) as a yellow oil which was used in the next step without further purification. LC/MS (m/z) ES+=378.4 (M+1).

To an ice cold solution of benzyl (S)-5-(1-(tert-butwry)-2-ethoxy-2-oxoethyl)-6-iodo-4,7-dimethylisoindoline-2-carboxylate (135 mg, 0.24 mmol), Pd(PPh3)4(56 mg, 0.0478 mmol) in THF (1 mL) was added benzylzinc(II) bromide (1 M, 0.48 mL, 0.48 mmol) and the reaction mixture was heated to 65° C. After 1 h, the reaction mixture was quenched with the addition of sat. NH4Cl aq. solution and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-30% EtOAc in PE) to afford the title compound (122 mg, 97% yield) as a brown oil. LC/MS (m/z) ES+=530.7 (M+1).

A mixture of benzyl (S)-5-benzyl-6-(1-(tert-butwry)-2-ethoxy-2-oxoethyl)-4,7-dimethylisoindoline-2-carboxylate (115 mg, 0.16 mmol) and NaOH (193 mg, 4.8 mmol) in EtOH (2 mL) and H2O (1 mL) was stirred at 100° C. under N2atmosphere. After 2 h, the reaction mixture was cooled to ambient temperature and the resulting mixture was acidified with 1N HCl and extracted with DCM/i-PrOH (85:15). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound (80 mg, quant. yield) as a yellow oil which was used in the next step without further purification. LC/MS (m/z) ES+=368.5 (M+1).

A mixture of benzyl (S)-5-(1-(tert-butoxy)-2-ethoxy-2-oxoethyl)-6-iodo-4,7-dimethylisoindoline-2-carboxylate (1 g, 1.77 mmol), 2-allyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (386 mg, 2.3 mmol), Pd2(dba)3(324 mg, 0.35 mmol), PCy3(99 mg, 0.35 mmol) and K3PO4(1.1 g, 5.3 mmol) in DMF was stirred at 80° C. After 18 h, the reaction mixture was cooled to ambient temperature and partitioned between EtOAc and H2O. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the crude product which was purified by ISCO (silica gel, 0-20% EtOAc in PE) to afford the title compound (680 mg, 80% yield) as a colorless oil. LC/MS (m/z) ES+=480.6 (M+1).

A mixture of benzyl (S)-5-(1-(tert-butoxy)-2-ethoxy-2-oxoethyl)-4,7-dimethyl-6-(2-oxo-2-(piperidin-1-yl)ethyl)isoindoline-2-carboxylate (43 mg, 0.08 mmol) and 10% Pd/C (43 mg) in MeOH (4 mL) was hydrogenated under an atmosphere of H2(1 atm). After 1 h, the reaction mixture was filtered through a pad of Celite and the residue was concentrated under reduced pressure to give the crude title product (35 mg, quant. yield) as a brown oil which was used in the next step without further purification. LC/MS (m/z) ES+=431.4 (M+1).

To a solution of (S)-2-(tert-butoxy)-2-(2-(3-fluorobenzoyl)-4,7-dimethyl-6-(2-oxo-2-(piperidin-1-yl)ethyl)isoindolin-5-yl)acetic acid (35 mg, 0.08 mmol, crude product from the previous step) and 3-fluorobenzoic acid (25 mg, 0.16 mmol) in EtOAc (3 mL) was added propane phosphonic acid anhyrdide (141 mg, 0.2 mmol, 50% EtOAc solution) and Et3N (0.1, 0.49 mmol). After 1 h, the reaction mixture was quenched with sat. aq. NaHCO3and extracted with EtOAc. The organic layer was washed with half saturated aq. citric acid and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-30% EtOAc in PE) to afford the title compound (22 mg, 50% yield) as a yellow oil. LC/MS (m/z) ES+=553.8 (M+1).

Examples 5-7 were prepared in a manner similar to the procedures described for Example 4.

A mixture of benzyl (S)-5-(1-(tert-butoxy)-2-ethoxy-2-oxoethyl)-4,7-dimethyl-6-(4-methyl benzamido)isoindoline-2-carboxylate (80 mg, 0.14 mmol) and 10% Pd/C (80 mg) in MeOH (6 mL) was hydrogenated under a H2 atmosphere (1 atm). After 1 h, the resulting mixture was filtered through a pad of Celite and the residue was concentrated under reduced pressure to give the crude title product (50 mg, 81% yield) as a brown oil which was used in the next step without further purification. LC/MS (m/z) ES+=439.5 (M+1).

To a solution of ethyl (S)-2-(tert-butoxy)-2-(4,7-dimethyl-6-(4-methylbenzamido)isoindolin-5-yl)acetate (50 mg, 0.11 mmol, crude product from the previous step) and 3-fluorobenzoic acid (32 mg, 0.23 mmol) in EtOAc (3 mL) was added propane phosphonic acid anhyrdide (181 mg, 0.29 mmol, 50% EtOAc solution) and Et3N (87 mg, 0.86 mmol). After 2 h, the reaction mixture was quenched with sat. aq. NaHCO3and extracted with EtOAc. The organic layer was washed with half saturated aq. citric acid and brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-40% EtOAc in PE) to afford the title compound (35 mg, 55% yield) as a yellow oil. LC/MS (m/z) ES+=561.6 (M+1).

Examples 9-11 were prepared in a manner similar to the procedures described for Example 8.

An ice cold solution of ethyl (S)-2-(tert-butoxy)-2-(2-(3-fluorobenzoyl)-4,7-dimethyl-6-(4-methyl benzamido)isoindolin-5-yl)acetate (50 mg, 0.09 mmol) in THF (2 mL) was treated with NaH (60%, 18 mg, 0.45 mmol). After 30 min, Mel (126 mg, 0.89 mmol) was added and the reaction mixture was warmed to ambient temperature. After 1 h, the reaction mixture was quenched with sat. aq. NH4Cl and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-30% EtOAc in PE) to afford the title compound (30 mg, 59% yield) as a yellow oil. LC/MS (m/z) ES+=575.8 (M+1).

A mixture of (S,E)-benzyl 5-(1-(tert-butoxy)-2-ethoxy-2-oxoethyl)-6-(2-cyclohexylvinyl)-4,7-dimethylisoindoline-2-carboxylate (12 mg, 0.02 mmol) in NaOH (0.15 mL, 5 N) and EtOH (0.3 mL) was stirred at 100° C. After 18 h, the reaction mixture was cooled to ambient temperature and was neutralized with 1N HCl and extracted with DCM/i-PrOH (85:15). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give title compound (10 mg, quant. yield) which was used in the next step without further purification. LC/MS (m/z) ES+=386.4 (M+1).

The title compound was prepared from the known procedure as described inJ. Med. Chem.2008, 51, 4340-4345 and references therein.

A mixture of benzyl (S)-5-(1-(tert-butoxy)-2-ethoxy-2-oxoethyl)-6-iodo-4,7-dimethylisoindoline-2-carboxylate (10 g, 17.7 mmol), vinylBF3K (3.8 g, 28.3 mmol), Pd2(dba)3(3.2 g, 3.54 mmol), MePhos (1.3 g, 3.54 mmol) and K3PO4(11 g, 53.1 mmol) in DMF (110 mL) was stirred at 80° C. After 18 h, the reaction mixture was filtered through a pad of Celite and the filtrate was partitioned between EtOAc and H2O. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-30% EtOAc in PE) to afford the title compound (6.1 g, 67% yield) as a yellow solid. LC/MS (m/z) ES+=466.4 (M+1).

A mixture of benzyl (S)-5-(1-(tert-butoxy)-2-ethoxy-2-oxoethyl)-4,7-dimethyl-6-vinylisoindoline-2-carboxylate (300 mg, 0.64 mmol), NaIO4(413 mg, 1.93 mmol) and K2Os2O4(24 mg, 0.064 mmol) in THF (4 mL) and H2O (2 mL) was stirred at ambient temperature. After 18 h, the reaction mixture was quenched with sat. aq. Na2S2O3and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-40% EtOAc in PE) to afford the title compound (170 mg, 56% yield) as a yellow oil. LC/MS (m/z) ES+=490.6 (M+Na).

A mixture of (S,E)-benzyl 5-(1-(tert-butoxy)-2-ethoxy-2-oxoethyl)-6-(2-cyclohexylvinyl)-4,7-dimethylisoindoline-2-carboxylate (50 mg, 0.09 mmol) and NaOH (108 mg, 2.7 mmol) in EtOH (1.1 mL) and H2O (0.55 mL) was stirred at 100° C. under N2atmosphere overnight. After cooled down to r.t., the resulting mixture was neutralized with 1N HCl and extracted with DCM/i-PrOH (85:15). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give title compound (31 mg, 89% yield) which was used in the next step without further purification. LC/MS (m/z) ES+=388.3 (M+1).

A mixture of benzyl (S)-5-(1-(tert-butoxy)-2-ethoxy-2-oxoethyl)-6-iodo-4,7-dimethylisoindoline-2-carboxylate (150 mg, 0.27 mmol), (E)-(4-methylstyryl)boronic acid (87 mg, 0.53 mmol) Pd(dppf)Cl2(22 mg, 0.127 mmol), KOAc (80 mg, 0.81 mmol) in DMF (1.5 mL) was stirred at 80° C. After 18 h, the reaction mixture was cooled to ambient temperature and the resulting mixture was partitioned between EtOAc and H2O. The layers were separated and the organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (silica gel, 0-20% EtOAc in PE) to afford the title compound (120 mg, 82% yield) as a yellow solid. LC/MS (m/z) ES+=556.4 (M+1).

A mixture of benzyl (S,E)-5-(1-(tert-butoxy)-2-ethoxy-2-oxoethyl)-4,7-dimethyl-6-(4-methylstyryl) isoindoline-2-carboxylate (160 mg, 0.29 mmol) and NaOH (346 mg, 8.64 mmol) in EtOH (3 mL) and H2O (1.5 mL) was stirred at 100° C. After 18 h, the reaction mixture was cooled to ambient temperature and acidified with 6 N HCl and extracted with DCM/i-PrOH (85:15). The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure to give the title compound (110 mg, quant. yield) as a brown oil which was used in the next step without further purification. LC/MS (m/z) ES+=394.4 (M+1).

A mixture of 8-fluoro-6-iodo-5-methylchromane (2.5 g, 8.59 mmol), ethynyltrimethylsilane (4.2 g, 43 mmol), Pd(dppf)Cl2(600 mg, 0.859 mmol) and CuI (326 mg, 1.72 mmol) in Et3N (20 mL) was stirred at ambient temperature. After 18 h, the reaction mixture was filtered through a pad of Celite and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography ISCO (0-5% EtOAc in PE) to afford the title compound (2.0 g, 90% yield) as a yellow solid. LC/MS (m/z) ES+=263.1 (M+1).

A −30° C. solution of 6-ethynyl-8-fluoro-5-methylchromane (600 mg, 3.15 mmol) in THF (8 mL) was treated with n-BuLi (2.5 M, 1.86 mL, 4.7 mmol). After 30 min, iodomethane was added and the reaction mixture was warmed to ambient temperature. After 2 h, the reaction mixture was quenched with sat. aq. NH4Cl solution and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (0-10% EtOAc in PE) to afford the title compound (560 mg, 87% yield) as a white solid. LC/MS (m/z) ES+=205.3 (M+1).

A mixture of CuCl (20 mg, 0.196 mmol), PPh3(51.5 mg, 0.196 mmol) and t-BuONa (226 mg, 2.35 mmol) in THF was stirred at ambient temperature. After 30 min, a solution of 8-fluoro-5-methyl-6-(prop-1-yn-1-yl)chromane (400 mg, 1.96 mmol) was added, followed by the addition of MeOH (157 mg, 3.92 mmol). After 18 h, the resulting mixture was partitioned between EtOAc and H2O. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (silica gel, 0-5% EtOAc in PE) to afford the title compound (200 mg, 31% yield) as a white solid. LC/MS (m/z) ES+=333.4 (M+1).

To a solution of 6-ethynyl-8-fluoro-5-methylchromane (200 mg, 1.05 mmol) in THF (3 mL) was added catacolborane (235 mg, 2.10 mmol) and heated to 70° C. After 1.5 h, the resulting mixture was quenched with MeOH (1 mL) and partitioned between EtOAc and H2O. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography (silica gel, 0-10% EtOAc in PE) to afford the title compound (50 mg, 22% yield) as a white solid. LC/MS (m/z) ES−=281.4 (M+HCOOH-1).

Antiviral HIV activity and cytotoxicity values for compounds of the invention from Table 1 were measured in parallel in the HTLV-1 transformed cell line MT-4 based on the method previously described (Hazen et al., 2007, In vitro antiviral activity of the novel, tyrosyl-based human immunodeficiency virus (HIV) type 1 protease inhibitor brecanavir (GW640385) in combination with other antiretrovirals and against a panel of protease inhibitor-resistant HIV (Hazen et al., “In vitro antiviral activity of the novel, tyrosyl-based human immunodeficiency virus (HIV) type 1 protease inhibitor brecanavir (GW640385) in combination with other antiretrovirals and against a panel of protease inhibitor-resistant HIV”,Antimicrob. Agents Chemother.2007, 51: 3147-3154; and Pauwels et al., “Sensitive and rapid assay on MT-4 cells for the detection of antiviral compounds against the AIDS virus”,J. of Virological Methods1987, 16: 171-185).

Luciferase activity was measured 96 hours later by adding a cell titer glo (Promega, Madison, Wis.). Percent inhibition of cell protection data was plotted relative to no compound control. Under the same condition, cytotoxicity of the compounds was determined using cell titer Glo™ (Promega, Madison, Wis). ICsos were determined from a 10 point dose response curve using 3-4-fold serial dilution for each compound, which spans a concentration range >1000 fold.

These values are plotted against the molar compound concentrations using the standard four parameter logistic equation:

When tested in the MT4 assay compounds were found to have IC50values listed in Table 1.