Betulinic acid derivatives with HIV maturation inhibitory activity

Compounds having drug and bio-affecting properties, their pharmaceutical compositions and methods of use are set forth. In particular, betulinic acid derivaties that possess unique antiviral activity are provided as HIV maturation inhibitors, as represented by compounds of Formulas I, II and III:These compounds are useful for the treatment of HIV and AIDS.

FIELD OF THE INVENTION

The present invention relates to novel compounds useful against HIV and, more particularly, to compounds derived from betulinic acid and other structurally-related compounds which are useful as HIV maturation inhibitors, and to pharmaceutical compositions containing same, as well as to methods for their preparation.

BACKGROUND OF THE INVENTION

Each of these drugs can only transiently restrain viral replication if used alone. However, when used in combination, these drugs have a profound effect on viremia and disease progression. In fact, significant reductions in death rates among AIDS patients have been recently documented as a consequence of the widespread application of combination therapy. However, despite these impressive results, 30 to 50% of patients may ultimately fail combination drug therapies. Insufficient drug potency, non-compliance, restricted tissue penetration and drug-specific limitations within certain cell types (e.g. most nucleoside analogs cannot be phosphorylated in resting cells) may account for the incomplete suppression of sensitive viruses. Furthermore, the high replication rate and rapid turnover of HIV-1 combined with the frequent incorporation of mutations, leads to the appearance of drug-resistant variants and treatment failures when sub-optimal drug concentrations are present. Therefore, novel anti-HIV agents exhibiting distinct resistance patterns, and favorable pharmacokinetic as well as safety profiles are needed to provide more treatment options. Improved HIV fusion inhibitors and HIV entry coreceptor antagonists are two examples of new classes of anti-HIV agents further being studied by a number of investigators.

HIV attachment inhibitors are a further subclass of antiviral compounds that bind to the HIV surface glycoprotein gp120, and interfere with the interaction between the surface protein gp120 and the host cell receptor CD4. Thus, they prevent HIV from attaching to the human CD4 T-cell, and block HIV replication in the first stage of the HIV life cycle. The properties of HIV attachment inhibitors have been improved in an effort to obtain compounds with maximized utility and efficacy as antiviral agents. In particular, U.S. Pat. No. 7,354,924 and U.S. Pat. No. 7,745,625 are illustrative of HIV attachment inhibitors.

Another emerging class of compounds for the treatment of HIV are called HIV maturation inhibitors. Maturation is the last of as many as 10 or more steps in HIV replication or the HIV life cycle, in which HIV becomes infectious as a consequence of several HIV protease-mediated cleavage events in the gag protein that ultimately results in release of the capsid (CA) protein. Maturation inhibitors prevent the HIV capsid from properly assembling and maturing, from forming a protective outer coat, or from emerging from human cells. Instead, non-infectious viruses are produced, preventing subsequent cycles of HIV infection.

Certain derivatives of betulinic acid have now been shown to exhibit potent anti-HIV activity as HIV maturation inhibitors. For example, U.S. Pat. No. 7,365,221 discloses monoacylated betulin and dihydrobetuline derivatives, and their use as anti-HIV agents. As discussed in the '221 reference, esterification of betulinic acid (1) with certain substituted acyl groups, such as 3′,3′-dimethylglutaryl and 3′,3′-dimethylsuccinyl groups produced derivatives having enhanced activity (Kashiwada, Y., et al., J. Med. Chem. 39:1016-1017 (1996)). Acylated betulinic acid and dihydrobetulinic acid derivatives that are potent anti-HIV agents are also described in U.S. Pat. No. 5,679,828. Esterification of the hydroxyl in the 3 carbon of betulin with succinic acid also produced a compound capable of inhibiting HIV-1 activity (Pokrovskii, A. G., et al., “Synthesis of derivatives of plant triterpenes and study of their antiviral and immunostimulating activity,” Khimiya y Interesakh Ustoichivogo Razvitiya, Vol. 9, No. 3, pp. 485-491 (2001) (English abstract).

Other references to the use of treating HIV infection with compounds derived from betulinic acid include US 2005/0239748 and US 2008/0207573, as well as WO2006/053255, WO2009/100532 and WO2011/007230.

One HIV maturation compound that has been in development has been identified as Bevirimat or PA-457, with the chemical formula of C36H56O6and the IUPAC name of 3β-(3-carboxy-3-methyl-butanoyloxy) 1up-20(29)-en-28-oic acid.

What is now needed in the art are new compounds which are useful as HIV maturation inhibitors, as well as new pharmaceutical compositions containing these compounds.

SUMMARY OF THE INVENTION

The present invention provides compounds of Formulas I, II and III below, including pharmaceutically acceptable salts thereof, their pharmaceutical formulations, and their use in patients suffering from or susceptible to a virus such as HIV. The compounds of Formulas I, II and III are effective antiviral agents, particularly as inhibitors of HIV. They are useful for the treatment of HIV and AIDS.

One embodiment of the present invention is directed to a compound, including pharmaceutically acceptable salts thereof, which is selected from the group of:

a compound of formula I

a compound of formula II

and a compound of formula III

with the proviso that only one of R8or R9can be —COOR3;
R10and R11are independently selected from the group of —H, —C1-6alkyl, —C1-6substituted alkyl and —C3-6cycloalkyl;
R12is selected from the group of —C1-6alkyl, —C1-6alkyl-OH; —C1-6alkyl, —C1-6substituted alkyl, —C3-6cycloalkyl, and —COR7;
R13and R14are independently selected from the group of —H, —C1-6alkyl, —C3-6cycloalkyl, —C1-6substituted alkyl, —C1-6alkyl-Q3, —C1-6alkyl-C3-6cycloalkyl-Q3, and C1-6substituted alkyl-Q3;
Q3is selected from the group of -heteroaryl, substituted heteroaryl, —NR18R19, —CONR2R2, —COOR2, —OR2, and —SO2R3;
R15is selected from the group of —C1-6alkyl, —C3-6cycloalkyl, —C1-6substituted alkyl, —C1-6alkyl-Q3, —C1-6alkyl-C3-6cycloalkyl-Q3and —C1-6substituted alkyl-Q3;
R16is selected from the group of —H, —C1-6alkyl, —NR2R2, and —COOR3;
R17is selected from the group of —H, —C1-6alkyl, —COOR3, and -aryl;
R18and R19are independently selected from the group of —H, —C1-6alkyl, —C1-6substituted alkyl, —C1-6substituted alkyl-OR2, and —COR3;
R20and R21are independently selected from the group of —H, —C1-6alkyl, —C1-6substituted alkyl, aryl, heteroaryl, substituted aryl, substituted heteroaryl, —C1-6alkyl-Q2, and —COOR3,
or R20and R21are taken together with the adjacent N to form a cycle selected from the group of:

and
R22is selected from H and —COR3.

In a further embodiment, there is provided a method for treating mammals infected with a virus, especially wherein said virus is HIV, comprising administering to said mammal an antiviral effective amount of a compound which is selected from the group of compounds of Formulas I, II and III, and one or more pharmaceutically acceptable carriers, excipients or diluents. Optionally, the compound of Formulas I, II and III can be administered in combination with an antiviral effective amount of another-AIDS treatment agent selected from the group consisting of: (a) an AIDS antiviral agent; (b) an anti-infective agent; (c) an immunomodulator; and (d) other HIV entry inhibitors.

Another embodiment of the present invention is a pharmaceutical composition comprising one or more compounds of Formulas I, II, and III, and one or more pharmaceutically acceptable carriers, excipients, and/or diluents; and optionally in combination with another AIDS treatment agent selected from the group consisting of: (a) an AIDS antiviral agent; (b) an anti-infective agent; (c) an immunomodulator; and (d) other HIV entry inhibitors.

In another embodiment of the invention there is provided one or more methods for making the compounds of Formulas I, II, and III herein.

Also provided herein are intermediate compounds useful in making the compounds of Formulas I, II and III herein.

The present invention is directed to these, as well as other important ends, hereinafter described.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Since the compounds of the present invention may possess asymmetric centers and therefore occur as mixtures of diastereomers, the present disclosure includes the individual diastereoisomeric forms of the compounds of Formulas I, II and III in addition to the mixtures thereof.

Definitions

Unless otherwise specifically set forth elsewhere in the application, one or more of the following terms may be used herein, and shall have the following meanings:

“H” refers to hydrogen, including its isotopes, such as deuterium.

The term “C1-6alkyl” as used herein and in the claims (unless specified otherwise) mean straight or branched chain alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, amyl, hexyl and the like.

“C1-C4fluoroalkyl” refers to F-substituted C1-C4alkyl wherein at least one H atom is substituted with F atom, and each H atom can be independently substituted by F atom;

As used herein, a “heteroaryl” group refers to a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Unless otherwise indicated, the heteroaryl group may be attached at either a carbon or nitrogen atom within the heteroaryl group. It should be noted that the term heteroaryl is intended to encompass an N-oxide of the parent heteroaryl if such an N-oxide is chemically feasible as is known in the art. Examples, without limitation, of heteroaryl groups are furyl, thienyl, benzothienyl, thiazolyl, imidazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, benzothiazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, pyrrolyl, pyranyl, tetrahydropyranyl, pyrazolyl, pyridyl, pyrimidinyl, quinolinyl, isoquinolinyl, purinyl, carbazolyl, benzoxazolyl, benzimidazolyl, indolyl, isoindolyl, pyrazinyl. diazinyl, pyrazine, triazinyl, tetrazinyl, and tetrazolyl. When substituted the substituted group(s) is preferably one or more selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroalicycloxy, thioalkoxy, thiohydroxy, thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy, cyano, halogen, nitro, carbonyl, O-carbamyl, N-carbamyl, C-amido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, sulfonamido, trihalomethyl, ureido, amino, and —NRxRy, wherein Rxand Ryare as defined above.

As used herein, a “heteroalicyclic” group refers to a monocyclic or fused ring group having in the ring(s) one or more atoms selected from the group consisting of nitrogen, oxygen and sulfur. Rings are selected from those which provide stable arrangements of bonds and are not intended to encompass systems which would not exist. The rings may also have one or more double bonds. However, the rings do not have a completely conjugated pi-electron system. Examples, without limitation, of heteroalicyclic groups are azetidinyl, piperidyl, piperazinyl, imidazolinyl, thiazolidinyl, 3-pyrrolidin-1-yl, morpholinyl, thiomorpholinyl and its S oxides and tetrahydropyranyl. When substituted the substituted group(s) is preferably one or more selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroalicycloxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy, cyano, halogen, nitro, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, C-thioamido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, silyl, guanyl, guanidino, ureido, phosphonyl, amino and —NRxRy, wherein Rxand Ryare as defined above.

An “alkyl” group refers to a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms (whenever a numerical range; e.g., “1-20”, is stated herein, it means that the group, in this case the alkyl group may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc. up to and including 20 carbon atoms). More preferably, it is a medium size alkyl having 1 to 10 carbon atoms. Most preferably, it is a lower alkyl having 1 to 4 carbon atoms. The alkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more individually selected from trihaloalkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, heteroaryloxy, heteroalicycloxy, thiohydroxy, thioalkoxy, thioaryloxy, thioheteroaryloxy, thioheteroalicycloxy, cyano, halo, nitro, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, C-thioamido, N-amido, C-carboxy, O-carboxy, sulfinyl, sulfonyl, sulfonamido, trihalomethanesulfonamido, trihalomethanesulfonyl, and combined, a five- or six-member heteroalicyclic ring.

An “alkenyl” group refers to an alkyl group, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond.

An “alkynyl” group refers to an alkyl group, as defined herein, having at least two carbon atoms and at least one carbon-carbon triple bond.

A “hydroxy” group refers to an —OH group.

An “alkoxy” group refers to both an —O-alkyl and an —O-cycloalkyl group as defined herein.

An “aryloxy” group refers to both an —O-aryl and an —O-heteroaryl group, as defined herein.

A “heteroaryloxy” group refers to a heteroaryl-O— group with heteroaryl as defined herein.

A “heteroalicycloxy” group refers to a heteroalicyclic-O— group with heteroalicyclic as defined herein.

A “thiohydroxy” group refers to an —SH group.

A “thioalkoxy” group refers to both an S-alkyl and an —S-cycloalkyl group, as defined herein.

A “thioaryloxy” group refers to both an —S-aryl and an —S-heteroaryl group, as defined herein.

A “thioheteroaryloxy” group refers to a heteroaryl-S— group with heteroaryl as defined herein.

A “thioheteroalicycloxy” group refers to a heteroalicyclic-S— group with heteroalicyclic as defined herein.

A “carbonyl” group refers to a —C(═O)—R″ group, where R″ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon), as each is defined herein.

An “aldehyde” group refers to a carbonyl group where R″ is hydrogen.

A “thiocarbonyl” group refers to a —C(═S)—R″ group, with R″ as defined herein.

A “keto” group refers to a —CC(═O)C— group wherein the carbon on either or both sides of the C═O may be alkyl, cycloalkyl, aryl or a carbon of a heteroaryl or heteroalicyclic group.

A “trihalomethanecarbonyl” group refers to a Z3CC(═O)— group with said Z being a halogen.

A “C-carboxy” group refers to a —C(═O)O—R″ groups, with R″ as defined herein.

An “O-carboxy” group refers to a R″C(—O)O-group, with R″ as defined herein.

A “carboxylic acid” group refers to a C-carboxy group in which R″ is hydrogen.

A “trihalomethyl” group refers to a —CZ3, group wherein Z is a halogen group as defined herein.

A “trihalomethanesulfonyl” group refers to an Z3CS(═O)2— groups with Z as defined above.

A “trihalomethanesulfonamido” group refers to a Z3CS(═O)2NRx— group with Z as defined above and Rxbeing H or (C1-6)alkyl.

A “sulfinyl” group refers to a —S(═O)—R″ group, with R″ being (C1-6)alkyl.

A “sulfonyl” group refers to a —S(═O)2R″ group with R″ being (C1-6)alkyl.

A “S-sulfonamido” group refers to a —S(═O)2NRXRY, with RXand RYindependently being H or (C1-6)alkyl.

A “N-sulfonamido” group refers to a R″S(═O)2NRx— group, with Rxbeing H or (C1-6)alkyl.

A “O-carbamyl” group refers to a —OC(═O)NRxRygroup, with RXand RYindependently being H or (C1-6)alkyl.

A “N-carbamyl” group refers to a RxOC(═O)NRygroup, with Rxand Ryindependently being H or (C1-6)alkyl.

A “O-thiocarbamyl” group refers to a —OC(═S)NRxRygroup, with Rxand Ryindependently being H or (C1-6)alkyl.

A “N-thiocarbamyl” group refers to a RxOC(═S)NRy— group, with Rxand Ryindependently being H or (C1-6)alkyl.

An “amino” group refers to an —NH2group.

A “C-amido” group refers to a —C(═O)NRxRygroup, with Rxand Ryindependently being H or (C1-6)alkyl.

A “C-thioamido” group refers to a —C(═S)NRxRygroup, with Rxand Ryindependently being H or (C1-6)alkyl.

A “N-amido” group refers to a RxC(═O)NRy— group, with Rxand Ryindependently being H or (C1-6)alkyl.

An “ureido” group refers to a —NRxC(═O)NRyRy2group, with Rx, Ry, and Ry2independently being H or (C1-6)alkyl.

A “guanidino” group refers to a —RxNC(═N)NRyRy2group, with Rx, Ry, and Ry2independently being H or (C1-6)alkyl.

A “amidino” group refers to a RxRyNC(═N)— group, with Rxand Ryindependently being H or (C1-6)alkyl.

A “cyano” group refers to a —CN group.

A “silyl” group refers to a —Si(R″)3, with R″ being (C1-6)alkyl or phenyl.

A “phosphonyl” group refers to a P(═O)(ORx)2with Rxbeing (C1-6)alkyl.

A “hydrazino” group refers to a —NRxNRyRy2group, with Rx, Ry, and Ry2independently being H or (C1-6)alkyl.

A “4, 5, or 6 membered ring cyclic N-lactam” group refers to

A “spiro” group is a bicyclic organic group with rings connected through just one atom. The rings can be different in nature or identical. The connecting atom is also called the spiroatom, most often a quaternary carbon (“spiro carbon”).

An “oxospiro” or “oxaspiro” group is a spiro group having an oxygen contained within the bicyclic ring structure. A “dioxospiro” or “dioxaspiro” group has two oxygens within the bicyclic ring structure.

Any two adjacent R groups may combine to form an additional aryl, cycloalkyl, heteroaryl or heterocyclic ring fused to the ring initially bearing those R groups.

It is known in the art that nitrogen atoms in heteroaryl systems can be “participating in a heteroaryl ring double bond”, and this refers to the form of double bonds in the two tautomeric structures which comprise five-member ring heteroaryl groups. This dictates whether nitrogens can be substituted as well understood by chemists in the art. The disclosure and claims of the present disclosure are based on the known general principles of chemical bonding. It is understood that the claims do not encompass structures known to be unstable or not able to exist based on the literature.

Pharmaceutically acceptable salts and prodrugs of compounds disclosed herein are within the scope of the invention. The term “pharmaceutically acceptable salt” as used herein and in the claims is intended to include nontoxic base addition salts. Suitable salts include those derived from organic and inorganic acids such as, without limitation, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, acetic acid, tartaric acid, lactic acid, sulfinic acid, citric acid, maleic acid, fumaric acid, sorbic acid, aconitic acid, salicylic acid, phthalic acid, and the like. The term “pharmaceutically acceptable salt” as used herein is also intended to include salts of acidic groups, such as a carboxylate, with such counterions as ammonium, alkali metal salts, particularly sodium or potassium, alkaline earth metal salts, particularly calcium or magnesium, and salts with suitable organic bases such as lower alkylamines (methylamine, ethylamine, cyclohexylamine, and the like) or with substituted lower alkylamines (e.g. hydroxyl-substituted alkylamines such as diethanolamine, triethanolamine or tris(hydroxymethyl)-aminomethane), or with bases such as piperidine or morpholine.

As stated above, the compounds of the invention also include “prodrugs”. The term “prodrug” as used herein encompasses both the term “prodrug esters” and the term “prodrug ethers”.

As set forth above, the invention is directed to a compound, including pharmaceutically acceptable salts thereof, which is selected from the group of:

a compound of formula I

a compound of formula II

and a compound of formula III

with the proviso that only one of R8or R9can be —COOR3;
R10and R11are independently selected from the group of —H, —C1-6alkyl, —C1-6substituted alkyl and —C3-6cycloalkyl;
R12is selected from the group of —C1-6alkyl, —C1-6alkyl-OH; —C1-6alkyl, —C1-6substituted alkyl, —C3-6cycloalkyl, and —COR7;
R13and R14are independently selected from the group of —H, —C1-6alkyl, —C3-6cycloalkyl, —C1-6substituted alkyl, —C1-6alkyl-Q3, —C1-6alkyl-C3-6cycloalkyl-Q3, and C1-6substituted alkyl-Q3;
Q3is selected from the group of -heteroaryl, substituted heteroaryl, —NR18R19, —CONR2R2, —COOR2, —OR2, and —SO2R3;
R15is selected from the group of —C1-6alkyl, —C3-6cycloalkyl, —C1-6substituted alkyl, —C1-6alkyl-Q3, —C1-6alkyl-C3-6cycloalkyl-Q3and —C1-6substituted alkyl-Q3;
R16is selected from the group of —H, —C1-6alkyl, —NR2R2, and —COOR3;
R17is selected from the group of —H, —C1-6alkyl, —COOR3, and -aryl;
R18and R19are independently selected from the group of —H, —C1-6alkyl, —C1-6substituted alkyl, —C1-6substituted alkyl-OR2, and —COR3;
R20and R21are independently selected from the group of —H, —C1-6alkyl, —C1-6substituted alkyl, aryl, heteroaryl, substituted aryl, substituted heteroaryl, —C1-6alkyl-Q2, and —COOR3,
or R20and R21are taken together with the adjacent N to form a cycle selected from the group of:

and
R22is selected from H and —COR3.

More preferred compounds include those wherein R1is isopropenyl.

Also preferred are compounds wherein X is phenyl. It is also preferred that Y is —COOH.

In certain embodiments, it is preferred that the compound of the invention has the Formula I. In these embodiments, it is also preferred that E1is —CHOR22. More preferably, E1is —CHOH or is —CO or is —CHF.

In certain embodiments, it is preferred that the compound of the invention has the Formula II. In these embodiments, it is preferred that E2and E3are each —CHOR22. It is also preferred that E2and E3together form a ketal. Also preferred is the embodiment wherein R3is methyl.

In certain embodiments, it is preferred that the compound of the invention has the Formula III. In these embodiments, it is also preferred that W is —(CH2)0-1NR4R5.

Preferred compounds, including pharmaceutically acceptable salts thereof, as part of the invention including the following:

Preferred compounds, including pharmaceutically acceptable salts thereof, as part of the invention also include the following:

The compounds above represent the mixture of diastereoisomers, and the two individual disastereomers. In certain embodiments, one of the specific diastereomers may be particularly preferred.

The compounds of the present invention, according to all the various embodiments described above, may be administered orally, parenterally (including subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques), by inhalation spray, or rectally, and by other means, in dosage unit formulations containing non-toxic pharmaceutically acceptable carriers, excipients and diluents available to the skilled artisan. One or more adjuvants may also be included.

Thus, in accordance with the present invention, there is further provided a method of treatment, and a pharmaceutical composition, for treating viral infections such as HIV infection and AIDS. The treatment involves administering to a patient in need of such treatment a pharmaceutical composition which contains an antiviral effective amount of one or more of the compounds of Formulas I, II, and III together with one or more pharmaceutically acceptable carriers, excipients or diluents. As used herein, the term “antiviral effective amount” means the total amount of each active component of the composition and method that is sufficient to show a meaningful patient benefit, i.e., inhibiting, ameliorating, or healing of acute conditions characterized by inhibition of HIV infection. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. The terms “treat, treating, treatment” as used herein and in the claims means preventing, inhibiting, ameliorating and/or healing diseases and conditions associated with HIV infection.

The pharmaceutical compositions of the invention may be in the form of orally administrable suspensions or tablets; as well as nasal sprays, sterile injectable preparations, for example, as sterile injectable aqueous or oleaginous suspensions or suppositories. Pharmaceutically acceptable carriers, excipients or diluents may be utilized in the pharmaceutical compositions, and are those utilized in the art of pharmaceutical preparations.

When administered orally as a suspension, these compositions are prepared according to techniques typically known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents known in the art. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents, and lubricants known in the art.

The injectable solutions or suspensions may be formulated according to known art, using suitable non-toxic, parenterally acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.

The compounds herein set forth can be administered orally to humans in a dosage range of about 1 to 100 mg/kg body weight in divided doses, usually over an extended period, such as days, weeks, months, or even years. One preferred dosage range is about 1 to 10 mg/kg body weight orally in divided doses. Another preferred dosage range is about 1 to 20 mg/kg body weight in divided doses. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

Also contemplated herein are combinations of the compounds of Formulas I, II and III herein set forth, together with one or more other agents useful in the treatment of AIDS. For example, the compounds of this disclosure may be effectively administered, whether at periods of pre-exposure and/or post-exposure, in combination with effective amounts of the AIDS antivirals, immunomodulators, antiinfectives, or vaccines, such as those in the following non-limiting table:

Additionally, the compounds of the disclosure herein set forth may be used in combination with HIV entry inhibitors. Examples of such HIV entry inhibitors are discussed in DRUGS OF THE FUTURE 1999, 24(12), pp. 1355-1362; CELL, Vol. 9, pp. 243-246, Oct. 29, 1999; and DRUG DISCOVERY TODAY, Vol. 5, No. 5, May 2000, pp. 183-194 andInhibitors of the entry of HIV into host cells. Meanwell, Nicholas A.; Kadow, John F., Current Opinion in Drug Discovery & Development (2003), 6(4), 451-461. Specifically the compounds can be utilized in combination with attachment inhibitors, fusion inhibitors, and chemokine receptor antagonists aimed at either the CCR5 or CXCR4 coreceptor. HIV attachment inhibitors are also set forth in U.S. Pat. No. 7,354,924 and U.S. Pat. No. 7,745,625.

It will be understood that the scope of combinations of the compounds of this application with AIDS antivirals, immunomodulators, anti-infectives, HIV entry inhibitors or vaccines is not limited to the list in the above Table but includes, in principle, any combination with any pharmaceutical composition useful for the treatment of AIDS.

Preferred combinations are simultaneous or alternating treatments with a compound of the present disclosure and an inhibitor of HIV protease and/or a non-nucleoside inhibitor of HIV reverse transcriptase. An optional fourth component in the combination is a nucleoside inhibitor of HIV reverse transcriptase, such as AZT, 3TC, ddC or ddI. A preferred inhibitor of HIV protease is REYATAZ® (active ingredient Atazanavir). Typically a dose of 300 to 600 mg is administered once a day. This may be co-administered with a low dose of Ritonavir (50 to 500 mgs). Another preferred inhibitor of HIV protease is KALETRA®. Another useful inhibitor of HIV protease is indinavir, which is the sulfate salt of N-(2(R)-hydroxy-1-(S)-indanyl)-2(R)-phenylmethyl-4-(S)-hydroxy-5-(1-(4-(3-pyridyl-methyl)-2(S)—N′-(t-butylcarboxamido)-piperazinyl))-pentaneamide ethanolate, and is synthesized according to U.S. Pat. No. 5,413,999. Indinavir is generally administered at a dosage of 800 mg three times a day. Other preferred protease inhibitors are nelfinavir and ritonavir. Another preferred inhibitor of HIV protease is saquinavir which is administered in a dosage of 600 or 1200 mg tid. Preferred non-nucleoside inhibitors of HIV reverse transcriptase include efavirenz. These combinations may have unexpected effects on limiting the spread and degree of infection of HIV. Preferred combinations include those with the following (1) indinavir with efavirenz, and, optionally, AZT and/or 3TC and/or ddI and/or ddC; (2) indinavir, and any of AZT and/or ddI and/or ddC and/or 3TC, in particular, indinavir and AZT and 3TC; (3) stavudine and 3TC and/or zidovudine; (4) tenofovir disoproxil fumarate salt and emtricitabine.

General Chemistry (Methods of Synthesis)

The present invention comprises compounds of Formulas I, II and III, their pharmaceutical formulations, and their use in patients suffering from or susceptible to HIV infection. The compounds of Formulas I, II and III also include pharmaceutically acceptable salts thereof. Procedures to construct compounds of Formulas I, II and III and intermediates useful for their synthesis are described after the Abbreviations.

Abbreviations

One or more of the following abbreviations, most of which are conventional abbreviations well known to those skilled in the art, may be used throughout the description of the disclosure and the examples:

LCMS=liquid chromatography mass spectroscopy

HPLC=high performance liquid chromatography

Rpm=revolutions per minute

The terms “C-3” and “C-28” refer to certain positions of a triterpene core as numbered in accordance with IUPAC rules (positions depicted below with respect to an illustrative triterpene: betulin):

The same numbering is maintained when referring to the compound series in schemes and general descriptions of methods.

EXAMPLES

The following examples illustrate typical syntheses of the compounds of Formulas I, II and III as described generally above. These examples are illustrative only and are not intended to limit the disclosure in any way. The reagents and starting materials are readily available to one of ordinary skill in the art.

Chemistry

Typical Procedures and Characterization of Selected Examples:

Unless otherwise stated, solvents and reagents were used directly as obtained from commercial sources, and reactions were performed under a nitrogen atmosphere. Flash chromatography was conducted on Silica gel 60 (0.040-0.063 particle size; EM Science supply).1H NMR spectra were recorded on Bruker DRX-500f at 500 MHz (or Bruker AV 400 MHz, Bruker DPX-300B, or Varian Gemini 300 at 300 MHz as stated). The chemical shifts were reported in ppm on the δ scale relative to δTMS=0. The following internal references were used for the residual protons in the following solvents: CDCl3(δH7.26), CD3OD (δH3.30), acetic-d4 (Acetic Acid d4) (δH11.6, 2.07), DMSO mix or DMSO-D6-CDCl3(δH2.50 and 8.25) (ratio 75%:25%), and DMSO-D6 (δH2.50). Standard acronyms were employed to describe the multiplicity patterns: s (singlet), br. s (broad singlet), d (doublet), t (triplet), q (quartet), m (multiplet), b (broad), app (apparent). The coupling constant (J) is in Hertz. All Liquid Chromatography (LC) data were recorded on a Shimadzu LC-10AS liquid chromatograph using a SPD-10AV UV-Vis detector with Mass Spectrometry (MS) data determined using a Micromass Platform for LC in electrospray mode.

Prep HPLC Methods

Analytical HPLC Methods

Preparation of (1R,3aS,4aS,4a1R,7aS,7a1R,8aR,12aS,12bR,14aR,14bR)-10-(4-carboxyphenyl)-4a1,6,6,7a1,9,9,12a-heptamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,4a,4a1,7a,7a1,8,8a,9,12,12a,12b,13,14,14a,14b-octadecahydro-1H-cyclopenta[1,2]chryseno[4,5-def][1,3]dioxepine-3a-carboxylic acid

Ingredients for SG-M2 medium are: Glucose monohydrate 22 g, Toasted Nutrisoy 5 g, Tastone154 5 g, K2HPO45 g, deionized water 1000 mL. After mixing, the pH was adjusted to 7.0, and then autoclaved. One culture vial (1 mL) ofBacillus megaterium(SC16644, ATCC14581) was used to inoculate a 500 mL flask containing 100 mL of sterile SG-M2 medium. The flask was shaken at 30° C. and 250 rpm. After 29 h of growth, the stage 1 culture was used to inoculate (0.4% inoculation) sixteen 4 L flasks each with 500 mL of SG-M2 medium. The flasks were shaken at 30° C. and 250 rpm for 17 h. To each 4 L flask containing 500 mL stage 2 culture ofBacillus megaterium(SC16644, ATCC14581) was added a solution of betulonic acid (50 mg) in 5 mL of DMSO. A total of 800 mg betulonic acid (prepared as described in WO2013169578) was added to sixteen flasks. Biotransformation was conducted by shaking the 4 L flasks at 30° C. and 250 rpm. Biotransformation was monitored by taking out samples each day and analyzing by HPLC (Method 6) and LCMS (Method 3).

After four days, a dihydroxy product (MW 486, M+32) was found to be the major product peak with 5 to 15% of unreacted betulinic acid in different flasks. Biotransformation mixtures from all flasks were combined and acidified to pH 4.0 with 6 N HCl. Another 800 mL aqueous 0.01 M HCl was used to rinse all empty flasks and combined with the biotransformation mixture.

The mixture was filtered through a pad (200 g) of celite. The filtrate was extracted with 2 L of EtOAc. The EtOAc phase contained no dihydroxy product or betulonic acid, but contained a small amount (estimated to be about 28 mg by HPLC) of a possible trihydroxy product (MW 502). This 2 L EtOAc extract was used below. The soft cake together with the celite was transferred into a 3-L beaker and stirred vigorously with 1.6 L MeOH with an overhead stirrer for 1 h. The MeOH extract was collected by filtration.

The cake was again extracted with 1 L MeOH in the same way. Finally, the cake was treated in the filter funnel with 200 mL MeOH. All MeOH extracts were combined (2.8 L). The MeOH extract was concentrated on a rotary evaporator at 30° C. to about 200 mL, mixed with 200 mL of brine, and extracted twice (1.2 L and 0.8 L) with the above EtOAc extract containing the trihydroxy compound. The combined EtOAc extract was washed twice with 500 mL of brine and filtered through a filter paper. Removal of solvent from the EtOAc solution gave a brown residue, which was further dried in a vacuum oven at room temperature overnight to give 4.2 g of brown solid. The 4.2 g of brown solid was heated with a mixture of MeOH (10 mL) and EtOAc (10 mL) at 40° C. to give a brown solution. The solution was concentrated on a rotary evaporator to about 10 mL and immediately subjected to chromatography on a silica gel column packed with heptanes. The column was eluted with a mixture of heptanes-EtOAc-HOAc in a ratio of 90:10:0.5. When the front yellow color band came out, collection of small fractions (10 mL each) was started. The unreacted betulonic acid was eluted first in the fractions 5-10. The ratio of heptanes-EtOAc-HOAc was changed to 80:20:0.5 and then to 50:50:0.5. The fractions were collected and combined as five components (Table 1, Components 01, 02, 03, 04 and 05). After the completion of chromatography, the silica gel was poured into a beaker and stirred with 300 mL MeOH for 1 h and the MeOH solution was separated (Component 05A). Table 1 contains details of the separation of biotransformation mixture.

The different components (01, 02, 03, 04, 05) and MeOH solution (05A) were analyzed by TLC, HPLC (method 6) and LCMS (method 3). The components 01, 03 and 05A contained no peaks related to betulonic acid and were discarded. Components 02 (Solid 94 mg, MW 470, AP 23) and 05 (Solid 308 mg, MW 502, AP 19) were kept for separation and isolation of biotransformation products.

Component 04 (Major product, MW 486, AP 38, 0.5 g solid) was dissolved in MeOH at 40° C. The solution was concentrated to about 10 mL. Water (90 mL) was added slowly when a precipitate was formed. The mixture was kept in an ice-bath for 1 h and then filtered providing 280 mg solid, MW 486, AP 50 (Table 1 Component 06).

The 280 mg solid, MW 486, AP 50 (Component 06) was dissolved in 100 mL EtOAc at 40° C. Heptane (100 mL) was added. The mixture was concentrated to about 20 mL. More heptane (100 mL) was added. The mixture was kept at room temperature for 1 h, and then in an ice-bath for 1 h, and filtered. The cake was dried in a vacuum oven at 30° C. overnight to give 146 mg off-white solid, HPLC retention time 9.9 min AP 87 (Component 08). LCMS and NMR indicated a dihydroxy-betulonic acid structure.

The filtrate was kept at room temperature overnight. The precipitate formed was filtered and gave 90 mg solid as the second crop, retention time 9.9 min product AP 35, a major impurity AP 62 (retention time 3.9 min, with strong UV 256 nm) (Table 1 Component 09). The second crop (Component 09) was subjected to silica gel (38 g) column chromatography and eluted with CH2Cl2containing 5% MeOH and 3% HOAc. Fractions 22-25 (Table 1, Component 11) gave the product (retention time 9.9 min in HPLC), 72 mg, AP 85.

To a solution of (1R,3aS,4aS,4a1R,7aS,7a1R,8aR,12aS,12bR,14aR,14bR)-benzyl 10-(4-(methoxycarbonyl)phenyl)-4a1,6,6,7a1,9,9,12a-heptamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,4a,4a1,7a,7a1,8,8a,9,12,12a,12b,13,14,14a,14b-octadecahydro-1H-cyclopenta[1,2]chryseno[4,5-def][1,3]dioxepine-3a-carboxylate (0.127 g, 0.173 mmol) in DCE (2 mL) was added palladium(II) acetate (9.70 mg, 0.043 mmol), triethylamine (0.039 mL, 0.276 mmol) and t-butyldimethylsilane (0.057 mL, 0.346 mmol). The mixture was flushed with nitrogen then was heated to 60° C. After heating the mixture for 6 h, it was cooled to rt and was filtered through a pad of silica gel and celite. The filtrate was concentrated under reduced pressure and was used in the next step with no with no additional purification. Rf=0.45, 10% EtOAc in hexanes, stained with Hanessian's stain.

Preparation of (1R,3aS,5S,5aR,5bR,6S,7aR,11aS,11bR,13aR,13bR)-9-(4-carboxyphenyl)-5,6-dihydroxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid

Preparation of 4-((1R,3aS,5S,5aR,5bR,6S,7aR,11aS,11bR,13aR,13bR)-3a-((2-(1,1-dioxidothiomorpholino)ethyl)amino)-5,6-dihydroxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoic acid

Step 1. Preparation of methyl 4-((1R,3aS,4aS,4a1R,7aS,7a1R,8aR,12aS,12bR,14aR,14bR)-3a-isocyanato-4a1,6,6,7a1,9,9,12a-heptamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,4a,4a1,7a,7a1,8,8a,9,12,12a,12b,13,14,14a,14b-octadecahydro-1H-cyclopenta[1,2]chryseno[4,5-def][1,3]dioxepin-10-yl)benzoate

Step 2. Preparation of methyl 4-((1R,3aS,4aS,4a1R,7aS,7a1R,8aR,12aS,12bR,14aR,14bR)-3a-amino-4a1,6,6,7a1,9,9,12a-heptamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,4a,4a1,7a,7a1,8,8a,9,12,12a,12b,13,14,14a,14b-octadecahydro-1H-cyclopenta[1,2]chryseno[4,5-def][1,3]dioxepin-10-yl)benzoate

Step 3. Preparation of methyl 4-((1R,3aS,4aS,4a1R,7aS,7a1R,8aR,12aS,12bR,14aR,14bR)-3a-((2-(1,1-dioxidothiomorpholino)ethyl)amino)-4a1,6,6,7a1,9,9,12a-heptamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,4a,4a1,7a,7a1,8,8a,9,12,12a,12b,13,14,14a,14b-octadecahydro-1H-cyclopenta[1,2]chryseno[4,5-def][1,3]dioxepin-10-yl)benzoate

Step 4. Preparation of methyl 4-((1R,3aS,5S,5aR,5bR,6S,7aR,11aS,11bR,13aR,13bR)-3a-((2-(1,1-dioxidothiomorpholino)ethyl)amino)-5,6-dihydroxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoate

To a solution of methyl 4-((1R,3aS,4aS,4a1R,7aS,7a1R,8aR,12aS,12bR,14aR,14bR)-3a-((2-(1,1-dioxidothiomorpholino)ethyl)amino)-4a1,6,6,7a1,9,9,12a-heptamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,4a,4a1,7a,7a1,8,8a,9,12,12a,12b,13,14,14a,14b-octadecahydro-1H-cyclopenta[1,2]chryseno[4,5-def][1,3]dioxepin-10-yl)benzoate (0.016 g, 0.021 mmol) in 1,4-dioxane (0.5 mL) was added 1N HCl (0.051 mL, 0.618 mmol). The mixture was heated to 75° C. for 8 h then cooled to 60° C. and stirred for an additional 14.5 h. The mixture was cooled to rt and purified by prep HPLC (method 3). The fractions containing the product were combined and concentrated under reduced pressure to give the title product (4.0 mg, 5.4 μmol, 26% yield) as a clear film. LCMS: m/e 737.6 (M+H)+, 1.82 min (method 1).

Step 5. To a solution of methyl 4-((1R,3aS,5S,5aR,5bR,6S,7aR,11aS,11bR,13aR,13bR)-3a-((2-(1,1-dioxidothiomorpholino)ethyl)amino)-5,6-dihydroxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoate (0.004 g, 5.43 μmol) in 1,4-dioxane (0.25 mL) was added NaOH (1N) (0.054 mL, 0.054 mmol). The mixture was heated to 60° C. for 14.5 h then was cooled to rt and purified by prep HPLC (method 1). The fractions containing the expected product were combined and concentrated under reduced pressure to give. Because the purity of the compound was not sufficient, it was purified a second time by prep HPLC (method 4). The fractions containing the product were combined and concentrated under reduced pressure to give the TFA salt of 4-((1R,3aS,5S,5aR,5bR,6S,7aR,11aS,11bR,13aR,13bR)-3a-((2-(1,1-dioxidothiomorpholino)ethyl)amino)-5,6-dihydroxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoic acid (1.1 mg, 1.3 μmol, 24% yield) as an off-white solid. LCMS: m/e 723.7 (M+H)+, 1.51 min (method 1).

Preparation of (1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-9-(4-carboxyphenyl)-1-isopropyl-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid

To a mixture of chromium(VI) oxide (752 mg, 7.52 mmol) in DCM (10 mL) was added pyfidine (1.216 mL, 15.04 mmol), the reaction mixture was stirred for 2 hours at 20° C. Then (1S,3aS,5aR,5bR,7aR,11aS,11bR,13aR,13bR)-benzyl 1-isopropyl-9-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylate (500 mg, 0.752 mmol) (prepared as described in WO201153319) was added and the reaction mixture was stirred for 30 h. The reaction was filtered and washed with 1 N HCl (10 mL) and sat. sodium bicarbonate (15 mL). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography with 0-15% ethyl acetate/hexanes to give the title product as a white solid. (210 mg, 41%). LCMS: m/e 679.3 (M+H)+, 3.50 min (method 2).

Step 2. Preparation of (1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-1-isopropyl-9-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid

A mixture of (1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-benzyl 1-isopropyl-9-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylate (270 mg, 0.398 mmol), palladium acetate (17.86 mg, 0.080 mmol) tert-butyldimethylsilane (60.1 mg, 0.517 mmol) and TEA (0.166 mL, 1.193 mmol) in dichloroethane (5 mL) was heated up at 60° C. for 3 h. The reaction mixture was filtered through a pad of celite, then concentrated under reduced pressure to provide the corresponding silyl ester intermediate. To this intermediate in tetrahydrofuran (5 mL) was added TBAF (693 mg, 1.988 mmol). The reaction mixture was stirred for 3 h at room temperature. The mixture was concentrated under reduced pressure and the residue was purified by flash chromatography with 0-40% ethyl acetate/hexanes to provide the title product as a white solid (150 mg, 64%). LCMS: m/e 589.6 (M+H)+, 2.61 min (method 1).

Step 3. Preparation of (1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-9-(4-carboxyphenyl)-1-isopropyl-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid

Example 5 and Example 6

Preparation of (1S,3aS,5aR,5bR,7aS,11S,11aS,11bS,13aR,13bR)-9-(4-carboxyphenyl)-11-hydroxy-1-isopropyl-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid and (1S,3aS,5aR,5bR,7aS,11R,11aS,11bS,13aR,13bR)-9-(4-carboxyphenyl)-11-hydroxy-1-isopropyl-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid

To a solution of (1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-benzyl 1-isopropyl-9-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylate (20 mg, 0.029 mmol) in methanol(1 mL) and dioxane (1 mL) was added sodium borohydride (22.29 mg, 0.589 mmol) at 0° C. The reaction mixture was stirred at 20° C. for 18 h. The reaction mixture was quenched with distilled water (3 mL) and extracted with ethyl acetate (3×4 mL). All the extracts were combined, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography with 0-20% ethyl acetate/hexanes to provide the desired product as a white solid (20 mg, 100%). LCMS: m/e 663.3 (M-18+H)+, 3.26, 3.39 min (method 2).

Step 2. Preparation of (1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-11-hydroxy-1-isopropyl-9-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid

A mixture of (1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-benzyl 11-hydroxy-1-isopropyl-9-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylate (20 mg, 0.029 mmol), palladium acetate (1.32 mg, 0.006 mmol) tert-butyldimethylsilane (10.25 mg, 0.088 mmol) and TEA (0.012 mL, 0.088 mmol) in dichloroethane (1 mL) was heated up at 60° C. for 3 h. The reaction mixture was filtered through a pad of celite, then concentrated under reduced pressure to provide the silyl ester intermediate. To this intermediate in dioxane (1 mL) was added TBAF (0.117 mL, 0.117 mmol). The reaction mixture was stirred for 3 h at room temperature and then concentrated under reduced pressure. The residue was purified by flash chromatography with 0-45% ethyl acetate/hexanes to provide the mixture of two isomers (15 mg, 86%). LCMS: m/e 589.4 (M−H)−, 2.65, 2.76 min (method 2).

Example 7 and Example 8

Preparation of (1S,3aS,5aR,5bR,7aS,11S,11aS,11bS,13aR,13bR)-9-(4-carboxyphenyl)-11-hydroxy-1-isopropyl-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid and (1S,3aS,5aR,5bR,7aS,11R,11aS,11bS,13aR,13bR)-9-(4-carboxyphenyl)-11-hydroxy-1-isopropyl-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid

A mixture of (1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-benzyl 11-fluoro-1-isopropyl-9-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylate (40 mg, 0.057 mmol), palladium acetate (2.6 mg, 0.011 mmol), triethylamine (0.024 mL, 0.172 mmol) and tert-butyldimethylsilane (19.99 mg, 0.172 mmol) in dichloroethane (1 mL) was heated up at 60° C. for 3 h. The reaction mixture was filtered through a pad of celite, then concentrated under reduced pressure to provide the silyl ester intermediate. To this intermediate in dioxane (1 mL) was added TBAF (0.229 mL, 0.229 mmol) and the reaction mixture was stirred for 3 h at room temperature. To the reaction mixture was added 2 mL distilled water. A white precipitate was observed, collected and dried to provide the title compound (30 mg, 88%). LCMS: m/e 591.4 (M−H)−, 3.05 min (method 2).

Step 3. A mixture of (1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-benzyl 11-fluoro-1-isopropyl-9-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylate (30 mg, 0.049 mmol) and oxalyl dichloride (0.247 mL, 0.494 mmol) in dichloromethane (1 mL) was stirred at rt for 3 h. The reaction mixture was concentrated under reduced pressure to provide the corresponding acid chloride.

To a solution of 4-(3-aminopropyl)thiomorpholine 1,1-dioxide (13.78 mg, 0.072 mmol), Hunig's Base (0.025 mL, 0.143 mmol) and DMAP (0.584 mg, 4.78 μmol) in dichloromethane (1 mL) was added a solution of the acid chloride from above in dichloromethane (1 mL). The reaction mixture was stirred at 20° C. for 3 h. The reaction mixture was quenched with distilled water (3 mL) and extracted with dichloromethane (3×4 mL). All the extracts were combined, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide 38 mg of a mixture of methyl 4-((1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-11-chloro-3a-((3-(1,1-dioxidothiomorpholino)propyl)carbamoyl)-1-isopropyl-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoate, LCMS: m/e 783.4 (M+H)+, 2.88 min (method 2) and methyl 4-((1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-3a-((3-(1,1-dioxidothiomorpholino)propyl)carbamoyl)-11-fluoro-1-isopropyl-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoate, LCMS: m/e 765.4 (M−H)−, 2.75 min (method 2).

Preparation of 4-((1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-3a-amino-1-isopropyl-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoic acid

Step 1. Preparation of methyl 4-((1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-3a-isocyanato-1-isopropyl-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoate

A mixture of (1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-1-isopropyl-9-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylic acid (150 mg, 0.255 mmol), diphenyl phosphorazidate (60.7 μl, 0.280 mmol) and triethylamine (107 μl, 0.764 mmol) in dioxane (5 mL) was refluxed at 100° C. for 16 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by flash chromatography with 0-30% ethyl acetate/hexanes to provide the desired final product as a white solid. (90 mg, 60%). LCMS: m/e 586.6 (M+H)+, 3.24 min (method 1).

Step 2. Preparation of methyl 4-((1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-3a-amino-1-isopropyl-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoate

Preparation of 4-((1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-3a-((2-(1,1-dioxidothiomorpholino)ethyl)amino)-1-isopropyl-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoic acid

Step 1. Preparation of methyl 4-((1S,3aS,5aR,5bR,7aS,11aS,11bS,13aR,13bR)-3a-((2-(1,1-dioxidothiomorpholino)ethyl)amino)-1-isopropyl-5a,5b,8,8,11a-pentamethyl-11-oxo-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysen-9-yl)benzoate

Preparation of (1S,3aS,5aR,5bR,7aR,8aS,9aS,10aR,10bR,12aR,12bR)-8a-(4-carboxyphenyl)-1-isopropyl-5a,5b,8,8,10a-pentamethylicosahydro-1H-cyclopenta[7,8]chryseno[2,3-b]oxirene-3a-carboxylic acid

To a mixture of (1S,3aS,5aR,5bR,7aR,11aS,11bR,13aR,13bR)-benzyl 1-isopropyl-9-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,11a-pentamethyl-2,3,3a,4,5,5a,5b,6,7,7a,8,11,11a,11b,12,13,13a,13b-octadecahydro-1H-cyclopenta[a]chrysene-3a-carboxylate (27 mg, 0.041 mmol) in dichloromethane (1 mL) at −78° C. was added 3-chlorobenzoperoxoic acid (27.3 mg, 0.122 mmol) and the mixture was stirred for 3 h at −78° C. The reaction mixture was quenched with distilled water (3 mL) and extracted with dichloromethane (3×4 mL). All the extracts were combined, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide the title compound as a white solid (20 mg, 72%). LCMS: m/e 681.4 (M+H)+, 3.37 min (method 2).

Step 2. Preparation of 4-((1S,3aS,5aR,5bR,7aR,8aS,9aS,10aR,10bR,12aR,12bR)-3a-((benzyloxy)carbonyl)-1-isopropyl-5a,5b,8,8,10a-pentamethylicosahydro-1H-cyclopenta[7,8]chryseno[2,3-b]oxiren-8a-yl)benzoic acid

A mixture of (1S,3aS,5aR,5bR,7aR,8aS,9aS,10aR,10bR,12aR,12bR)-benzyl 1-isopropyl-8a-(4-(methoxycarbonyl)phenyl)-5a,5b,8,8,10a-pentamethylicosahydro-1H-cyclopenta[7,8]chryseno[2,3-b]oxirene-3a-carboxylate (20 mg, 0.030 mmol) and sodium hydroxide (0.150 mL, 0.150 mmol) in dioxane (1 mL) was heated up at 78° C. for 3 h. The reaction mixture was quenched with distilled water (3 mL) and extracted with dichloromethane (3×2 mL). All the extracts were combined, dried over sodium sulfate, filtered and concentrated under reduced pressure to provide the desired product as a white solid (15 mg, 75%). LCMS: m/e 667.3 (M+H)+, 2.37 min (method 1).

HIV Cell Culture Assay—

MT-2 cells and 293T cells were obtained from the NIH AIDS Research and Reference Reagent Program. MT-2 cells were propagated in RPMI 1640 media supplemented with 10% heat inactivated fetal bovine serum, 100 μg/mL penicillin G and up to 100 units/mL streptomycin. The 293T cells were propagated in DMEM media supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 units/mL penicillin G and 100 μg/mL streptomycin. The proviral DNA clone of NL4-3was obtained from the NIH AIDS Research and Reference Reagent Program. A recombinant NL4-3virus, in which a section of the nef gene from NL4-3 was replaced with theRenillaluciferase gene, was used as a reference virus. In addition, residue Gag P373 was converted to P373S. Briefly, the recombinant virus was prepared by transfection of the altered proviral clone of NL4-3. Transfections were performed in 293T cells using LipofectAMINE PLUS from Invitrogen (Carlsbad, Calif.), according to manufacturer's instruction. The virus was titered in MT-2 cells using luciferase enzyme activity as a marker. Luciferase was quantitated using the Dual Luciferase kit from Promega (Madison, Wis.), with modifications to the manufacturer's protocol. The diluted Passive Lysis solution was pre-mixed with the re-suspended Luciferase Assay Reagent and the re-suspended Stop & Glo Substrate (2:1:1 ratio). Fifty (50) μL of the mixture was added to each aspirated well on assay plates and luciferase activity was measured immediately on a Wallac TriLux (Perkin-Elmer). Antiviral activities of inhibitors toward the recombinant virus were quantified by measuring luciferase activity in cells infected for 4-5 days with NLRluc recombinants in the presence serial dilutions of the inhibitor. The EC50data for the compounds is shown in Table 1.

The foregoing description is merely illustrative and should not be understood to limit the scope or underlying principles of the invention in any way. Indeed, various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the following examples and the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.