WO2011088561A1

WO2011088561A1 - Anti-viral compounds and compositions

Info

Publication number: WO2011088561A1
Application number: PCT/CA2011/000070
Authority: WO
Inventors: Xiaojian Yao; Zhujun Ao
Original assignee: University of Manitoba
Current assignee: University of Manitoba
Priority date: 2010-01-20
Filing date: 2011-01-20
Publication date: 2011-07-28
Anticipated expiration: 2012-07-20

Abstract

The present disclosure provides novel compounds as well as uses, methods, and compositions of treating, or reducing the risk of, HIV-infection.

Description

ANTI-VIRAL COMPOUNDS AND COMPOSITIONS

FIELD

The present disclosure provides compounds and compositions having an antiviral effect especially with respect to the HIV-1 virus.

BACKGROUND

Acquired immune deficiency syndrome (AIDS) is a slow degenerative disease of the immune and nervous systems resulting from HIV infection. This infectious disease has shown the potential to threaten the lives, welfare, and social stability of the world and has been placed as the fourth leading cause of death. Global estimates of the HIV epidemic indicate that there were estimated 31 million people living with HIV-1 and the estimated cumulative total of deaths from AIDS is approximately 12 million. It has been calculated that there are 16,000 new HIV infections per day, one tenth of these in children. Recent studies have clearly shown that about 5-20% of infants born to HIV- positive women acquire the infection through breastfeeding, and this mode may be responsible for 30-42% of HIV infection in infants and young children in Africa.

Even though the progress in the field of anti-HIV-1 chemotherapy in the past several years has been dramatic, the efficient protection against & treatment of HIV-1 infection still remains a challenge. The complete blockage of AIDS progression with current chemotherapy appears to be difficult due to a rapid occurrence of viral drug- resistance. Hence, in addition to developing an efficient AIDS vaccine, the discovery of new anti-HIV therapies is a priority as a part of the global strategy of controlling the spread of the HIV infection.

HIV-1 protease (PR), reverse transcriptase (RT) and integrase (IN) are three viral enzymes which are critical for HIV replication. Among them, the reverse transcriptase and integrase seem to be essential for HIV reverse transcription and integration. Soon after infection, HIV-1 reverse transcriptase catalyzes the conversion of viral RNA genome into double-stranded DNA, and the integrase mediates integration of newly synthesized viral DNA into host chromosomes.

Moreover, HIV-1 integrase has also shown to play crucial roles for HIV cDNA nuclear import and host chromatin targeting prior to its integration. In contrast to RT and IN, HIV-1 protease (PR) acts on the late stage of HIV replication and is the key enzyme required for HIV maturation during and after virus budding. Due to critical roles of these viral enzymatic molecules for HIV infection, extensive studies have focused on elucidation of their functional roles and mechanisms involved, and after 25 years of research, some 25 anti-HIV compounds have been licensed for the treatment of HIV infection. However, the rapid emergence of drug resistance as well as issues associated with drug toxicity remain important challenges in the fight against HIV. It is also urgent to develop new anti-HIV agents with novel targets to be used as new HIV prevention and therapy.

SUMMARY

The present disclosure provides novel compounds as well as uses, methods, and compositions of treating, or reducing the risk of, HIV-infection. The present uses, methods, and compositions utilize the compounds according to the formula:

4 (I) wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom;

each of the 1, 3, 4, 5, and 6 positions on Ring A may be independently selected from H, or a group containing 1-6 carbon atoms;

each of the 1, 2, and 4 positions on Ring B may be independently selected from H, or a group containing 1-6 carbon atoms; and each of the 1, 2, 3, and 4 positions on Ring C may be independently selected from H, or a group containing 1-6 carbon atoms. An embodiment of the present disclosure provides uses, methods, and compositions of treating, or reducing the risk of, HIV-infection. The present uses, methods, and compositions utilize the compounds according to the formula:

wherein X is a halogen such as CI.

As used herein, "HIV" refers to human immunodeficiency virus, the lentivirus that causes acquired immunodeficiency syndrome (AIDS). The HIV virus has two known forms HIV-1 and HIV-2. HIV-1 is the most prevalent accounting for approximately 80% of all HIV infections.

As used herein, "treatment" refers to the remediation or amelioration of the symptoms and/ or underlying causes of a disease.

As used herein, "microbiocide" refers to agents that disinfect by destroying, neutralizing, or otherwise inhibiting the growth of disease-carrying microorganisms.

This summary does not necessarily describe all features of the invention. Other aspects, features and advantages of the invention will be apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the structure of various compounds according to the present disclosure including 4-chloro-3-{[(2,5-dimemylphenyl)amino] sulfonyl}-N-(2- pyridinylmethyl) benzamide (Compound 1).

Figure 2 shows the inhibition of HIV infection by 4-chloro-3-{[(2,5- dimemylphenyl)aniino]sulfonyl}-N-(2-pyridinylmethyl) benzamide. C8166 cells were infected with VSV-G pseudotyped HIV-1 (pNL4.3VSV-G/luc+)virus (100 pg of HIV p24) in the presence or absence of the compound. At 48 hrs post-infection equal amounts of cells were harvested and the luciferase activity was measured.

Figure 3 shows the effect of 4-cUoro-3-{[(2,5-dimetJiylphenyl)amino]sulfonyl}- N-(2-pyridinylmethyl) benzamide on the wild type HIV infection. C8166 cells were infected with a HIV-1 (pNL4.3-GFP virus) (100 pg of HIV p24) in the presence (6) or absence (7) of the compound. Cells were infected with HIV in the presence of the compound which was either removed after two hours or maintained in the culture during viral infection. After 4-days of infection and the levels of HIV-infected (GFP-positive) cells and HlV-induced syncytia formation were counted.

Figure 4 shows that 4-chloro-3-{[(2,5-dimethylphenyl)amino]sulfonyl}-N-(2- pyridinylmethyl) benzamide blocked HIV-1 infection when it is administered after 2 hours of infection. C8166 cells were infected with HIV-1 virus (pNL4.3-GFP) in the presence or absence of the compound. Cells were infected with HIV for two hours the compound was then added to the culture. HIV infection at different time intervals was monitored by the measurement of HIV p24 production and the presence of HIV infected (GFP-positive) cells.

Figure 5 shows an in-vitro HIV-1 reverse transcription assay, a HIV-1 integration assay, and a HIV-1 DNA synthesis analysis. The results indicate Comp. 1 specifically inhibits reverse transcriptase, but not integrase

Figure 6 shows different modifications of Comp. 1 to attenuate anti-HlV activity. HIV p24 ELISA (OD₄₅₀) assays for 3 and 6 days post-infection in the presence of various compounds are shown.

Figure 7 shows the effect of changing CI of compound 1 for Br or F. AH0110 (Br replacing CI) achieved a better anti-HIV effect , while F seems to decrease the anti- HIV activity.

Figure 8 shows the effect of AH 110 on W nuclear import and replication. A) C8166 T cells were infected with HTV pNL4.3 for 8 hrs and then AH 110 (5 ug/ml) was added. After 24 hrs, compound was maintained or removed from the medium (as indicated). At 48 hrs, HIV Gagp24 was measured in the supernatants. B) C8166 T cells were infected with HIV pNL4.3 for 8 hrs, then the compound was added and the cells were cultured until 20 hours and total HIV DNA (upper panel), 2-LTR cycles (middle panel) and integrated DNA (low panel) were measured by real-time PCR analysis. Results indicate that AH 110 may affect HIV nuclear translocation in addition to affecting reverse transcriptase, since the detected levels of 2-LTR DNA (a marker of nuclear import of viral DNA) was significantly reduced even large amount of reverse transcripted viral DNA present in the cells

Figure 9 shows the effect of AH0110 at inhibiting different drug resistant viruses.

C8166 T cells were infected with different drug resistant HIV-1 in the presence or absence of AH0110. After 4 days post-infection, HIV infection status was monitored by measurement of the levels of HIVp24 antigen in the supernatant. Results indicate that AH0110 is able to inhibit AZT-, 3TC- and Reltagravir resistant HIV viruses.

DETAILED DESCRIPTION

The present disclosure provides the use of a compound according to Formula I for the treatment of viral infection. For example, the compound according to Formula I may be used for the treatment of HIV infection. The present compounds may be used for treating a variety of retroviruses or other viruses whose replication requires reverse transcription such as Hepadnaviruses (e.g. Hepatitis B).

The present disclosure provides the use of a compound according to Formula I for the manufacture of a medicament. For example, the medicament may be for the treatment and prevention of HIV infection.

The present disclosure provides a composition comprising a compound according to Formula I and an anti-viral agent. For example, the anti-viral agent may be selected from such nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV integrase inhibitors, and/ or anti-HIV siRNA approaches.

The present disclosure provides a method for the treatment of HIV infection, said method comprising

to a HIV-infected subject a suitable dose of a compound according to Formula I.

The present disclosure provides a composition comprising a compound according to Formula I to constitute an anti-HIV component in a microbiocide which can be used, for example, by females to block the sexual transmission of HIV.

The present disclosure provides a prophylactic method for reducing the likelihood of HIV infection, said method comprising administering to a subject at risk of HIV infection a suitable dose of a compound according to Formula I. The present disclosure provides a method of reducing the risk of HIV- transmission from, for example, a nursing mother to her baby.

The present disclosure provides a composition comprising a therapeutically effective amount of a compound according to Formula I and a pharmaceutically acceptable carrier.

The present disclosure provides a solid composition comprising a therapeutically effective amount of a compound according to Formula I and a pharmaceutically acceptable carrier.

The present disclosure provides a liquid composition comprising a therapeutically effective amount of a compound according to Formula I and a pharmaceutically acceptable carrier.

The present disclosure provides a compound having the formula:

⁴ (I) wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom;

each of the 1, 3, 4, 5, and 6 positions on Ring A may be independently selected from H, or a group containing 1 -6 carbon atoms;

each of the 1, 2, and 4 positions on Ring B may be independently selected from H, or a group containing 1-6 carbon atoms; and each of the 1, 2, 3, and 4 positions on Ring C may be independendy selected from H, or a group containing 1-6 carbon atoms.

X may be selected from any suitable halogen such as, for example, CI, F, or Br. X may be CI.

Y may be selected from carbon or any suitable heteroatom such as, for example, N, S, O, or P. Y may be selected from carbon or nitrogen.

Each of the 1, 3, 4, 5, and 6 positions on Ring A may be independendy selected from H or a group containing 1-6 carbon atoms. It is preferred that at least one of positions 1, 3, 4, 5, and 6 is H. More preferably at least two of positions 1, 3, 4, 5, and 6 are H. More preferably at least three of positions 1, 3, 4, 5, and 6 are H. It is preferred that at least one of positions 1, 3, 4, 5, and 6 is a C1-C4 alkyl group. More preferably at least two of positions 1, 3, 4, 5, and 6 are C1-C4 alkyl group. Positions 1 and 4 on Ring A n ay, for example, be selected from groups containing 1-4 carbon atoms such as, methyl, ethyl, methyl alcohol, ethyl alcohol and the like. Positions 3, 5, and 6 on the ring may, for example, be H.

Each of the 1, 2, and 4 positions on Ring B may be independently selected from H or a group containing 1-6 carbon atoms. Preferably at least two of positions 1, 2, and 4 are H. More preferably positions 1, 2, and 4 are H.

Each of the 1, 2, 3, and 4 positions on Ring C may be independendy selected from H or a group containing 1-6 carbon atoms. Position 4 may, for example, be selected from groups containing 1-4 carbon atoms such as, methyl, ethyl, methyl alcohol, ethyl alcohol, CF₃, and the like. Two of positions 1, 2, 3 and 4 may be linked through a group containing 3-6 carbon atoms. For example, position 1, 2, 3 and 4 may be linked by propyl, butyl, pentyl, or hexyl group. The groups may contain one or more heteroatoms such as N, S, O, or P. For example, positions 1, 2, 3 and 4 may be linked by a -0-C-0-, - O-C-C-O-, -C-0-C-, or the like. Preferably at least two of positions 1, 2, 3, and 4 are H. Preferably at least three of positions 1, 2, 3, and 4 are H. Preferably, all of positions 1, 2, 3 and 4.

The group linking Ring A to Ring B may be altered in various ways. For example, the N and/ or S may be substituted for another heteroatom or for carbon.

The group linking Ring B to Ring C may be altered in various ways. For example, the N may be substituted for another heteroatom or for carbon. The present disclosure provides a compound having the formula (I) :

(I) wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom;

each of the 1, 3, 4, 5, and 6 positions on Ring A may be independently H, or a group containing 1-6 carbon atoms except that position 1 and 4 cannot both be a CH₃ group;

each of the 1, 2, and 4 positions on Ring B may be independently H, or a group containing 1-6 carbon atoms; and

each of the 1, 2, 3, and 4 positions on Ring C may be independently H, or a group containing 1-6 carbon atoms.

The present disclosure provides a compound having the formula (I) :

(I) wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom; each of the 1, 3, 5, and 6 positions on Ring A may be independendy H, or a group containing 1-6 carbon atoms;

position 4 of Ring A is H, or a group containing 2-6 carbon atoms;

each of the 1, 2, and 4 positions on Ring B may be independendy H, or a group containing 1-6 carbon atoms; and

each of the 1, 2, 3, and 4 positions on Ring C may be independendy H, or a group containing 1-6 carbon atoms.

The present disclosure provides a compound having the formula (I) :

wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom;

each of the 1 and 4 positions on Ring A may be independendy H, or a group containing 1-6 carbon atoms;

each of the 3, 5 and 6 positions on Ring A may be independendy H, or a group containing 1-6 carbon atoms provided at least one of positions 3, 5, or 6 is a group containing 1-6 carbon atoms;

each of the 1, 2, and 4 positions on Ring B may be independendy H, or a group containing 1-6 carbon atoms; and each of the 1, 2, 3, and 4 positions on Ring C may be independendy H, or a group containing 1-6 carbon atoms.

The present compound may be 4-[halo]-3-{[(2,5- a^ethylphenyl)amino]sulfonyl}-N-(2-pyridinylmethyl) benzamide. For example, the present compound may be 4-chloro-3-{[(2,5-a½iemylphenyl)amino]sulfonyl}-N-(2- pyridinylmethyl) benzamide, 4-bromo-3- { [(2,5-dimemylphenyl)amino] sulfonyl} -N-(2- pyridinylmethyl) benzamide, 4-fluro-3- { [(2,5-dimemylphenyl)amino] sulfonyl} -N-(2- pyridinylmethyl) benzamide.

The present disclosure provides different salts, hydrates, clathrates, solvates, acids etc. of compounds herein.

The compounds of the present disclosure may be formulated in a wide variety of oral administration dosage forms and carriers. Oral adrriinistration can be in the form of tablets, coated tablets, dragees, hard and soft gelatine capsules, solutions, emulsions, syrups, or suspensions. Compounds of the present disclosure may be efficacious when administered by other routes such as, for example, continuously (e.g. intravenous drip), topically, parenterally, intramuscularly, intravenously, subcutaneously, transdermally (which may include a penetration enhancement agent), buccally, nasally, via inhalation and/ or suppository administration, among other routes of administration.

A compound or compounds of the present disclosure, as well as their pharmaceutically useable salts, together with one or more conventional excipients, carriers, or diluents, may be placed into the form of pharmaceutical compositions and unit dosages. The pharmaceutical compositions and unit dosage forms may be comprised of conventional ingredients in conventional proportions, with or without additional active ingredients, and the unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended dosage regimen to be employed. The pharmaceutical compositions may be employed as solids, such as tablets or filled capsules, semisolids, powders, sustained release formulations, or liquids such as solutions, suspensions, emulsions, elixirs, or filled capsules for oral use; or in the form of suppositories for rectal or vaginal administration (including a microbiocide as a feasible method for women to protect themselves against HIV sexual transmission), or in the form of sterile injectable solutions for parenteral use. A typical preparation will contain from about 5% to about 95% active compound or compounds (w/w). The term "preparation" or "dosage form" is intended to include both solid and liquid formulations of the active compound and one skilled in the art will appreciate that an active ingredient can exist in different preparations depending on the target organ or tissue and on the desired dose and pharmacokinetic parameters.

The term "excipient" as used herein refers to a compound that is useful in preparing a pharmaceutical composition, generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes excipients that are acceptable for veterinary use as well as human pharmaceutical use. The term "excipient" as used herein includes both one and more than one such excipient.

Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier may be one or more substances which may also act as diluents, flavouring agents, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, and encapsulating materials. In powders, the carrier generally is a finely divided solid which is a mixture with the finely divided active component. In tablets, the active component generally is mixed with the carrier having the necessary bmding capacity in suitable proportions and compacted in the shape and size desired. Suitable carriers include, but are not limited to, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, wax, cocoa butter, and the like. Solid form preparations may contain, in addition to the active component, colorants, flavours, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

Liquid formulations also are suitable for oral administration include liquid formulation including emulsions, syrups, elixirs, aqueous solutions, aqueous suspensions. These include solid form preparations which are intended to be converted to liquid form preparations shortly before use.

Emulsions may be prepared in solutions, for example, in aqueous propylene glycol solutions or may contain emulsifying agents such as lecithin, sorbitan monooleate, or acacia. Aqueous solutions may be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilizing, and thickening agents. Aqueous suspensions may be prepared by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents.

The compounds of the present disclosure may be formulated for parenteral administration (e. g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion, or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.

When desired, formulations can be prepared with enteric coatings adapted for sustained or controlled release administration of the active ingredient. For example, the compounds of the present disclosure can be formulated in transdermal or subcutaneous drug delivery devices. These delivery systems are advantageous when sustained release of the compound is necessary and where patient compliance with a treatment regimen is important.

Suitable formulations along with pharmaceutical carriers, diluents and excipients are described in Remington : The Science and Practice of Pharmacy 2005, edited by Philip P. Gerbino, Lippincott Williams & Wilkins, 21st edition. A skilled formulation scientist may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration without rendering the compositions of the present disclosure unstable or compromising their therapeutic activity.

The modification of the present compounds to render them more soluble in water or other vehicle, for example, may be easily accomplished by minor modifications (salt formulation, esterification, etc.), which are well within the ordinary skill in the art. It is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in patients.

The term "therapeutically effective amount" as used herein means an amount required to significantly inhibit viral load in vivo and to reduce related symptoms of the disease in an individual. The dose may be adjusted to the individual requirements in each particular case. That dosage can vary within wide limits depending upon numerous factors such as the severity of the disease to be treated, the age and general health condition of the patient, other medicaments with which the patient is being treated, the route and form of administration and the preferences and experience of the medical practitioner involved.

In embodiments of the disclosure, the active compound or a salt can be administered in combination with other antiviral agents, such as nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, HIV protease inhibitors, HIV fusion inhibitors, HIV integrase inhibitors, and/or anti-HIV siRNA approach. When the active compound or its derivative or salt are administered in combination with another antiviral agent the activity may be increased over the parent compound, and reduced frequency for drug resistance of the virus. When the treatment is combination therapy, such administration may be concurrent or sequential with respect to that of the nucleoside derivatives. Administration of two or more agents at the same time can be achieved by a single formulation containing two or more active ingredients or by substantially simultaneous administration of two or more dosage forms with a single active agent.

It will be understood that references herein to treatment extend to prophylaxis as well as to the treatment of existing conditions. Furthermore, treatment of a HIV infection, as used herein, also includes treatment or prophylaxis of a disease or a condition associated with or mediated by HIV infection, or the clinical symptoms thereof

The term "solvate" as used herein means a compound of the disclosure or a salt, thereof, that further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. Preferred solvents are volatile, non-toxic, and/ or acceptable for administration to humans in trace amounts.

The term "hydrate" as used herein means a compound of the disclosure or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

The term "clafhrate" as used herein means a compound of the disclosure or a salt thereof in the form of a crystal lattice that contains spaces (e. g., channels) that have a guest molecule (e. g. , a solvent or water) trapped within

It is contemplated that any embodiment discussed in this specification can be implemented or combined with respect to any other embodiment, method, composition or aspect of the invention, and vice versa.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise specified, all patents, applications, published applications and other publications referred to herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference. Citation of references herein is not to be construed nor considered as an admission that such references are prior art to the present invention.

Use of examples in the specification, including examples of terms, is for illustrative purposes only and is not intended to limit the scope and meaning of the embodiments of the invention herein. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to," and the word "comprises" has a corresponding meaning.

The use of "a" or "an" entity herein refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms "a" (or "an"), "one or more", and "at least one" can be used interchangeably herein.

The invention includes all embodiments, modifications and variations substantially as hereinbefore described and with reference to the examples and figures. It will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims. Examples of such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way.

The present invention will be further illustrated in the following examples. However it is to be understood that these examples are for illustrative purposes only, and should not be used to limit the scope of the present invention in any manner.

EXAMPLES

A sample of each of the compounds shown in Figure 1 was obtained

(ChemBridge Corp., San Diego CA, USA). Each compound was dissolved in DMSO at a stock concentration of 5mg/ ml. A HIV-1 p24 ELISA Kit was purchased from the AIDS Vaccine Program of the Frederick Cancer Research and Development Center. A

Luciferase assay system was purchased from Promega Com. The CD4⁺ C8166 T cell line was maintained in RPMI-1640 medium containing 10% FCS and 1% penicillin and streptomycin. To generate pNL4.3 GFP+/env+ virus stock, the HIV-1 pNL4.3-GFP proviral plasmid was transfected into 293T cells. The vesicular stomatitis virus G (VSV-G) glycoprotein pseudotyped single cycle replicating virus (pNL4.3VSV-G/Luc+) was produced in 293T by co-transfected HIV-1 provirus pNL4.3ABgl/Luc+ with a VSV-G expresser. Supernatants were collected at 48 h post-transfection and subjected to ultra- centrifugation (32,000 rpm for 1 hour at 4°C) to concentrate virus particles. The virus quantity was determined by measuring the Gag-p24 in HIV-1 p24 ELISA Kit.

To infect CD4+ C8166 T cells, viruses (100pg of HIVp24) were incubated with

0.5xl0⁶ cells at 37°C for 2 hours in the presence or absence of the compound. Then, the cells were washed and incubated with fresh medium in the presence or absence of the compound. At different time points, viral production levels were monitored by measurement of HIV-1 Gag-p24 antigen in each infected culture supernatant by HIV-1 Gag-p24 ELISA. To evaluate the infection mediated by pNL4.3-GFP HIV-1 virus, infected cells were fixed with PBS-4% Para formaldehyde and observed under fluorescence microscopy. The HIV syncytia formation was observed under microscopy. To check the virus induced luciferase activity, lxlO⁶ cells from each sample were collected at 48 h post-infection and lysed in 50 μΐ of luciferase lysis buffer (Promega). 10 μΐ of cell lysate were subjected to the luciferase assay by using a POLARstar OPTIMA (BMG LABTECH, Germany).

It was observed that the presence of the compound was able to completely block HIV infection after cells were infected with a VSV-G pseudotyped pNL4.3-Luc virus (Fig. 2) and the wild type HIV (pNL-4.3-GFP virus) (Fig. 3 and 4). The ID50 dose for VSV-G pseudotyped pNL4.3-Luc virus was less than 1.25 ug/ml (Fig. 2). Also, the results showed that the compound was able to block HIV infection when compound was added after two hours of HIV infection (Figure 3 and 4). These results indicate that 1) the compound is effective even after virus enters into the cells; and 2) the compound acts on the early stage of HIV replication and may target one critical step required for establishment of HIV infection, which may, for example, be the reverse transcription, nuclear import or integration step.

Compounds 2, 3, 4, and 5 also had an inhibitory effect on HIV infection although the effect was less marked than with Compound 1.

Claims

1. Use of a com ound having the formula (I) for the treatment of HIV infection:

(I) wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom;

each of the 1, 3, 4, 5, and 6 positions on Ring A may be independently H, or a group containing 1-6 carbon atoms;

A use according to Claim 1 wherein X is CI.

Use of a compound having the formula (I) for reducing the risk of a subject becomin infected with HIV:

wherein;

X is a halogen; Y may be a hetero atom or a carbon atom;

each of the 1, 3, 4, 5, and 6 positions on Ring A may be independendy H, or a group containing 1-6 carbon atoms;

of a compound having the formula (I) for the manufacture of a medicament:

4 (I) wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom;

each of the 1 , 2, and 4 positions on Ring B may be independendy H, or a group containing 1-6 carbon atoms; and

each of the 1 , 2, 3, and 4 positions on Ring C may be independendy H, or a group containing 1-6 carbon atoms.

A use according to Claim 4 wherein the medicament is a treatment or prophylactic for HIV infection.

A composition comprising a pharmaceutically acceptable carrier and a compound according to formula (I):

wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom;

7. A composition according to Claim 6 wherein the pharmaceutically acceptable carrier is suitable for rectal and/ or vaginal application.

A method of treating HIV infection said method comprising administering to a HIV-infected sub ect a compound according to the formula I:

⁴ (I) wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom;

9. A method of reducing the risk of HIV infection, said method comprising administering to a subject at risk of HIV infection a compound according to the formula I:

X is a halogen;

Y may be a hetero atom or a carbon atom;

each of the 1, 3, 4, 5, and 6 positions on Ring A may be independendy H, or a group containing 1 -6 carbon atoms;

A compound having the formula (I) :

4 (I) wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom;

11. A com ound having the formula (I) :

4 (I) wherein;

X is a halogen;

Y may be a hetero atom or a carbon atom;

each of the 1, 3, 5, and 6 positions on Ring A may be independendy H, or a group containing 1-6 carbon atoms;

position 4 of Ring A is H, or a group containing 2-6 carbon atoms; each of the 1, 2, and 4 positions on Ring B may be independendy H, or a group containing 1-6 carbon atoms; and

each of the 1, 2, 3, and 4 positions on Ring C may be independendy H, or a group containing 1-6 carbon atoms,

com ound having the formula (I) :

4 (I) wherein;

X is a halogen; Y may be a hetero atom or a carbon atom; each of the 1 and 4 positions on Ring A may be independendy H, or a group containing 1-6 carbon atoms; each of the 3, 5 and 6 positions on Ring A may be independendy H, or a group containing 1-6 carbon atoms provided at least one of positions 3, 5, or 6 is a group containing 1-6 carbon atoms; each of the 1, 2, and 4 positions on Ring B may be independendy H, or a group containing 1-6 carbon atoms; and each of the 1, 2, 3, and 4 positions on Ring C may be independendy H, or a group containing 1 -6 carbon atoms. Use of a com ound having the formula:

wherein X is a halogen; for the treatment of HIV infection.

14. A use according to Claim 13 wherein X is CI.

15. Use of a com ound having the formula:

wherein X is a halogen; for reducing the risk of a subject becoming infected with HIV.

Use of a com ound having the formula:

wherein X is a halogen; for the manufacture of a medicament.

17. A use according to Claim 16 wherein the medicament is a treatment or prophylactic for HIV infection.

18. A composition comprising a pharmaceutically acceptable carrier and a compound having the formula:

wherein X is a halogen

19. A composition according to Claim 17 wherein the pharmaceutically acceptable carrier is suitable for rectal and/ or vaginal application.

20. A method of treating HIV infection said method comprising administering to a HIV-infected sub ect a compound according to the formula I:

wherein X is a halogen.

A method of reducing the risk of HIV infection, said method comprising administering to a subject at risk of HIV infection a compound according to the formula I:

wherein X is a halogen