EA045041B1 - NEW TRITERPENE DERIVATIVES AS HIV INHIBITORS - Google Patents

Description

This application claims the benefit of Indian Provisional Application No. 201941005217 filed February 2019, which is incorporated herein by reference in its entirety.

Field of technology to which the invention relates

The present invention relates to new triterpene derivatives and related compounds, compositions useful for the therapeutic treatment of viral diseases and specifically diseases mediated by HIV.

BACKGROUND OF THE INVENTION

More than 20 years ago, the human immunodeficiency virus (HIV) was recognized as the causative agent of acquired immunodeficiency syndrome (AIDS) (Science 1983, 220, 868-871; N. Eng. J. Med. 1984, 311, 12921297). AIDS is characterized by the destruction of the immune system, specifically CD4+ T cells. HIV is a retrovirus, and the HIV life cycle includes several important steps, starting with the attachment of the virus to the host cell membrane and ending with the release of daughter virions from the cell.

The naturally occurring compound betulinic acid, isolated from Syzygium clavifolium and several other plant species, has been found to have anti-HIV activity. Chemical modifications have been undertaken by several research groups in an attempt to identify potent anti-HIV agents by creating semisynthetic analogues of betulinic acid, leading to the discovery of bevirimate as a compound with a new mechanism of action (J. Nat. Prod. 1994, 57(2): 243-7 ; J. Med. Chem. 1996, 39 (5), 1016). Further studies showed that bevirimat acts by interfering with Gag processing (Proc. Natl. Acad. Sci. USA 2003, 100 (23): 13555-60; Antimicrob. Agents. Chemother. 2001, 45 (4), 1225-30; J Virol 2004, 78 (2): 922-9; J Biol Chem 2005, 280 (51): 42149-55; J Virol 2006, 80 (12): 5716-22) and is the first in a maturation inhibitor in its class with high activity against HIV-1. Bevirimat has entered phase 2 clinical trials; in clinical trials, despite optimal plasma concentrations, not all patients treated with bevirimat experienced a significant reduction in viral load. Patients who did not respond to treatment were reported to have more frequent Gag core polymorphisms near the SP-1 capsid cleavage site than patients who responded to treatment. (HIV gag polymorphism determines treatment response to bevirimat. XVII international HIV drug resistance work shop June 10-14, 2008, Sitges, Spain)

Encouraged by these advances, medicinal chemists began to intensively study betulinic acid derivatives and related compounds for their therapeutic effects. For example, publication WO2017/149518 discloses novel C-3 triterpene C-17 amino derivatives as HIV inhibitors; WO2014/105926 discloses novel proline derivatives of betulinic acid as HIV inhibitors; WO2014/130810 describes the preparation of C3-alkyl and alkenyl modified betulinic acid derivatives useful for the treatment of HIV; WO2014/123889 describes the preparation of triterpenoid derivatives for use as HIV maturation inhibitors; WO2013/160810 discloses novel betulinic acid derivatives as HIV inhibitors; WO2013/169578 describes C-17 bicyclic amine triterpenoids with HIV maturation inhibitory activity and their preparation; WO2013/123019 describes C-3 cycloalkenyl triterpenoids with HIV maturation inhibitory activity; WO2013/043778 describes novel betulinic acid derivatives with antiviral activity; WO2013/035943 describes betulinic acid and its derivatives having anti-aging activity; WO2012/106190 describes the preparation of C17- and C3-modified triterpenoids with HIV maturation inhibitory activity; CN102399254 describes new derivatives of pentacyclic triterpenoids, their method of preparation and use for the prevention and treatment of diabetes, cardiovascular diseases, cerebrovascular diseases and tumors; WO2011/007230 describes the preparation of lupeol-type triterpene derivatives as antiviral agents; WO2010/032123 describes the preparation of triterpenoid compounds for pharmaceutical use; Journal of Medicinal Chemistry (2010), 53(1), 178-190 describes structure-activity relationship studies of betulinic acid, a novel and selective TGR5 agonist, and its synthetic derivatives: potential impact on diabetes; WO2009/100532 describes the preparation of 17b-lupane derivatives for the treatment of HIV infection; CN101367861 describes a method for the preparation and use of 2-hydroxy-3-deoxy-pentacyclic triterpene compounds and derivatives; WO2008/138200 describes the preparation of lupane derivatives as N-methyl-D-aspartate (NMDA) receptor and MC receptor antagonists exhibiting neuroprotective and memory enhancing activities; WO2008/127364 describes the preparation of betulinic acid derivatives for use in antiviral and anticancer pharmaceutical compositions.

Some additional references disclose related compounds of betulinic acid. For example, WO2006/053255 describes the preparation of betulin derivatives for use in pharmaceutical compositions that inhibit the transmission of viral infection; WO2004/089357 describes an antifungal composition of triterpene and essential oil; Bioorganic & Medicinal Chemistry Letters (2003), 13(20), 3549-3552 describes lupane triterpenes and derivatives with antiviral activity; Russian Journal of Bioorganic Chemistry (2003), 29(6), 594-600 describes the synthesis and antiviral activity of ureides and carbamates of betulinic acid and its derivatives; Journal of Medicinal Chemistry

- 1 045041 (1996), 39(5), 1056-68 describes betulinic acid derivatives: a new class of specific inhibitors of human immunodeficiency virus type 1 with a new mechanism of action; Oxidation Communications (1987), 10(3-4), 305-12 describes oxidative decarboxylation-II: oxidative decarboxylation of acetyl betulinic acid.

Given the global level of the AIDS epidemic, there is an urgent and ongoing need for new effective drugs to treat HIV-infected patients, disease conditions and/or disorders mediated by HIV, through the discovery of new compounds with new structures and/or mechanisms of action.

The essence of the invention

The present invention relates to a compound of formula (I) to.

H <form - I where R1 is selected from ^o '°. ^He . ⁰ > ^he , or

R2 is selected from C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C3-C8 cycloalkyl; wherein the optional substituent is C1-C6 alkyl;

R3 represents hydrogen or C1-C6alkyl;

R4 is selected from optionally substituted C1-C6alkyl or -C(O)ORa; wherein the optional substituent is selected from halogen, hydroxy, alkoxy, -OC(O)CH2 alkoxy, -OSi(Ra)3, -N(Ra)(Rb), -S(Ra) or -O-CH2-P(O) (ORa)2;

. ί ) φ -0 ring ^x represents o''° or ^o '

Ra represents hydrogen or C1-C6 alkyl; And

Rb is selected from hydrogen, alkyl, -C(O)alkoxy, or -S(O)2 alkyl, or pharmaceutically acceptable salts, pharmaceutically acceptable stereoisomers, prodrugs, or combinations thereof.

In accordance with one embodiment, the present invention relates to a connection

Formulas (1), where R1 represents

According to another embodiment, the present invention provides a compound of formula (I) wherein R1 is

According to yet another embodiment, the present invention provides a compound of formula (I) wherein R2 is isopropylene.

According to yet another embodiment, the present invention provides a compound of formula (I) wherein R2 is methylcyclopropyl.

According to yet another embodiment, the present invention provides a compound of formula (I) wherein R3 is hydrogen.

According to yet another embodiment, the present invention provides a compound of formula (I) wherein R3 is methyl.

According to yet another embodiment, the present invention provides a compound of formula (I) wherein R4 is methyl, isopropyl, -C(O)OH, ⁿ > hydroxyethyl, hydroxymethyl, fluoromethyl, fluoroethyl,

According to yet another embodiment, the present invention provides a compound of formula (I), wherein the ring

represents ^{ο o}

- 2 045041

According to another embodiment, the present invention provides a compound of formula (I), wherein the ring

represents

Accordingly, in yet another aspect, the present invention provides a compound of formula (IA):

where R2, R3, R4 and ring

have the meanings given in formula (I); or pharmaceutically acceptable salts, pharmaceutically acceptable stereoisomers, prodrugs, or combinations thereof.

In accordance with one embodiment, the present invention provides a compound of formula (IA) wherein R2 is isopropylene.

According to another embodiment, the present invention provides a compound of formula (IA) wherein R2 is methylcyclopropyl.

According to yet another embodiment, the present invention provides a compound of formula (IA) wherein R3 is hydrogen.

According to yet another embodiment, the present invention provides a compound of formula (IA) wherein R3 is methyl.

According to yet another embodiment, the present invention provides a compound of formula (IA) wherein R4 is methyl,

Y6-°-SiX n

isopropyl, -C(O)OH, ¹ hydroxyethyl, hydroxymethyl, fluoromethyl, fluoroethyl,

According to yet another embodiment, the present invention provides a compound of formula (IA) wherein the ring is ^o

According to yet another embodiment, the present invention provides a ^compound of formula (IA) wherein the ring is '

Accordingly, in yet another aspect, the present invention provides a compound of formula (IB):

where, R2, R3, and R4 have the same meaning as defined in formula (I); or pharmaceutically acceptable salts, pharmaceutically acceptable stereoisomers, prodrugs, or combinations thereof.

In accordance with one embodiment, the present invention provides a compound of formula (IB) wherein R2 is isopropylene.

According to another embodiment, the present invention provides a compound of formula (IB) wherein R2 is methylcyclopropyl.

- 3 045041

According to yet another embodiment, the present invention provides a compound of formula (IB) wherein R3 is hydrogen.

According to yet another embodiment, the present invention provides a compound of formula (IB) wherein R3 is methyl.

According to yet another embodiment, the present invention provides a compound I of formula (IB) wherein R4 is methyl, isopropyl, -C(O)OH, ^n- hydroxymethyl, fluoromethyl, fluoroethyl, 'hydroxyethyl,

The following are representative compounds which are for illustrative purposes only and are not intended to limit the scope of the invention (the nomenclature was created from Chem Bio Draw Ultra 13.0 version):

(1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11aR, 11 bR, 13aR, 13bR)-3a-((2-(1,1dioxidothiomorpholino)-2- methylpropyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1,1dioxidothiomorpholino)propyl) amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11aR, 11 bR, 13aR, 13bR)-3a-(((R)-2-(1,1dioxidothiomorpholino)- 3-methylbutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11aR, 11 bR, 13aR,13bR)-3a-(((R)-3-(( TpeTbutyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl) oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11aR, 11 bR, 13aR, 13bR )-3a-(((R)-2-(1,1dioxidothiomorpholino)-3-hydroxypropyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a] chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR, 13bR)-3a-(((S)-2-(1,1dioxidothiomorpholino)-3-methylbutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a ]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutan-1-carboxylic acid,

- 4 045041 (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-(((2R,3R)- 2-(1, 1dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl) -2,2dimethylcyclobutane-1-carboxylic acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-( ((S)-2-(1,1 dioxidothiomorpholino)-3-hydroxypropyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysene-9 -yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR , 13bR)-3a-(((S)-2-(1,1 dioxidothiomorpholino)-3-fluoropropyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta [a]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-(((R)-2-(1,1dioxidothiomorpholino)-3-fluoropropyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl) icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR, 7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-(((S)-2-(4(isopropylsulfonyl)piperazin-1-yl)propyl)amino)-5a,5b,8,8,11a -pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-(((2R,3S)-2-(1,1 dioxidothiomorpholino)-3-fluorobutyl)amino)-5a ,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1 R,3S) -3-((((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-(((2S,3S)-2-(1,1dioxidothiomorpholino)-3 -hydroxybutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid , (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-(((2R,3R)-3hydroxy- 2-(4-(isopropylsulfonyl)piperazin-1-yl)butyl)amino)-5a,5b,8,8,11apentamethyl-1-1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy ) carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR, 13bR )-3a-(((2R,3R)-2-(1,1dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-ene 2-yl )icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR ,7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-(((R)-2-Kap6oKcn-2(1,1-dioxidothiomorpholino)ethyl)amino)-5a,5b,8,8,11a -pentamethyl-1-(1-

- 5 045041 methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1 R,3S)-3-((((1 R,3aS ,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-(((2R,3R)-2-(1, 1dioxidothiomorpholino)-3-(2-(2-methoxyethoxy)acetoxy) butyl)amino)-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid, (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)-13-(1, 1dioxidothiomorpholino)-12-methyl-2,5,8,11-tetraoxatetradecane-14-yl)amino)-5a,

5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid, (1 R,3S )-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-(((2R,3R)-2-(1, 1dioxidothiomorpholino)- 3-(2-(2-methoxyethoxy)acetoxy)butyl)amino)-5a,5b,8,8,11apentamethyl-1-(prop-1-en-2-yl)icosahydro-1 H-cyclopenta[a]chrysene -9-yl)oxy)carbonyl)-

2,2-dimethylcyclobutane-1-carboxylic acid,

4-(((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2-(1,1dioxidothiomorpholino)-3-hydroxybutyl)amino) -5a,5b,8,8,11a-pentamethyl-1-(prop-1-en2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)-2,2-dimethyl-4-oxobutane acid, (1 R,3S)-3-((((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13aR, 13bR)-3a-(((15R, 16R)-16 -(1,1 dioxidothiomorpholino)-15-methyl-13-oxo-2,5,8,11,14-pentaoxaheptadecan-17-yl) amino)-5a,5b,8,8,11a-pentamethyl-1-( prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysen9-yl)oxy)carbonyl)-2,2-dimethylcyclobutan-1-carboxylic acid, (1R,3S)-3-(((( 1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)-13-(1,1dioxidothiomorpholino)-12-methyl-2,5,8,11 -tetraoxatetradecane-14-yl)amino)-5a,

5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl) oxy)carbonyl)-2,2-dimethylcyclobutan-1 - carboxylic acid, or pharmaceutically acceptable salts and prodrugs of the compounds are also contemplated.

The present invention also provides a pharmaceutical composition that includes at least one compound described herein and at least one pharmaceutically acceptable excipient (such as a pharmaceutically acceptable carrier or diluent). In particular, the pharmaceutical composition contains a therapeutically effective amount of at least one compound described herein. The compound(s) present in the composition may be associated with a pharmaceutically acceptable excipient (such as a carrier or diluent), or may be diluted with a carrier or enclosed in a carrier, which may be in the form of a capsule, sachet, or other container.

The compounds and pharmaceutical compositions described herein are useful in the treatment of diseases, conditions and/or disorders mediated by viral infections.

The present invention also provides a method of treating a disease, condition and/or disorder mediated by viral infections in a subject in need thereof by administering to the subject one or more of the compounds described herein in a therapeutically effective amount to treat the infection, specifically in the form of a pharmaceutical composition .

The present invention also provides methods for preparing the compounds described herein.

The invention relates to a method for preventing; alleviating or treating an HIV-mediated disease, disorder or syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the present invention. The invention also provides a method wherein the HIV-mediated disease, disorder or syndrome is similar to AIDS, AIDS-related complex or syndrome characterized by symptoms such as persistent generalized lymphadenopathy, fever and weight loss, or a retroviral infection genetically associated with AIDS.

The potential of the compounds of the present invention to inhibit HIV can be demonstrated using any one or more methodologies known in the art, for example, using the assays described by Mossman T, December 1983, Journal of immunological methods, 65 (1-2), 55-63 and SPCCole, cancer chemotherapy and Pharmacology, 1986, 17, 259-263.

- 6 045041

Detailed Description of the Invention

The present invention also relates to new triterpene derivatives and related compounds that can be used as antiviral, particularly as anti-HIV compounds, and methods for synthesizing these compounds and their pharmaceutically acceptable salts together with pharmaceutically acceptable carriers, excipients or diluents that can also be used to treat diseases, conditions and/or disorders mediated by viral infections.

The following definitions apply to the terms used herein: The term alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to eight carbon atoms, and which is attached to the rest of the molecule by a single bond eg methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl and 1,1-dimethylethyl (tert-butyl).

The term alkenyl refers to a hydrocarbon chain, which may be straight or branched, containing a specified number of carbon atoms, having at least one -C=C-, for example, a C2-C6 alkenyl group may have from 2 to 6 (inclusive) -C=C- atoms in it. Examples of C2-C6 alkenyl groups include, but are not limited to, ethylene, prop-1-ene, but-1-ene, but-2-ene, pent-1-ene, pent-2-ene, hex-1-ene , hex-2-ene, etc.

The term alkoxy refers to a straight or branched hydrocarbon chain with an oxygen radical, consisting of carbon and hydrogen atoms, containing saturation or unsaturation, having from one to eight carbon atoms and which is attached through an oxygen atom to the rest of the molecule by a single bond. One of the hydrogen atoms in the alkoxy group may be further replaced by one or more alkoxy groups to further increase the length of the alkoxy group, for example, methyloxy, ethyloxy, isopropoxy, n-propyloxy, tert-butyloxy, 1-methylethyloxy (isopropyloxy), n-butyloxy, n-pentyloxy , 1,1-dimethylethyloxy (tert-butyloxy)

The term cycloalkyl refers to a non-aromatic mono- or polycyclic ring system containing from 3 to about 12 carbon atoms, such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, perhydronaphthyl, adamantyl and norbornyl groups, bridged cyclic groups and spirobicyclic groups, for example, spiro(4,4)non-2-yl.

The terms halogen or halo include fluorine, chlorine, bromine or iodine.

The term hydroxy refers to -OH.

The term optionally substituted refers to the replacement of one or more hydrogen radicals in a given structure with a radical of a specified substituent, including, but not limited to: hydroxy, halo, carboxyl, cyano (CN), nitro, oxo (=O), thio (=S), alkyl, methylsulfonyl, haloalkyl, alkoxy, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, amino, -C(O)O-alkyl, heterocyclyl, heteroaryl, heterocyclylalkyl, heteroarylalkyl, alkylthio, arylthio, aryloxy, aminocarbonyl, alkoxycarbonyl, alkylamino, arylamino , acyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid and aliphatic. It is understood that the substituent may be further substituted.

The term prodrug refers to a derivative compound, a derivative that, when administered to warm-blooded animals, such as humans, is converted into a compound (drug). Enzymatic and/or chemical hydrolytic cleavage of the compounds of the present invention occurs in such a way that the tested dosage form (carboxylic acid parent drug) is released and the fragment or fragments removed remain non-toxic or are metabolized in such a way that non-toxic metabolic products are produced. For example, a carboxylic acid group can be esterified, for example, with a methyl group or ethyl group to form an ester. When an ester is administered to a subject, the ester is cleaved enzymatically or non-enzymatically, by reduction, oxidation, or hydrolytically to reveal an anionic group. The anionic group can be esterified with moieties (eg acyloxymethyl esters) which are cleaved to form an intermediate which subsequently decomposes to form the active compound. A discussion of the use of prodrugs is presented by T. Higuchiand W. Stella, Pro-drugsas Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers Drug Design, ed Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

The term treatment or therapy for a condition, disease, disorder or disorder includes:

(1) preventing or delaying the onset of clinical symptoms of a condition, disease, disorder or disorder developing in a subject who may be affected by or predisposed to the condition, disease, disorder or disorder but is not yet experiencing or exhibiting clinical manifestations or subclinical symptoms of the condition, diseases, disorders or impairments;

(2) inhibition of a condition, disease, disorder or disorder, i.e. stopping or reducing the progression of a condition, disease, disorder or disorder, or at least one of these

- 7 045041 clinical or subclinical symptom; or (3) alleviation of a condition, disease, disorder or disorder, i.e. providing regression of a condition, disease, disorder or disorder or at least one of its clinical or subclinical symptoms.

The benefit to the subject receiving the treatment is either statistically significant or at least tangible to the subject or clinician.

The term subject includes mammals (especially humans) and other animals such as domestic animals (eg, pets, including cats and dogs) and non-domesticated animals (eg, wild animals).

The term "therapeutically effective amount" means an amount of a compound that, when administered to a subject to treat a condition, disease, disorder, or disorder, is sufficient to effect such treatment. The therapeutically effective amount will vary depending on the compound, condition, disease, disorder or disorder and its severity, as well as the age, weight, physical condition and response of the subject receiving treatment. The compounds of the present invention may form salts. Non-limiting examples of pharmaceutically acceptable salts forming part of this invention include salts derived from inorganic bases, salts of organic bases, salts of chiral bases, salts of natural amino acids and salts of non-natural amino acids. Certain compounds of the present invention may exist in stereoisomeric forms (eg, diastereomers, enantiomers, racemates, and combinations thereof). With regard to all compounds described by formula (I), the present invention extends to these stereoisomeric forms and mixtures thereof. To the extent that the synthesis or resolution of specific stereoisomers is known in the prior art, the different stereoisomeric forms of the present invention can be separated from each other by methods known in the art, or a given isomer can be obtained by stereospecific or asymmetric synthesis. Tautomeric forms and mixtures of the compounds described herein are also contemplated.

Pharmaceutical compositions.

The pharmaceutical compositions of the present invention include at least one compound described herein, and at least one pharmaceutically acceptable excipient (such as a pharmaceutically acceptable carrier or diluent). In particular, the pharmaceutical compositions provided include the compound(s) described herein in an amount sufficient to treat a viral infection in a subject.

Intended subjects include, for example, a living cell and a mammal, including a human. The compound of the present invention may be associated with a pharmaceutically acceptable excipient (such as a carrier or diluent) or be diluted with a carrier, or enclosed in a carrier, which may be in the form of a capsule, sachet, or other container.

Examples of suitable carriers include, but are not limited to, water, saline solutions, alcohols, polyethylene glycols, peanut oil, olive oil, gelatin, lactose, white clay (terra alba), sucrose, dextrin, magnesium carbonate, sugar, amylose, magnesium stearate, talc, gelatin, agar, pectin, gum acacia, stearic acid, lower alkyl cellulose esters, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, fatty acid esters and polyoxyethylene.

The carrier or diluent may include a sustained release material, such as, for example, glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

The pharmaceutical composition may also include one or more pharmaceutically acceptable auxiliary agents, wetting agents, emulsifying agents, suspending agents, preservatives, osmotic salts, buffers, sweeteners, flavorings, coloring agents, or any combination of the foregoing. The pharmaceutical composition of the present invention can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a subject using procedures known in the art.

The pharmaceutical compositions described herein can be prepared, for example, as described in Remington: The Science and Practice of Pharmacy, 20th Ed., 2003 (Lippincott Williams & Wilkins). For example, the active compound may be mixed with a carrier or diluted with a carrier, or enclosed in a carrier, which may be in the form of an ampoule, capsule or sachet. When the carrier serves as a diluent, it may be a solid, semi-solid or liquid material that acts as a carrier, excipient or vehicle for the active compound.

The pharmaceutical compositions may be in conventional forms, for example capsules, tablets, solutions, suspensions, injections or topical products. In addition, the pharmaceutical composition of the present invention can be formulated to provide the desired release profile.

The route of administration may be any route that effectively transports the active compound to a suitable or desired site of action. Suitable routes of administration include,

- 8 045041 without limitation, oral, nasal, pulmonary, buccal, subcutaneous, intradermal, transdermal, parenteral, rectal, depot, subcutaneous, intravenous, intraurethral, intramuscular, intranasal, ophthalmic (for example, with ophthalmic solution) or topical (for example, such as topical ointment). The oral route is particularly suitable.

Solid oral compositions include, but are not limited to, tablets, capsules (soft or hard gelatin), dragees (containing the active ingredient in powder or granule form), lozenges and medicated lozenges. Tablets, dragees or capsules containing talc and/or a carbohydrate carrier, binder or the like are particularly suitable for oral administration. Examples of carriers for tablets, dragees or capsules include lactose, corn starch and/or potato starch. A syrup or elixir may be used in cases where a sweetened carrier can be used.

A typical tablet that can be prepared by conventional tabletting methods.

Liquid formulations include, but are not limited to, syrups, emulsions, soft gelatin, and sterile injectable liquids such as aqueous or non-aqueous liquid suspensions or solutions. For parenteral use, injectable solutions or suspensions are particularly suitable, especially aqueous solutions with the active compound dissolved in polyhydroxylated castor oil.

Screening methods.

Anti-HIV activity) and cytotoxicity of the compounds of the present invention can be measured in parallel, by methods published in the literature.

The cytotoxic effect of the compounds can be analyzed by measuring cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasium bromide (MTT) staining. Cells (5x103 cells/well) are incubated in 96-well plates in the presence or absence of compounds. At the end of treatment, 20 μl of MTT (5 mg/ml in PBS) was added to each well and incubated for an additional 4 hours at 37°C. The purple-blue MTT formazan precipitate is dissolved in a triplex reagent containing 10% SDS, 5% isobutanol and 10 mmol/L HCl. Mitochondrial activity, which reflects cell growth and viability, is assessed by measuring absorbance at 570 nm on a microtiter plate.

The effect of compounds on HIV replication in Sup-T1 cells can be determined by the method published by Roda Rani et al., 2006 (Archives of Biochemistry and Biophysics, Volume 456, Issue 1, 1 December 2006, Pages 79-92).

Briefly, 1x106 Sup-T1 cells with 100% cell viability were seeded in RPMI 1640, 0.1% FBS, four 12-well plates. Increasing concentrations of Epap-1 peptides are added to cells and infected with HIV1 93 IN 101, each at a final virus concentration equivalent to 2 ng p24 per ml. Infected cells are incubated at 37°C and 5% CO2 in an incubator for 2 hours. After 2 hours, the cells are pelleted at 350 g for 10 minutes, the supernatant is removed and the cells are maintained in RPMI 1640 medium containing 10% FBS. Cells are resuspended in the same medium with increasing concentrations of Epap-1 peptides and incubated for 96 hours. Peptides are added to the cells every 24 hours. Supernatants were collected after 96 hours and analyzed using a P24 Antigen Capture Assay Kit (SAIC Fredrick). Infection in the absence of Erap-1 is considered 0% inhibition. Azidothymidine (AZT) is used as a positive control.

The effect of a compound on viral entry and quantification of the introduced virus can be assessed in terms of GFP expression by the following methods published in J. Virol. 72, 6988 (1998), by Cecilia et al., and Analytical Biochemistry Volume 360, Issue 2, 15 January 2007, Pages 315317 (Dyavar S. Ravi and Debashis Mitra).

Briefly, cells were seeded into wells of 24-well plates 1 day before the experiment. Cells are transfected with the Tat reporter. The viral inoculum is adjusted to 1000-4000 TCID 50/ml in assay medium (DMEM, 10% FCS, glutamine and antibiotics), and 50 µl aliquots are incubated with serial dilutions of compounds (50 µl) for 1 hour at 37°C. At the appropriate time, reporter expression is quantified, and the calculated inhibitory doses are relative to the concentration of these agents in this preincubation mixture.

Other relevant references useful for screening for anti-HIV activity are: Averett, D.R.1989. Anti-HIV compound assessment by two novel high capacity assays. J. Virol. Methods 23: 263-276; Schwartz, O., et al. I998; A rapid and simple colorimetric test for the study of anti HIV agents. AIDS Res. and Human Retroviruses, 4(6):441–447; Daluge, S. M., et al. 1994. 5-Chloro-2',3'-deoxy3'fluorouridine (935U83), a selective anti human immunodeficiency virus agent with an improved metabolic and toxicological profile; Antimicro. Agents and Chemotherapy, 38(7): 1590-1603; H. Mitsuya and S. Border, Inhibition of the in vitro infectivity and cytopathic effect of human T -lymph tropic virus type lymphadenopathy associated virus (HLTV-111/LAV) by 2,3'-dideoxynucleosides, Proc. Natl. Acad. Sci. USA,83,1911-15(1986); Pennington et al., Peptides 1990; Meek T.D et al., Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues, Nature, 343, p90 (1990); Weislow et al., J. Natl. Cancer Inst. 81, 577-586, 1989; T.

- 9 045041

Mimoto et al., J. Med. Chern., 42, 1789-1802, 1999; Uckun et al 1998, Antimicrobial Agents and Chemotherapy 42:383; for P24 antigen assay Erice et al., 1993, Antimicrob. Ag. Chemotherapy 37: 385-383; Koyanagi et al., Int. J. Cancer, 36, 445-451, 1985; Balzarini et al. AIDS (1991), 5, 21-28; Connor et al., Journal of virology, 1996, 70, 5306-5311; Popik et al., Journal of virology, 2002, 76, 4709-4722; Harrigton et al., Journal of Virology Methods, 2000, 88, 111-115; Roos et al., Virology 2000, 273, 307-315; Fedyuk N.V. et al; Problems of Virology 1992, (3)P135; Mosmann T, December 1983, Journal of immunological methods, 65 (1-2), 55-63; SPC Cole, cancer chemotherapy and Pharmacology, 1986, 17, 259-263, Antiviral methods and protocols (Eds: D Kinchington and R. F. Schinazi) Humana Press Inc., 2000, HIV protocols (Eds: N. L. Michael and J. H. Kim) Humana Press Inc , 1999, DAIDS Virology manual from HIV laboratories, Publication NIH-97-3838, 1997, 4. HIV-1 p24antigen capture assay, enzyme immunoassay for detection of Human immunodeficiency Virus Type 1 (HIV-1) p24 in tissue culture media - Advanced bio science laboratories, Inc kit procedure.

Methods of treatment.

The present invention relates to compounds and pharmaceutical compositions thereof that are useful for the treatment of diseases, conditions and/or disorders mediated by viral infections. The connection between the therapeutic effect and the antiviral effect is illustrated. For example, publications WO01//07646, WO01/65957 or WO03/037908; US Patent Publications No. US 4,598,095 or US 2002/0068757; EP Publications No. EP 0989862 or EP 0724650;

Bioorganic & Medicinal Chemistry Letters, 16, (6), 1712-1715, 2006; and the references cited therein, all of which are incorporated herein by reference in their entirety and for the stated purpose.

The present invention also provides a method of treating a disease, condition and/or disorder mediated by viral infections in a subject in need thereof by administering to the subject a therapeutically effective amount of a compound or pharmaceutical composition of the present invention.

Diseases, conditions and/or disorders that are believed to be mediated by viral infections include, but are not limited to, HIV infection, HBV infection (hepatitis B virus infection), HCV infection (hepatitis B virus infection C), retroviral infection genetically associated with HIV, AIDS, inflammatory diseases, respiratory disorders (including adult respiratory distress syndrome (ARDS), bronchitis, chronic bronchitis, chronic obstructive pulmonary disease, cystic fibrosis, asthma, emphysema, rhinitis and chronic sinusitis , inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), multiple sclerosis, rheumatoid arthritis, transplant rejection (specifically, but not limited to kidney and lung allografts), endometriosis, type I diabetes, renal diseases, chronic pancreatitis, inflammatory conditions of the lungs, chronic heart failure and bacterial infections (specifically, but not limited to, tuberculosis).

The compounds of the present invention may provide more beneficial than additive effects in the prevention or treatment of the above diseases when appropriately used in combination with available drugs. In addition, the administered dose may be reduced compared to administration of a single drug, or the side effects of coadministered drugs other than antivirals may be avoided or reduced.

Methods of obtaining.

The compounds described in the present invention can be prepared by methods known in the art. In addition, the compounds described herein can be prepared by the following reaction sequences depicted in the schemes below. In addition, in the following diagrams where specific bases, acids, reagents, solvents, coupling agents, etc. are mentioned, it is understood that other bases, acids, reagents, solvents, coupling agents, etc. may also be used. known in the art, and therefore included in the present invention. Changes in reaction conditions, such as temperature and/or reaction time, which may be used as is known in the art, are also within the scope of the present invention. All stereoisomers of the compounds in these diagrams, unless otherwise indicated, are also included in the scope of the present invention.

The compounds of the present invention can be synthesized from naturally occurring betulinic acid. Key intermediates required for the synthesis of the analogues are both commercially available and can be prepared by methods published in the literature. For example, the key intermediates in the present invention were prepared by modification of procedures published in Journal of organic chemistry 2010, 75, 1285-1288; Journal of organic chemistry 2000, 65, 3934-3940; Tetrahedron: asymmetry 2008, 19, 302-308; or Tetrahedron: asymmetry 2003, 14, 217223.

In another embodiment, the present invention relates to a method for preparing the compounds of general formula (I) presented in the following general schemes. One skilled in the art will appreciate that the following general schemes can be adapted to prepare the compounds of general formula (I) and the pharmaceutically acceptable salts of the present invention. In this case, all symbols/variables are previously defined unless otherwise specified.

- 10 045041

Scheme-1.

General synthesis procedures

The compounds of formula (I) (wherein R1, R3 and R4 are the same as defined above; P1 and P2 are protecting groups such as acetyl, benzyl, etc.) can be prepared as described in Scheme 1. C- The 3 hydroxy compounds of formula (i) can be protected using a reagent such as acetic anhydride, with acetic acid at reflux (or) with acetic anhydride under alkaline conditions using a reagent such as triethylamine or an N,N-diisopropylethylamine catalyst, such as 4-dimethylaminopyridine, under reflux conditions, in solvents such as tetrahydrofuran (THF), and the like. The C-28 acid compounds of formula (ii) can be converted to the ester compounds of formula (iii) using a reagent such as benzyl bromide and bases such as potassium carbonate or sodium carbonate in solvents such as acetonitrile or THF under boiling conditions with reflux condenser. The C-3 acetyl and C-28 ester compounds of formula (iii) can be converted to the C-20 cyclopropyl compounds of formula (iv) in the presence of cyclopropanating agents such as diethylzinc or dimethylzinc, with diiodomethane in solvents such as dichloromethane or toluene and etc. C-28 ester compounds of formula (iv) can be deprotected to C-28 acid compounds of formula (v) using (i) a catalyst such as Pd/C in solvents such as ethyl acetate and methanol and the like. , in an atmosphere of hydrogen under pressure (or) (ii) a catalyst like Pd/C and a reagent like ammonium formate, in solvents like ethanol and THF. C-28 acid compounds of formula (v) can be converted to C-17 isocyanate compounds of formula (vi) in the presence of a reagent such as diphenylphosphoryl azide (DPPA) and the like, in the presence of bases such as triethylamine (TEA) or N ,N-diisopropylethylamine and the like, in solvents such as toluene or 1,4-dioxane and the like. C-17 isocyanate compounds of formula (vi) can be converted to C-17 amine hydrochloride compounds of formula (vii) in the presence of reagents such as concentrated HCl and the like, in solvents such as 1,4-dioxane and the like . C-17 amine hydrochloride compounds of formula (vii) can be converted to C-17 amine-protected compounds of formula (viii) in the presence of reagents such as di-tert-butyl dicarbonate [(Boc)2O] and the like, in the presence of bases such as sodium bicarbonate (NaHCO3) or sodium carbonate and the like, in solvents such as 1,4-dioxane: H2O and the like. C-3 hydroxy compounds of formula (viii) can be coupled with compounds of formula (ix) to produce C-3 ester compounds of formula (x) with a reagent such as EDC.HCl or the like and a catalyst such as 4-dimethylaminopyridine (DMAP ) etc., in solvents such as dichloromethane (DCM) or THF, etc. C-17 amine protected compounds of formula (x) can be converted to C-17 amine compounds of formula (xi) in the presence of reagents such as 4N HCl in 1,4-dioxane and the like, in solvents such as 1 ,4-dioxane, etc. C-17 amino compounds of formula (xi) can be coupled with hydroxy compounds of formula (xii) in situ to produce the corresponding C-17 substituted amino compounds of formula (xiii) in the presence of reagents such as triflic anhydride, 2,6-lutidine and bases, such as triethylamine or N,Ndiisopropylethylamine and the like, in solvents such as dichloromethane and the like. C-3-protected compounds of formula (xiii) can be deprotected to yield the corresponding acidic compounds

- 11 045041 formula (I) of the present invention in the presence of reagents such as KOH or NaOH, in a combination of solvents such as methanol, THF, H2O and the like.

Scheme-2.

The compounds of formula (I) (wherein R1, R3 and R4 are the same as defined above; P1 and P2 are protecting groups such as acetyl, benzyl, etc.) can be prepared as described in Scheme 2. C- The 3 hydroxy compounds of formula (i) can be protected using a reagent such as acetic anhydride, with acetic acid at reflux (or) with acetic anhydride under alkaline conditions using a reagent such as triethylamine or N,N-diisopropylethylamine and a catalyst such as 4-dimethylaminopyridine, under reflux conditions, in solvents such as tetrahydrofuran (THF), and the like. The C-28 acid compounds of formula (ii) can be converted to the C-17 isocyanate of the compound of formula (iii) in the presence of a reagent such as diphenylphosphoryl azide (DPPA) and the like, in the presence of bases such as triethylamine (TEA) or N ,Ndiisopropylethylamine and the like, in solvents such as toluene or 1,4-dioxane and the like. C-17 isocyanate compounds of formula (iii) can be converted to C-17 amine hydrochloride compounds of formula (iv) in the presence of reagents such as conc. HCl, etc. in solvents such as 1,4-dioxane and the like. C-17 amine hydrochloride compounds of formula (iv) can be converted to C-17 amine-protected compounds of formula (v) in the presence of reagents such as di-tert-butyl dicarbonate [(Boc)2O] and the like, in the presence bases such as sodium bicarbonate (NaHCO3) or sodium carbonate and the like, in solvents such as 1,4-dioxane:H2O and the like. C-3 hydroxy compounds of formula (v) can be coupled with compounds of formula (vi) to produce C-3 ester compounds of formula (vii) with a reagent such as EDC.HCl or the like and a catalyst such as 4-dimethylaminopyridine (DMAP ) or the like, in solvents such as dichloromethane (DCM) or THF or the like. C17 amine-protected compounds of formula (vii) can be converted to C-17 amine compounds of formula (viii) in the presence of reagents such as 4N HCl in 1,4-dioxane or the like, in solvents such as 1,4-dioxane or similar. C-17 amino compounds of formula (xiii) can be coupled with hydroxy compounds of formula (ix) in situ to produce the corresponding substituted C-17 amino compounds of formula (x) in the presence of reagents such as triflic anhydride, 2,6-lutidine and bases, such as triethylamine or N,N-diisopropylethylamine and the like, in solvents such as dichloromethane and the like. CC-3 protected compounds of formula (x) can be deprotected to obtain the corresponding acidic compounds of formula (I) of the present invention in the presence of reagents such as KOH or NaOH, in a combination of solvents such as methanol, THF, H2O and the like.

- 12 045041

Scheme-3.

C-17 amino compounds of formula (xiv) can be coupled with hydroxy compounds of formula (xv) in situ to produce the corresponding substituted C-17 amino compounds of formula (xvi) in the presence of reagents such as triflate anhydride, 2,6-lutidine and bases, such as triethylamine or N,N-diisopropylethylamine or the like, in solvents such as dichloromethane or the like. CC-3 protected compounds of formula (xvi) can be deprotected to give the corresponding acidic compounds of formula (xvii) in the presence of reagents such as KOH or NaOH, in a combination of solvents such as methanol, THF, H2O and the like. The silyl ether group in the compounds of formula (xvii) can be deprotected to obtain the corresponding acidic compounds of the present invention of formula (I) using a reagent like TBAF in THF or the like and in solvents like THF and the like.

In an additional aspect, the compounds of the present invention may also contain non-naturally occurring proportions of isotopes of the atoms of one or more of the atoms that constitute such compounds. For example, the present invention also covers isotopically labeled variants of the present invention that are identical to those provided herein except for the fact that one or more atoms of the compound are replaced by an atom having an atomic mass or mass number different from the predominant atomic mass or mass number typically naturally occurring for that atom. As stated, all isotopes of any particular atom or element are included within the scope of the compounds of the invention and their uses. Examples of isotopes that can be included in the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine, such as 2H(D), 3H, nC, 13C, 14C, 13N, 15N , 150, 170, 180, 32Р, 33Р, 35S, 18F, 36С1, 123I and 125I. Specific isotopes are -CD3 or -C(D2)-. The isotopically labeled compounds of the present invention can generally be prepared by following procedures similar to those disclosed in the diagrams and/or examples herein below, by replacing the isotopically labeled reagent with a non-isotopically labeled reagent.

Abbreviations: The abbreviations used throughout the specification may be summarized herein below with their specific meaning: 1H NMR (proton nuclear magnetic resonance); Hz (hertz); MHz (megahertz); DMSO-d6 (deuterated dimethyl sulfoxide); δ (delta); ppm (ppm, parts per million); s (singlet); d (doublet); dd(double doublet(you)); t (triplet); m (multiplet); J (spin-spin interaction constant, coupling); JAB (spin-spin coupling constant); ABq (AVquartet); brs (wide singlet); ml (milliliter); °C (degrees Celsius); mole (mole(s)); mmol (millimol(s)); N (normal solution); g (gram(s)); pH (strength of hydrogen); Pd/C (palladium on activated carbon); eq(equivalent(s)); psi (pounds per square inch); ESI-MS (electrospray ionization mass spectrometry); ES-MS (electrospray mass spectrometry); m/z (mass to charge ratio); MH- (maximum peak of the mass spectrum, corresponding to undissociated molecules minus hydrogen-); M+Na+ (maximum n+IR mass spectrum, corresponding to undissociated molecules plus sodium+); M+H+ (maximum peak of the mass spectrum corresponding to undissociated molecules plus hydrogen+); DCM (dichloromethane); DMF (N,N-dimethylformamide); THF (tetrahydrofuran); Na ₂ SO ₄ (sodium sulfate); HCl (hydrochloric acid); TLC (thin layer chromatography); % (percent); TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl); KBr (potassium bromide); DIBAL-H (diisobutylaluminum hydride); DAST (diethylaminosulfur trifluoride); TBAF (tetra-n-butylammonium fluoride); NaOH (sodium hydroxide); KOH (potassium hydroxide); EDC.HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride); MTBE (methyl tert-butyl ether); HBTU (N,N,N',N'-tetramethyl-O-(1H-benzotriazol-1yl)uronium hexafluorophosphate); h(hour(s)); H (hydrogen); APCI-MS (atmospheric pressure chemical ionization mass spectroscopy); DMAP (4-dimethylaminopyridine); TBDMSCI (tert-butyldimethylsilyl chloride).

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Experimental part of the invention

The present invention is further illustrated by the following examples, which should in no way be construed as limiting the scope of the disclosure of the present invention, but rather are intended to be illustrative only. On the contrary, it should be clearly understood that one can resort to various other embodiments, modifications and equivalents thereof, which, after reading the description of the present invention, can be suggested by any person skilled in the art without deviating from the essence of the present invention. Thus, one skilled in the art will understand how the experiments and examples can be further implemented as described by various changes to the following examples, substituents, reagents, or conditions.

Intermediates

Intermediate 1. Preparation of 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3aamino-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl) )icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3benzyl(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

Stage 1. Synthesis of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-acetoxy-5a,5b,8,8,11a-pentamethyl1-(prop-1-ene-2 -yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxylic acid:

To betulinic acid (200 g, 0.438 mol, 1.0 eq.) in a round bottom flask were added acetic acid (1600 ml) and acetic anhydride (173 g, 1.696 mol, 3.87 eq.). The reaction mixture was stirred and heated to 130-140°C for about 4 hours. The reaction progress was monitored by TLC to determine that the starting material was consumed. Approximately 1100 ml of acetic acid was distilled off under vacuum. The flask was cooled to room temperature, methanol (1400 ml) was slowly added and stirred at room temperature for 12-16 hours. The solid was filtered, washed with methanol (200 ml) and dried in vacuum (50-55°C) to obtain the title compound as a white solid (182 g, 83.32% yield).

Step 2. Synthesis of benzyl(1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-acetoxy-5a,5b,8,8,11apentamethyl-1-(prop-1-ene- 2-yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxylate:

To a stirred solution of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-acetoxy

5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxylic acid (step 1, 180 g, 0.361 mol, 1 .0 eq.) in acetonitrile (1800 ml) was added potassium carbonate (99.75 g, 0.721 mol, 2.0 eq.). The reaction mixture was stirred at room temperature and benzyl bromide (42.5 mL, 0.361 mol, 1.0 eq.) was added dropwise over 30 minutes. The reaction mixture was stirred and heated until condensation appeared for about 6 hours. The reaction progress was monitored by TLC to determine that the starting material was consumed. The reaction mixture was cooled to room temperature, water (2700 ml) was added and stirred for 2 hours. Ethyl acetate (2700 ml) was added and stirred for 30 minutes. The organic layer was separated and the aqueous layer was again extracted twice with ethyl acetate (2x900 ml). The combined organic layers were washed with water (1800 ml) and brine (1800 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by treatment with methanol (720 ml), stirred and heated to 60-65°C for 30 minutes. The reaction mixture was cooled to room temperature and stirred for 2 hours. The mixture was filtered, washed with methanol (180 ml) and dried in vacuum (55-60°C) to obtain the title compound (180 g, 84.7% yield) as a solid.

Step 3. Synthesis of benzyl(1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-acetoxy-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl)icosahydro- 3aH-cyclopenta[a]chrysene-3a-carboxylate:

- 14 045041

To a stirred solution benzyl(1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-acetoxy5a,5b,8,8,11a-pentamethyl-1-(prop-1-ene-2 -yl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxylate (step 2, 120 g, 0.204 mol, 1.0 eq.) in DCM (1200 ml) at -25 to -35°C under nitrogen was added diethylzinc (75.61 g, 408 ml, 0.612 mol, 3.0 eq., 1.5 M in toluene). The reaction mixture was stirred at a temperature of -25 to -35°C for 1 hour. Diiodomethane (81.5 mL, 1.02 mol, 5.0 eq.) was added dropwise slowly at -25 to -35°C over about 45 minutes. The reaction mixture was stirred at a temperature of -25 to -35°C for about 3 hours. The reaction mixture was slowly warmed to room temperature and stirred for about 14 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was cooled to 0-5°C, quenched with saturated ammonium chloride solution (600 ml), 1 NHCl (240 ml) was slowly added to adjust the pH (3 to 4) and stirred for 30 minutes. The organic layer was separated and the aqueous layer was extracted with DCM (600 ml). The combined organic layers were washed with 10% sodium bicarbonate solution, water (1800 ml) and brine (1800 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. Diisopropyl ether (360 ml) was added to the resulting solid and stirred at room temperature for 3 hours. The solid was filtered, washed with diisopropyl ether (120 ml) and dried in vacuo (55-60°C) to obtain the title compound (100 g, 81.4% yield) as a white solid.

Step 4. Synthesis of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-acetoxy-5a,5b,8,8,11a-pentamethyl1-(1-methylcyclopropyl)icosahydro-3aH -cyclopenta[a]chrysene-3a-carboxylic acid:

To a suspension of 10% Pd/C (15 g, 50% humidity) in ethyl acetate benzyl(1R,3aS,5aR,5bR,7aR,9S, 11 aR, 11bR,13aR, 13bR)-9-acetoxy-5a,5b,8 8.11 a (350 ml) pentamethyl 1-(1-methylcyclopropyl)icosahydro-3aH-cyclopenta[a]chrysene-3a-carboxylate was added (step 3, 35 g, 58.13 mmol, 1.0 eq.) , dissolved in methanol (200 ml). The reaction mixture was hydrogenated on a Parr shaker at 50 psi for 3 hours. TLC showed the consumption of the starting material. The reaction mixture was filtered through a pad of celite and the filtrate was evaporated under reduced pressure to obtain the title compound (30 g) as a solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.51-4.46 (m, 1H), 2.25-2.21 (m, 1H), 2.13-0.78 (m, 24H), 2.04 (s, 3H), 0.98 (s, 3H), 0.91 (s, 6H), 0.86 ( s, 3H), 0.85 (s, 3H), 0.84 (s, 3H), 0.38-0.34 (m, 2H), 0.27-0.24 (m, 2H); ESI-MS: m/z 511.45 (M-H).

Step 5. Synthesis of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-isocyanato-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl)icosahydro-1 N-cyclopenta[a]chrysene-9-ylacetate:

To a stirred solution of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-acetoxy5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-3aH- cyclopent[a]chrysene-3a-carboxylic acid (step 4, 20 g, 39.00 mmol, 1.0 eq.) in toluene (200 ml) was added diphenylphosphoryl azide (22.36 ml, 97.50 mmol, 2.5 eq. .) and triethylamine (13.70 ml, 97.50 mmol, 2.5 eq.). The reaction mixture was heated until condensation appeared for 1 hour. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, diluted with water (250 ml) and extracted with DCM (2x200 ml). The combined organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was triturated with methanol (100 ml), filtered and dried in vacuo to give the title compound (19 g, 95.5% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.45 (m, 1H), 2.04 (s, 3H), 2.03-0.78 (m, 25H), 1.04 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H) , 0.85 (s, 3H), 0.84 (s, 3H), 0.38-0.26 (m, 4H).

Step 6. Synthesis of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl1-(1-methylcyclopropyl)icosahydro-1H -cyclopenta[a]chrysene-9-ol hydrochloride:

- 15 045041

To a stirred solution of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-isocyanato5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H -cyclopenta[a]chrysene-9-ylacetate (step 5, 19 g, 37.27 mmol, 1.0 eq.) in 1,4-dioxane (190 ml) was added with concentrated HCl (57 ml) at 0°C. The reaction mixture was stirred at 50-60°C overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure and distilled with acetonitrile (100 ml) to obtain the title compound (20 g) as a solid, which was used as such in the next step without further purification. ES-MS: m/z 442.20 (M+H) ⁺ .

Step 7. Synthesis of tert-butyl((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-hydroxy-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl )icosahydro-3aH-cyclopenta[a]chrysen-3a-yl)carbamate:

To a stirred solution of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino

5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)ixahydro-1H-cyclopenta[a]chrysen-9-ol hydrochloride (step 6, 20 g, 41.823 mmol, 1.0 eq.) c To 1,4-dioxane (200 ml) a saturated solution of sodium bicarbonate (100 ml) and tert-butyl dicarbonate (14.41 ml, 62.734 mmol, 1.5 eq.) was added at 0°C. The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, diluted with water and extracted with DCM (2x200 ml). The combined organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 10% ethyl acetate in hexanes as the eluent to give the title compound (9 g, 44.57% yield over two steps) as an off-white solid. Ή NMR (300 MHz, CDCl ₃ ): δ ppm 3.23-3.16 (m, 1H), 2.54-2.51 (m, 1H), 2.42-2.25 (m, 2H), 2.0-1.80 (m, 2H), 1.73-0.67 (m, 20H), 1.42 (s, 9H ), 1.01 (s, 3H), 0.97 (s, 6H), 0.91 (s, 3H), 0.85 (s, 3H), 0.77 (s, 3H), 0.40-0.25 (m, 4H); ES-MS: m/z 564.17 (M+Na)+.

Step 8. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((tert-butoxycarbonyl)amino)-5a,5b,8, 8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9yl)(1R,3S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution tert-butyl((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9hydroxy-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl )icosahydro-3 aH-cyclopenta[a] chrysene-3ail)carbamate (step 7, 9 g, 16.609 mmol, 1.0 eq.) in dichloromethane (90 ml) was added (1S,3R)-3((benzyloxy)carbonyl )-2,2-dimethylcyclobutane-1-carboxylic acid (prepared according to the procedure described in WO2011/007230A2, 8.71 g, 33.21 mmol, 2.0 eq.), 1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride (EDC.HCl) (9.55 g, 49.82 mmol, 3.0 eq.) and 4-(dimethylamino)pyridine (0.81 g, 6.643 mmol, 0.4 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with water (100 ml) and extracted with dichloromethane (2x100 ml). The combined organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using 10% ethyl acetate:hexane as eluent to give the title compound (11 g, 84.29% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 7.34 (m, 5H), 5.15, 5.09 (ABq, JAB = 12.3 Hz, 2H), 4.44 (dd, J= 11.1, 4.5 Hz, 1H), 4.17 (s, 1H), 2.84-2.73 (m, 2H) , 2.69-2.58 (m, 1H), 2.57-2.51 (m, 1H), 2.42-2.23 (m, 2H), 2.07-1.81 (m, 3H), 1.72-0.76 (m, 20H), 1.42 (s, 9H), 1.34 (s, 3H), 1.0 (s, 3H), 0.96 (s, 6H), 0.91 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.85 (s, 3H ), 0.40-0.25 (m, 4H); ES-MS: m/z 786.47 (M+H) ⁺ .

Step 9. Synthesis of 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a- 16 045041 pentamethyl-1-( 1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3-benzyl(1S,3R)-2,2dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11αR,11bR,13aR,13bR)-3a-((tertbutoxycarbotnyl)amino)-5a,5b,8,8,11a -pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)(1R,3S)-2,2-dimethylcyclobutane-1,3-dicarboxylate (step 8, 11 g, 13, 99 mmol, 1.0 eq.) in 1,4-dioxane (11 ml) 4N HCl in 1,4-dioxane (33 ml) was added at 0°C. The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, the residue was made basic with saturated sodium bicarbonate solution and extracted with dichloromethane (3x100 ml). The combined organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure to give the title compound (9 g, 93.84% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 7.34 (m, 5H), 5.15, 5.09 (ABq, JAB = 12.3 Hz, 2H), 4.45 (m, 1H), 2.81-2.73 (m, 2H), 2.65-2.62 (m, 1H), 2.08-1.88 ( m, 3H), 1.74-0.78 (m, 23H), 1.34 (s, 3H), 1.03 (s, 3H), 0.96 (s, 6H), 0.91 (s, 3H), 0.87 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H), 0.37-0.22 (m, 4H); ES-MS: m/z 686.45 (M+H)+.

Intermediate 2. Preparation of tert-butyl(2-methyl-1-oxopropan-2-yl)carbamate:

Step 1. Synthesis of tert-butyl(1-hydroxy-2-methylpropan-2-yl)carbamate:

Triethylamine (1.57 mL, 11.217 mmol, 0.1 equiv.) and di-tert-butyl dicarbonate (30.92 ml, 134.60 mmol, 1.2 equiv.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, water (100 ml) was added and the compound was extracted with DCM (2x50 ml). The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was triturated with n-hexane, the solid was filtered and dried in vacuo to give the title compound (14.0 g, 66% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.66 (s, 1H), 4.04 (s, 1H), 3.58 (d, J=6.3 Hz, 2H), 1.43 (s, 9H), 1.25 (s, 6H); ES-MS: m/z 189.80 (M+H)+.

Step 2. Synthesis of tert-butyl(2-methyl-1-oxopropan-2-yl)carbamate:

To a stirred solution of tert-butyl (1-hydroxy-2-methylpropan-2-yl)carbamate (step 1.4 g, 21.134 mmol, 1.0 eq.) in dichloromethane (50 ml) was added TEMPO (0.098 g, 0.634 mmol , 0.03 eq.), KBr (0.251 g, 2.1134 mmol, 0.1 eq.), sodium bicarbonate (10.82 g, 128.91 mmol, 6.1 eq.), 10% NaOCl (26 ml) and then water (1.05 ml). The reaction mixture was stirred at room temperature for about 30 minutes. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with dichloromethane (2x50 ml) and washed with water (50 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to obtain the title compound (7.0 g) as a colorless liquid. The resulting compound was used as such in the next step without further purification; 1H NMR (300 MHz, CDCl ₃ ): δ ppm 9.43 (s, 1H), 4.98 (s, 1H), 1.44 (s, 9H), 1.33 (s, 6H); ES-MS: m/z 187.78 (M+H) ⁺ .

Intermediate 3. Preparation of (S)-4-(1-chloropropan-2-yl)thiomorpholine-1,1-dioxide:

Step 1. Synthesis of (S)-4-(1-hydroxypropan-2-yl)thiomorpholine-1,1-dioxide:

- 17 045041

Divinyl sulfone ( 2.67 ml, 26.62 mmol, 2.0 eq.) and triethylamine (5.61 ml, 39.93 mmol, 3.0 eq.). The reaction mixture was heated to 85°C for about 3 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure and the crude compound was purified by silica gel column chromatography using 5% methanol in DCM as eluent to give the title compound (2.0 g, 80% yield) as an off-white solid . ¹ H NMR (300 MHz, CDCl ₃ ):

δ ppm 3.51-3.34 (m, 2H), 3.22-3.16 (m, 2H), 3.13-3.06 (m, 4H), 3.03-2.90 (m, 3H), 2.60 (s, 1H), 0.97 (d, J=6.9 Hz, 3H); ES-MS: m/z 193.97 (M+H)+.

Step 2. Synthesis of (S)-4-(1-chloropropan-2-yl)thiomorpholine-1,1-dioxide:

To a stirred solution of (S)-4-(1-hydroxypropan-2-yl)thiomorpholine-1,1-dioxide (step 1, 1.3 g, 6.72 mmol, 1.0 eq.) in 1,2- dichloroethane (13 ml) was added thionyl chloride (1.47 ml, 20.18 mmol, 3.0 eq.). The reaction mixture was heated until condensation appeared for about 3 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, diluted with dichloromethane (20 ml), washed with saturated sodium bicarbonate solution and water. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 3% methanol in dichloromethane as eluent to give the title compound (0.925 g, 64.91% yield) as a colorless liquid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.06-3.99 (m, 1H), 3.12-3.05 (m, 8H), 2.84-2.77 (m, 1H), 2.72-2.66 (m, 1H), 1.51 (d, J=6.6 Hz, 3H); ES-MS: m/z 212.34 (M+H)+.

Intermediate 4. Preparation of (R)-4-(1-hydroxy-3-methylbutan-2-yl)thiomorpholine-1,1 dioxide:

Triethylamine ( 12.25 mL, 87.207 mmol, 3.0 eq.), followed by divinyl sulfone (6.869 g, 58.139 mmol, 2.0 eq.). The reaction mixture was heated until condensation appeared for about 3 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure. The crude compound was purified by silica gel column chromatography using 2% methanol in dichloromethane as eluent to give the title compound (3.6 g, 55.9% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 3.69 (m, 1H), 3.46-3.39 (m, 1H), 3.32-3.14 (m, 4H), 3.05-3.03 (m, 4H), 2.45-2.38 (m, 1H), 1.85-1.73 (m, 1H ), 1.02 (d, J=6.6 Hz, 3H), 0.89 (d, J=6.6 Hz, 3H).

Intermediate 5. Preparation of methyl (R)-2-(1,1-dioxidothiomorpholino)-3hydroxypropanoate:

To a stirred solution of methyl D-serinate hydrochloride (4.0 g, 25.70 mmol, 1.0 eq.) in 1,4dioxane (20 ml) and ethanol (20 ml) was added triethylamine (14.34 ml, 102.8 mmol , 4.0 equiv.), followed by divinyl sulfone (5.15 ml, 51.4 mmol, 2.0 equiv.). The reaction mixture was heated to 85°C for 3 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 5-10% methanol in dichloromethane to give the title compound (5.8 g, 95% yield) as a white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 3.90-3.84 (m, 1H), 3.81-3.73 (m, 1H), 3.76 (s, 3H), 3.54 (dd, J=8.1, 6.0 Hz, 1H), 3.40-3.30 (m, 2H), 3.17- 3.13 (m, 1H), 3.09-3.06 (m, 5H), 2.48 (s, 1H).

Intermediate 6. Preparation of (R)-4-(1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2-yl)thiomorpholine-1,1-dioxide:

- 18 045041

Step 1. Synthesis of methyl (S)-2-(1,1-dioxidothiomorpholino)-3-hydroxypropanoate:

To a stirred solution of methyl L-serinate hydrochloride (4 g, 25.707 mmol, 1.0 eq.) in 1,4dioxane (40 mL) and ethanol (40 mL) was added triethylamine (14.3 mL, 102.828 mmol, 4.0 eq. ) and divinyl sulfone (6 g, 51.414 mmol, 2.0 eq.). The reaction mixture was stirred at 85°C for about 3 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a 40-80% ethyl acetate in hexane gradient to give the title compound (5 g, 81.96% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 3.91-3.83 (m, 1H), 3.81-3.73 (m, 1H), 3.76 (s, 3H), 3.54 (dd, J=8.1, 6.0 Hz, 1H), 3.403.31 (m, 2H), 3.19- 3.02 (m, 6H), 2.42 (s, 1H); ES-MS: m/z 238.04 (M+H)+.

Step 2. Synthesis of methyl (S)-3-((tert-butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)propanoate:

To a stirred solution of methyl (S)-2-(1,1-dioxidothiomorpholino)-3-hydroxypropanoate (step 1, 2.5 g, 10.536 mmol, 1.0 eq.) in DCM (37.5 ml) at 0° Triethylamine (4.4 mL, 31.609 mmol, 3.0 eq), 4-(dimethylamino)pyridine (0.064 g, 0.525 mmol, 0.05 eq) was added followed by tert-butyldimethylsilyl chloride (2.5 g, 16.858 mmol , 1.6 eq.). The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water and the aqueous layer was separated. The organic layer was washed with saturated ammonium chloride solution, 5% sodium bicarbonate solution, water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-40% ethyl acetate in hexane to give the title compound (3 g, 81% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 3.93-3.90 (m, 2H), 3.72 (s, 3H), 3.53-3.49 (m, 1H), 3.29-3.24 (m, 4H), 3.08-3.02 (m, 4H), 0.87 (s, 9H), 0.05 (s, 6H).

Step 3. Synthesis of (R)-4-(1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2-yl)thiomorpholine-1,1 dioxide:

To a stirred solution of methyl (S)-3-((tert-butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)propanoate (step 2, 3.5 g, 9.956 mmol, 1.0 eq.) in methanol ( 70 ml) at 0°C, sodium borohydride (7.53 g, 199.129 mmol, 20 eq.) was added in portions. The reaction mixture was stirred at room temperature for about 2 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was neutralized with 1N HCl, the organic phase was evaporated under reduced pressure and the aqueous layer was extracted with ethyl acetate (2x100 ml). The combined organic layer was washed with water (100 ml), brine (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-10% methanol in dichloromethane to give the title compound (3 g, 93.7% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 3.73-3.68 (m, 1H), 3.67-3.57 (m, 1H), 3.56-3.47 (m, 1H), 3.46-3.33 (m, 2H), 3.183.01 (m, 7H), 2.99-2.89 (m , 1H), 0.89 (s, 9H), 0.06 (s, 6H); ES-MS: m/z 323.64 (M+H) ⁺ .

Intermediate 7. Preparation of (S)-4-(1-hydroxy-3-methylbutan-2-yl)thiomorpholine-1,1 dioxide:

- 19 045041

To a stirred solution of (S)-2-amino-3-methylbutan-1-ol (2.0 g, 19.398 mmol, 1.0 eq.) in ethanol (10 ml) and 1,4-dioxane (10 ml) was added divinyl sulfone (3.89 ml, 38.797 mmol, 2.0 equiv.) and triethylamine (8.178 ml, 58.19 mmol, 3.0 equiv.). The reaction mixture was heated until condensation appeared for about 5 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure. The residue was purified by silica gel column chromatography using 2% methanol in DCM as eluent to give the title compound (4.0 g, 95.2% yield) as an off-white solid. 1H NMR (300 MHz, CDCls): δ ppm 3.74-3.68 (m, 1H), 3.45-3.38 (m, 1H), 3.32-3.14 (m, 4H), 3.05-3.02 (m, 4H), 2.47-2.36 (m, 2H), 1.83-1.75 (m , 1H), 1.02 (d, J=6.6 Hz, 3H), 0.89 (d, J=6.6 Hz, 3H); ES-MS: m/z 221.85 (M+H) ⁺ .

Intermediate 8. Preparation of 4-((2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1hydroxybutan-2-yl)thiomorpholine-1,1-dioxide:

Stage 1. Synthesis of methyl-B-threoninate:

Thionyl chloride (18.38 mL, 251.84 mmol, 3.0 eq) was added to a stirred solution of L-threonine (10 g, 83.94 mmol, 1.0 eq.) in methanol (100 mL) at 0°C. . The reaction mixture was heated until condensation appeared for about 3 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated under reduced pressure, ethyl acetate (100 ml) and triethylamine (35.39 ml, 251.84 mmol, 3.0 eq.) were added to the reaction mixture, and a precipitate formed. The resulting precipitate was collected by filtration and the filtrate was evaporated under reduced pressure to obtain the title compound (8 g) as an oil. The resulting compound was used as such in the next step without further purification. 1H NMR (300 MHz, DMSO-^): 5 ppm 4.62 (s, 1H), 3.85-3.82 (m, 1H), 3.61 (s, 3H), 3.16 (d, J=3.9 Hz, 1H), 1.08 (d, J=6.3 Hz, 3H); ES-MS: m/z 134.07 (M+H)+.

Step 2. Synthesis of methyl-(2S,3R)-2-(1,1-dioxidothiomorpholino)-3-hydroxybutanoate:

Triethylamine (7.91 ml, 56.31 mmol, 3.0 equiv.) and divinyl sulfone (5.61 ml, 37.55 mmol, 2.0 equiv.). The reaction mixture was heated until condensation appeared for about 3 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure. The residue was purified by silica gel column chromatography using 2% methanol in DCM as eluent to give the title compound (4 g, 85.1% yield) as a solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.13-3.96 (m, 1H), 3.76 (s, 3H), 3.42-2.90 (m, 9H), 1.21 (d, J=6.0 Hz, 3H); ES-MS: m/z 252.26 (M+H) ⁺ .

Step 3. Synthesis of methyl (2S,3R)-3-((tert-butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)butanoate:

To a stirred solution of methyl (2S,3R)-2-(1,1-dioxidothiomorpholino)-3-hydroxybutanoate (step 2, 1.5 g, 5.976 mmol, 1.0 eq.) in DCM (15 ml) and DMF (10 mL) imidazole (0.813 g, 11.952 mmol, 2.0 eq.) and tert-butyldimethylsilyl chloride (1.35 g, 8.964 mmol, 1.5 eq.) were added. The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with water (20 ml) and extracted with DCM (2x30 ml). The combined organic layer was separated, dried over Na ₂ SO ₄ , filtered and

- 20 045041 was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 1% methanol in DCM as eluent to give the title compound (0.6 g, 27.52% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ):

δ ppm 4.39 (m, 1H), 3.73 (s, 3H), 3.64-3.56 (m, 2H), 3.30-3.24 (m, 3H), 3.06-3.03 (m, 4H), 1.26 (d, J=6.3 Hz, 3H), 0.86 (s, 9H), 0.08 (s, 3H), 0.04 (s, 3H); ES-MS: m/z 388.10 (M+Na)+.

Step 4. Synthesis of 4-((2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1-hydroxybutan-2-yl)thiomorpholine1,1-dioxide:

To a stirred solution of methyl (2S,3R)-3-((tert-butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)butanoate (step 3, 0.6 g, 1.641 mmol, 1.0 eq.) DIBAL-H (6.5 mL, 9.847 mmol, 6.0 eq., 1.5 M in toluene) was added to DCM (12 mL) at -78°C. The reaction mixture was stirred at the same temperature for about 4 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was quenched with saturated ammonium chloride solution and extracted with ethyl acetate (2x30 ml). The organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 2% methanol in DCM as eluent to give the title compound (0.442 g, 79.92% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.11-4.03 (m, 1H), 3.71-3.64 (m, 2H), 3.55-3.39 (m, 4H), 3.19-3.08 (m, 4H), 2.81-2.74 (m, 1H), 2.49 (s, 1H ), 1.30 (d, J=6.0 Hz, 3H), 1.0 (s, 9H), 0.20 (s, 6H); ESI-MS: m/z 360.05 (M+Na) ⁺ .

Intermediate 9. Preparation of (S)-4-(1-((tert-butyldimethylsilyl)oxy)-3hydroxypropan-2-yl)thiomorpholine-1,1-dioxide:

Step 1. Synthesis of methyl (R)-3-((tert-butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)propanoate:

To a stirred solution of methyl (R)-2-(1,1-dioxidothiomorpholino)-3-hydroxypropanoate (intermediate 5, 4.4 g, 18.54 mmol, 1.0 eq.) in DCM (45 ml) at At 0°C, triethylamine (7.7 mL, 55.63 mmol, 3.0 eq), 4-(dimethylamino)pyridine (0.113 g, 0.92 mmol, 0.05 eq) and tert-butyldimethylsilyl chloride (4.4 g, 29.67 mmol, 1.6 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with DCM (150 ml), washed with water (100 ml) and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-40% ethyl acetate in hexane to give the title compound (5 g, 77% yield) as an off-white solid. 1H NMR (300 MHz, CDC13): δ ppm 3.93-3.90 (m, 2H), 3.72 (s, 3H), 3.51 (t, J=5.7 Hz, 1H), 3.26-3.24 (m, 4H), 3.06-3.02 (m, 4H), 0.87 (s, 9H), 0.05 (s, 6H); ES-MS: m/z 352.55 (M+H) ⁺ .

Step 2. Synthesis of (S)-4-(1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2-yl)thiomorpholine-1,1 dioxide:

To a stirred solution of methyl (R)-3-((tert-butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)propanoate (step 1, 5 g, 14.223 mmol, 1.0 eq.) in methanol (100 ml) at 0°C add in portions

- 21 045041 sodium borohydride (10.76 g, 284.47 mmol, 20.0 eq.) was added. The reaction mixture was stirred at room temperature for about 2 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was cooled to 0°C, neutralized with 1N HCl and the organic phase was evaporated under reduced pressure. The aqueous layer was extracted with ethyl acetate (2x150 ml). The combined organic layer was washed with water (150 ml) and brine (150 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-10% methanol in dichloromethane to give the title compound (4 g, 87% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 3.75-3.62 (m, 1H), 3.61-3.55 (m, 1H), 3.54-3.46 (m, 1H), 3.42-3.33 (m, 2H), 3.17-3.10 (m, 2H), 3.10-3.02 (m , 5H), 3.0-2.90 (m, 1H), 2.50 (s, 1H), 0.90 (s, 9H), 0.06 (s, 6H); ES-MS: m/z 324.56 (M+H)+.

Intermediate 10. Preparation of (S)-4-(1-fluoro-3-hydroxypropan-2-yl)thiomorpholine-1,1 dioxide:

Step 1. Synthesis of (S)-4-(1-((tert-butyldimethylsilyl)oxy)-3-fluoropropan-2-yl)thiomorpholine-1,1 dioxide:

To a stirred solution of (R)-4-(1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2yl)thiomorpholine-1,1-dioxide (intermediate 6, 7.5 g, 23.291 mmol, 1.0 eq.) in DCM (150 ml) at 0°C was added DAST (3 ml, 23.291 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature for about 1 hour. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was quenched with saturated sodium carbonate solution and extracted with DCM. The organic layer was washed with water, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-20% ethyl acetate in hexane to give the title compound (1.8 g, 23.8% yield) as a colorless liquid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.67-4.64 (m, 1H), 4.51-4.49 (m, 1H), 3.79-3.71 (m, 2H), 3.25-3.22 (m, 3H), 3.14-2.87 (m, 6H), 0.90 (s, 9H ), 0.07 (s, 6H); ES-MS: m/z 326.46 (M+H) ⁺ .

Step 2. Synthesis of (S)-4-(1-fluoro-3-hydroxypropan-2-yl)thiomorpholine-1,1-dioxide:

To a stirred solution of (S)-4-(1-((tert-butyldimethylsilyl)oxy)-3-fluoropropan-2yl)thiomorpholine-1,1-dioxide (step 1, 1.8 g, 5.538 mmol, 1.0 eq. .) in THF (36 ml) at 0°C was added 1.0 M TBAF in THF (6.64 ml, 6.646 mmol, 1.2 eq.). The reaction mixture was stirred at room temperature for about 4 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with DCM, washed with saturated sodium bicarbonate solution and water. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-4% methanol in dichloromethane to give the title compound (0.950 g, 81.9% yield) as a colorless liquid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.65-4.63 (m, 1H), 4.49-4.47 (m, 1H), 3.66-3.64 (m, 2H), 3.39-3.30 (m, 2H), 3.21-3.12 (m, 3H), 3.11-3.05 (m , 4H), 2.20 (s, 1H); ES-MS: m/z 212.34 (M+H) ⁺ .

Intermediate 11. Preparation of (R)-4-(1-fluoro-3-hydroxypropan-2-yl)thiomorpholine-1,1 dioxide:

Step 1. Synthesis of (R)-4-(1-((tert-butyldimethylsilyl)oxy)-3-fluoropropan-2-yl)thiomorpholine-1,1 dioxide:

- 22 045041

To a stirred solution of (S)-4-(1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2yl)thiomorpholine-1,1-dioxide (intermediate 9.6 g, 18.633 mmol, 1.0 eq. ) in DCM (120 ml) at 0°C was added DAST (1.83 ml, 18.633 mmol, 1.0 eq.). The reaction mixture was stirred at 0°C for about 1 hour, brought to room temperature and stirred for about 1 hour. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was cooled to 0°C, quenched with saturated sodium carbonate solution and extracted with DCM (100 ml). The organic layer was washed with water, brine (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-20% ethyl acetate in hexane to give the title compound (1.3 g, 21.5% yield) as a colorless liquid. Ή NMR (300 MHz, CDCl ₃ ): δ ppm 4.67-4.64 (m, 1H), 4.51-4.49 (m, 1H), 3.75 (dd, J=6.0, 1.2 Hz, 2H), 3.25-3.22 (m, 4H), 3.04-2.90 (m, 5H), 0.89 (s, 9Н), 0.06 (s, 6Н); ES-MS: m/z 326.53 (M+H)+.

Step 2. Synthesis of (R)-4-(1-fluoro-3-hydroxypropan-2-yl)thiomorpholine-1,1-dioxide:

To a stirred solution of (R)-4-(1-((tert-butyldimethylsilyl)oxy)-3-fluoropropan-2yl)thiomorpholine-1,1-dioxide (step 1, 1.3 g, 4.0 mmol, 1, 0 eq.) in THF (26 ml) at 0°C was added TBAF (4.8 ml, 4.80 mmol, 1.2 eq., 1.0 M in THF). The reaction mixture was stirred at room temperature for about 4 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with DCM (100 ml), washed with saturated sodium bicarbonate solution and water. The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-3% methanol in dichloromethane to give the title compound (0.650 g, 77% yield) as a colorless liquid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.65-4.63 (m, 1H), 4.49-4.47 (m, 1H), 3.66-3.64 (m, 2H), 3.39-3.30 (m, 2H), 3.22-3.12 (m, 3H), 3.11-3.03 (m , 4H), 2.19 (s, 1H); ES-MS: m/z 212.34 (M+H) ⁺ .

Intermediate 12. Preparation of (S)-2-(4-(isopropylsulfonyl)piperazin-1-yl)propan1-ol:

Step 1. Synthesis of tert-butyl-4-(isopropylsulfonyl)piperazine-1-carboxylate:

A mixture of tert-butylpiperazine-1-carboxylate (5 g, 26.84 mmol, 1.0 eq) and DCM (100 ml) was cooled to -25°C, triethylamine (3.25 g, 32.21 mmol, 1. 2 eq.), and then isopropylsulfonyl chloride (4.4 g, 30.87 mmol, 1.15 eq.) was slowly added dropwise. The reaction mixture was slowly warmed to room temperature and stirred overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was cooled to 0°C, neutralized with 1N HCl solution and extracted with DCM (3x400 ml). The combined organic layer was washed with water (100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (6.9 g, 87.92% yield) as a yellow solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 3.48-3.46 (m, 4H), 3.33-3.30 (m, 4H), 3.23-3.14 (m, 1H), 1.46 (s, 9H), 1.34 (d, J=6.9 Hz, 6H); ES-MS: m/z 315.45 (M+Na)+.

Stage 2. Synthesis of 1-(isopropylsulfonyl)piperazine: .—. o HN^__ _z N-^°

To a stirred solution of tert-butyl 4-(isopropylsulfonyl)piperazine-1-carboxylate (step 1, 6.9 g, 23.60 mmol, 1.0 eq.) in 1,4-dioxane (30 ml) at 0° 4N HCl/1,4-dioxane (30 ml) was added. The reaction mixture was stirred at room temperature for about 3 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, saturated sodium bicarbonate solution was added and extracted with DCM (3x400 ml). The combined organic layers were washed with water (150 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-10% methanol in dichloromethane. Fractions containing the expected product were combined and concentrated under reduced pressure to give the title compound (2.8 g, 62.2% yield) as a yellow semi-solid. 1H NMR (300 MHz, CDCb): δ ppm 3.35-3.32 (m, 4H), 3.23-3.14 (m, 1H), 2.92-2.89 (m, 4H), 1.34 (d, J=6.9 Hz, 6H); ES-MS: m/z 193.42 (M+H)+.

Step 3. Synthesis of methyl-(S)-2-(4-(isopropylsulfonyl)piperazin-1-yl)propanoate:

To a stirred solution of methyl (R)-2-hydroxypropanoate (1.461 g, 14.042 mmol, 1.5 eq) in DCM (40 ml) at -78°C was added trifluoromethanesulfonic anhydride (2.67 ml, 15.915 mmol, .7 eq.). The reaction mixture was stirred at -78°C for about 45 minutes, during which time 2,6lutidine (1.85 ml, 15.915 mmol, 1.7 eq.) was added and stirred at the same temperature for about 45 minutes. A solution of 1-(isopropylsulfonyl)piperazine (step 2, 1.8 g, 9.361 mmol, 1.0 eq) in DCM (10 mL) was added to the reaction mixture, followed by the addition of triethylamine (3.9 mL, 28.085 mmol, 3. 0 equiv.) at 78°C. The reaction mixture was allowed to stir at 0°C for about 1 hour and stirred at room temperature for about 3 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was cooled to 0°C, quenched with saturated sodium bicarbonate solution and extracted with DCM (3x250 ml). The combined organic layers were washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (1.8 g, 69.2% yield) as a pale yellow oil. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 3.71 (s, 3H), 3.43-3.31 (m, 5H), 3.23-3.14 (m, 1H), 2.71-2.59 (m, 4H), 1.34 (d, J=6.9 Hz, 6H), 1.30 (d, J=7.2 Hz, 3H); ES-MS: m/z 279.40 (M+H)+.

Step 4. Synthesis of (S)-2-(4-(isopropylsulfonyl)piperazin-1-yl)propan-1-ol:

To a stirred solution of methyl (S)-2-(4-(isopropylsulfonyl)piperazin-1-yl)propanoate (step 3, 1.8 g, 6.466 mmol, 1.0 eq.) in methanol (100 ml) was added in portions at 0°C sodium borohydride (6.11 g, 161.65 mmol, 25.0 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was cooled to 0°C, the pH was adjusted to 6.0 with 1N HCl and evaporated under reduced pressure. The reaction mixture was cooled to 0°C, saturated sodium bicarbonate solution was added and extracted with DCM (3x250 ml). The combined organic layers were washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (1.0 g, 61.8% yield) as a liquid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 3.55-3.39 (m, 6H), 3.24-3.13 (m, 1H), 3.01-2.90 (m, 1H), 2.87-2.78 (m, 2H), 2.65-2.57 (m, 2H), 1.35 (d, J =6.6 Hz, 6H), 0.99 (d, J=6.9 Hz, 3H); ES-MS: m/z 251.58 (M+H) ⁺ .

Intermediate 13. Preparation of 4-((2R,3S)-3-fluoro-1-hydroxybutan-2-yl)thiomorpholine 1,1-dioxide:

Step 1. Synthesis of methyl (2R,3S)-2-(1,1-dioxidothiomorpholino)-3-fluorobutanoate:

To a stirred solution of methyl (2S,3R)-2-(1,1-dioxidothiomorpholino)-3-hydroxybutanoate (intermediate 8, step 2, 15 g, 59.76 mmol, 1.0 eq.) in DCM (150 ml) at 0°C, DAST (8.68 ml, 65.73 mmol, 1.1 eq.) was added. The reaction mixture was stirred at the same temperature for about 30 minutes and stirred at room temperature for about 1 hour. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was cooled to 0°C, saturated sodium bicarbonate solution was added and extracted with DCM (2x200 ml). The organic layer was washed with water, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using

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1% methanol in dichloromethane as eluent to give the title compound (8 g, 52.94% yield) as a liquid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 5.22-5.13 (m, 0.5N), 5.06-4.98 (m, 0.5N),

3.77 (s, 3H), 3.44-3.22 (m, 5H), 3.10-2.96 (m, 4H), 1.45 (d, J=6.3 Hz, 1.5H), 1.37 (d, J=6.3 Hz, 1.5H) ; ES-MS:

m/z 254.46 (M+H) ⁺ .

Step 2. Synthesis of 4-((2R,3S)-3-fluoro-1-hydroxybutan-2-yl)thiomorpholine-1,1-dioxide:

Methyl-(2R,3S)-2-(1,1-dioxidothiomorpholino) was added to a stirred suspension of lithium aluminum hydride (3.59 g, 94.86 mmol, 3.0 eq) in THF (160 mL) at 0°C. -3-fluorobutanoate (step 1, 8 g, 31.62 mmol, 1.0 eq.). The reaction mixture was stirred at 0°C for about 1 hour and then stirred at room temperature for about 2 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was cooled to 0°C, slowly quenched with water (3.59 ml), 10% NaOH solution (3.59 ml), followed by water (10.59 ml) and stirred for about 2 hours. The resulting precipitates were filtered and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-2% methanol in dichloromethane to give the title compound (1 g, 14.06% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.57-4.51 (m, 0.5H), 4.40-4.33 (m, 0.5H), 3.94-3.83 (m, 2H), 3.30-3.22 (m, 3H), 3.18-3.0 (m, 6H), 1.17 (d , J=6.9 Hz, 3H); ES-MS: m/z 226.48 (M+H) ⁺ .

Intermediate 14. Preparation of 4-((2S,3S)-3-((tert-butyldimethylsilyl)oxy)-1hydroxybutan-2-yl)thiomorpholine-1,1-dioxide:

Step 1. Synthesis of methyl-(2R,3S)-2-(1,1-dioxidothiomorpholino)-3-hydroxybutanoate:

Divinyl sulfone (19.18 mL, 191, 62 mmol, 2.5 eq.), followed by triethylamine (53.4 mL, 383.25 mmol, 5.0 eq.). The reaction mixture was heated until condensation appeared for about 6 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-6% methanol in dichloromethane to give the title compound (10 g, 51.9% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.0-3.96 (m, 1H), 3.76 (s, 3H), 3.40-3.33 (m, 2H), 3.18-2.99 (m, 7H), 1.20 (d, J=6.0 Hz, 3H); ES-MS: m/z 252.45 (M+H) ⁺ .

Step 2. Synthesis of methyl (2R,3S)-3-((tert-butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)butanoate:

To a stirred solution of methyl (2R,3S)-2-(1,1-dioxidothiomorpholino)-3-hydroxybutanoate (step 1, 5.0 g, 19.89 mmol, 1.0 eq.) in DCM (40 ml) and DMF (20 ml) at 0°C, imidazole (2.7 g, 39.78 mmol, 2.0 eq.) and tert-butyldimethylsilyl chloride (3.29 g, 21.88 mmol, 1.1 eq.) were added. . The reaction mixture was stirred at room temperature for about 72 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated under reduced pressure, diluted with water (200 ml) and extracted with ethyl acetate (3x100 ml). The combined organic layer was washed with water (2x200 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane to give the title compound (4.5 g, 61.89% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.414.37 (m, 1H), 3.69 (s, 3H), 3.62-3.52 (m, 2H), 3.29-3.20 (m, 3H), 3.11-2.99 (m, 4H), 1.22 (d, J=6.3 Hz, 3H), 0.82 (s, 9H), 0.04 (s, 3H), 0.01 (s, 3H); ES-MS: m/z 366.50 (M+H)+.

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Step 3. Synthesis of 4-((2S,3S)-3-((tert-butyldimethylsilyl)oxy)-1-hydroxybutan-2-yl)thiomorpholine1,1-dioxide:

To a stirred solution of methyl (2R,3S)-3-((tert-butyldimethylsilyl)oxy)-2-(1,1dioxidothiomorpholino)butanoate (step 2, 2.8 g, 7.65 mmol, 1.0 eq.) DIBAL-H (15.3 mL, 22.97 mmol, 3.0 eq., 1.5 M in toluene) was slowly added to DCM (56 mL) at -78°C. The reaction mixture was stirred at -78°C for about 30 minutes and left to stir at room temperature for about 1 hour. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was quenched with aqueous ammonium chloride and extracted with DCM (3x80 ml). The combined organic layer was washed with water (80 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-3% methanol in dichloromethane to give the title compound (1.8 g, 69.7% yield) as a solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.02-3.92 (m, 1H), 3.57-3.48 (m, 2H), 3.44-3.32 (m, 3H), 3.14-3.0 (m, 6H), 2.43 (s, 1H), 1.19 (d, J=6.0 Hz, 3H), 0.88 (s, 9H), 0.1 (s, 6H); ES-MS: m/z 338.57 (M+H)+.

Intermediate 15. Preparation of (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-2-(4(isopropylsulfonyl)piperazin-1-yl)butan-1-ol:

Step 1. Synthesis of methyl-O-(tert-butyldimethylsilyl)-L-threoninate:

o Ύγ ^Ν Η ₂ -Si. //

Triethylamine (124.7 mL, 887.32 mmol, 7 .0 eq.), 4(dimethylamino)pyridine (1.54 g, 12.676 mmol, 0.1 eq.) and tert-butyldimethylsilyl chloride (38.21 g, 253.53 mmol, 2.0 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with water (200 ml) and extracted with DCM (2x200 ml). The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-1% methanol in dichloromethane to give the title compound (20 g, 63.77% yield) as a colorless liquid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.34-4.27 (m, 1H), 3.72 (s, 3H), 3.29 (d, J=2.7 Hz, 1H), 1.62 (brs, 2H), 1.26 (d, J=6.3 Hz, 3H), 0.86 (s , 9Н), 0.04 (s, 6Н); ES-MS: m/z 248.37 (M+H) ⁺ .

Step 2. Synthesis of methyl (2S,3R)-2-(4-benzylpiperazin-1-yl)-3-((tert-butyldimethylsilyl)oxy)butanoate:

To a stirred solution of methyl O-(tert-butyldimethylsilyl)-L-threoninate (step 1, 20 g, 80.83 mmol, 1.0 eq.) and N-benzyl-2-chloro-N-(2-chloroethyl) ethan-1-amine hydrochloride (prepared as described in E. JM C, 2014, 75, pp: 11-20) (32.56 g, 121.24 mmol, 1.5 eq.) in methanol (20 ml) , N,N-diisopropylethylamine (100 ml) was added. The reaction mixture was heated at 90-100°C overnight. The reaction mixture was evaporated under reduced pressure, diluted with water (200 ml) and extracted with DCM (2x200 ml). The organic layer was separated, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using 2% methanol in dichloromethane as eluent to obtain the title compound (18 g, 54.86% yield) as a colorless liquid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.32-7.30 (m, 5H), 4.23-4.14 (m, 1H), 3.67 (s, 3H), 3.49 (s, 2H), 3.07 (d, J=6.6 Hz, 1H), 2.83-2.76 (m, 2H),

- 26 045041

2.64-2.58 (m, 2H), 2.45 (m, 4H), 1.19 (d, J=6.3 Hz, 3H), 0.85 (s, 9H), 0.04 (s, 6H); APCI-MS: m/z 407.3 (M+H) ⁺ .

Step 3. Synthesis of methyl-(2S,3R)-3-((tert-butyldimethylsilyl)oxy)-2-(piperazin-1-yl)butanoate:

To a stirred suspension of 10% Pd/C (9 g, 50% humidity) in methanol (360 ml) was added methyl (2S,3R)-2-(4-benzylpiperazin-1-yl)-3-((tert-butyldimethylsilyl) hydroxy)butanoate (step 2, 18 g,

44.33 mmol, 1.0 eq.) and ammonium formate (55.90 g, 886.6 mmol, 20 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was filtered through a pad of celite and washed with methanol. The filtrate was evaporated under reduced pressure, diluted with DCM, washed with water, dried over sodium sulfate, filtered and evaporated under reduced pressure to give the title compound (12 g, 85.7% yield), which was used as such in the next step without further purification . Ή NMR (300 MHz, CDCl ₃ ): δ ppm 4.36-4.30 (m, 1H), 3.71 (s, 3H), 3.37-3.29 (m, 2H), 3.23-3.11 (m, 5H), 3.05 -2.97 (m, 2H), 1.21 (d, J=6.3 Hz, 3H), 0.83 (s, 9H), 0.04 (s, 6H); APCI-MS: m/z 317.3 (M+H) ⁺ .

Step 4. Synthesis of methyl-(2S,3R)-3-((tert-butyldimethylsilyl)oxy)-2-(4-(isopropylsulfonyl)piperazin-1-yl)butanoate:

To a stirred solution of methyl (2S,3R)-3-((tert-butyldimethylsilyl)oxy)-2-(piperazin-1yl)butanoate (step 3, 12.0 g, 37.97 mmol, 1.0 eq.) triethylamine (21.18 mL, 151.88 mmol, 4.0 eq) followed by isopropylsulfonyl chloride (8.12 g, 56.96 mmol, 1.5 eq) was added to DCM (180 mL) at 0°C. . The reaction mixture was brought to room temperature and stirred overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with water (100 ml) and extracted with DCM (3x100 ml). The combined organic layer was washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-3% methanol in dichloromethane to give the title compound (8.0 g, 50% yield) as a liquid. Ή NMR (300 MHz, CDCl ₃ ): δ ppm 4.30-4.26 (m, 1H), 3.69 (s, 3H), 3.34-3.31 (m, 4H), 3.203.12 (m, 2H), 3.03-2.96 (m, 2H), 2.77-2.70 (m, 2H ), 1.34 (d J=6.9 Hz, 6H), 1.22 (d, J=6.3 Hz, 3H), 0.85 (s, 9H), 0.05 (s, 3H), 0.02 (s, 3H); ES-MS: m/z 423.1 (M+H)+.

Step 5. Synthesis of (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-2-(4-(isopropylsulfonyl)piperazin-1yl)butan-1-ol:

To a stirred solution of methyl (2S,3R)-3-((tert-butyldimethylsilyl)oxy)-2-(4-(isopropylsulfonyl)piperazin-1-yl)butanoate (step 4, 5.0 g, 11.82 mmol , 1.0 eq.) in DCM (75 ml) at -78°C was added DIBAL-H (19.7 ml, 29.57 mmol, 2.5 eq., 1.5 M in toluene). The reaction mixture was stirred at -78°C for about 4 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was quenched with a saturated ammonium chloride solution and stirred for about 10 minutes. The mixture was filtered and the filtrate was extracted with DCM (3x100 ml). The combined organic layer was washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-3% methanol in dichloromethane to give the title compound (3.7 g, 80% yield) as an off-white solid. 1H NMR (300 MHz, CDC13): δ ppm 3.99-3.95 (m, 1H), 3.54-3.26 (m, 6H), 3.22-3.13 (m, 1H), 3.04-2.96 (m, 2H), 2.82-2.75 (m, 2H), 2.63-2.56 (m , 1H), 1.34 (d, J=6.9 Hz, 6H), 1.15 (d, J=6.3 Hz, 3H), 0.89 (s, 9H), 0.086 (s, 3H), 0.081 (s, 3H); ES-MS: m/z 395.46 (M+H) ⁺ .

Intermediate 16. Preparation of 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a- 27 045041 amino-5a,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3-benzyl(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

Step 1. Synthesis of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-isocyanato-5a,5b,8,8,11apentamethyl-1 -(prop-1-ene-2 -yl)icosahydro-1 H-cyclopenta[a]chrysene-9-ylacetate:

To a stirred solution of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-acetoxy5a,5b,8,8,11a-pentamethyl-1-(prop-1-ene-2- diphenylphosphoryl azide (28.684 g, 104.26 mmol, 2.0 eq.) and triethylamine (15.82 g, 156.39 mmol, 3.0 eq.). The reaction mixture was heated to 80°C for 1.5 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, diluted with water (500 ml) and extracted with DCM (3x500 ml). The combined organic layer was washed with water (200 ml) and brine (200 ml). The organic layer was dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude compound was treated with methanol (78 mL), stirred at room temperature for 15 minutes, filtered and dried in vacuo to give the title compound (23 g, 89.1% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.74 (d, J=1.5 Hz, 1H), 4.63 (t, J=1.5 Hz, 1H), 4.45 (m, 1H), 2.57-2.50 (m, 1H), 2.15-2.02 (m, 1H), 2.04 (s, 3H), 1.88-0.77 (m, 23H), 1.68 (s, 3H), 1.05 (s, 3H), 0.93 (s, 3H), 0.86 (s, 3H), 0.84 (s, 3H), 0.84 (s, 3H).

Step 2. Synthesis of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl1-(prop-1-ene-2 -yl)icosahydro-1H-cyclopenta[a]chrysen-9-ol hydrochloride:

To a stirred solution of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-isocyanato5a,5b,8,8,11a-pentamethyl-1-(prop-1-ene-2- yl)icosahydro-1H-cyclopenta[a]chrysen-9-ylacetate (step 1, 23 g, 46.394 mmol, 1.0 eq.) in 1,4-dioxane (230 ml) at 0°C, add concentrated hydrochloric acid ( 69 ml). The reaction mixture was heated at 60°C overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was concentrated under reduced pressure. The resulting compound was treated with heptane (69 ml), stirred for 15 minutes, the solid was filtered and dried in vacuo to give the title compound (20 g, 93% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.67 (s, 3H), 4.72 (s, 1H), 4.62 (s, 1H), 3.09-2.94 (m, 1H), 2.60 (m, 1H), 2.11-1.99 (m, 1H), 1.91-1.79 ( m, 2H), 1.76-0.62 (m, 21H), 1.66 (s, 3H), 1.03 (s, 3H), 0.93 (s, 3H), 0.87 (s, 3H), 0.78 (s, 3H), 0.65 (s, 3H).

Stage 3. Synthesis of tert-butyl-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-hydroxy5a,5b,8,8,11a-pentamethyl-1-(prop- 1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysen-3a-yl)carbamate:

To a stirred solution of (1 R,3aS,5aR,5bR,7aR,9S, 11aR,11bR, 13aR,13bR)-3a-amino

5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-1H-cyclonenta[a]chrysene-9-ol hydrochloride (step 2, 20 g, 43.087 mmol, 1, 0 eq.) in 1,4-dioxane (200 ml) at 0° C. was added a saturated solution of sodium bicarbonate (200 ml) and tert-butyl dicarbonate (14.1 g, 64.631 mmol, 1.5 eq.). Reaction mixture

- 28 045041 was stirred at room temperature for 6 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with water (400 ml) and extracted with DCM (3x500 ml). The combined organic layer was washed with water (400 ml) and brine (200 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-3% ethyl acetate in hexane. Fractions containing the expected product were combined and concentrated under reduced pressure to give the title compound (16 g, 70.48% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.732 (d, J=1.5 Hz, 1H), 4.628 (d, J=1.5 Hz, 1H), 4.33 (s, 1H), 3.22-3.17 (m, 1H), 2.58-2.54 (m, 1H), 2.50 -2.35 (m, 2H), 2.06-1.92 (m, 1H), 1.70 (s, 3H), 1.66-0.69 (m, 21H), 1.46 (s, 9H), 1.03 (s, 3H), 0.99 (s , 3H), 0.97 (s, 3H), 0.85 (s, 3H), 0.78 (s, 3H); ESI-MS: m/z 550.45 (M+Na)+.

Step 4. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((tert-butoxycarbonyl)amino)-5a,5b,8, 8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-1 H-cyclopenta[a]chrysen-9yl)(1R,3S)-2,2-dimethylcyclobutan-1,3-dicarboxylate :

To a stirred solution of tert-butyl-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9hydroxy-5a,5b,8,8,11 a-pentamethyl-1 -(prop- 1 -en-2-yl)icosahydro-3 aH-cyclopenta[a] chrysen-3 a-yl) carbamate (step 3, 16 g, 30.312 mmol, 1.0 eq.) in DCM (240 ml) at 0° (1S,3R)-3((benzyloxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid (prepared as described in WO 2011/007230 A2, 11.92 g, 45.469 mmol, 1.5 eq) was added .), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC.HCl) (14.52 g, 75.781 mmol, 2.5 eq.) and 4-(dimethylamino)pyridine (1.11 g, 9.093 mmol, 0.3 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with water (500 ml) and extracted with DCM (3x500 ml). The combined organic layer was washed with water (200 ml) and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-3% ethyl acetate in hexane. Fractions containing the expected product were combined and concentrated under reduced pressure to give the title compound (16 g, 68.37% yield) as an off-white solid. 1H NMR (300 MHz, CDCI3): δ ppm 7.36-7.32 (m, 5H), 5.14, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.70 (s, 1H), 4.60 (s, 1H) , 4.43 (dd, J=11.4, 4.5 Hz, 1H), 4.30 (s, 1H), 2.84-2.51 (m, 4H), 2.48-2.31 (m, 2H), 2.07-0.76 (m, 23H), 1.68 (s, 3H), 1.43 (s, 9H), 1.34 (s, 3H), 1.01 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.85 (s, 6H), 0.84 ( s, 3H); ESI-MS: m/z 794.55 (M+Na) ⁺ .

Step 5. Synthesis of 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11apentamethyl-1-(prop-1- en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3-benzyl(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((tertbutoxycarbonyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl) (1R,3S)-2,2-dimethylcyclobutan-1,3-dicarboxylate ( step 4, 11.0 g, 14.24 mmol, 1.0 eq.) in 1,4-dioxane (55 ml) at 0°C was added 4N HCl in 1,4-dioxane (55 ml). The temperature of the reaction mixture was brought to room temperature and stirred for about 3 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated under reduced pressure, diluted with water (50 ml), made basic with aqueous sodium bicarbonate and extracted with DCM (3x100 ml). The combined organic layer was washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was treated with hexane (50 mL), stirred, filtered and dried in vacuo to give the title compound (7.0 g, 73.1% yield) as a solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.36-7.32 (m, 5H), 5.14, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.72 (s, 1H), 4.59 (s, 1H), 4.43 (dd, J=11.4, 4.5 Hz, 1H) , 2.84-2.48 (m, 4H), 2.07-1.98 (m, 2H), 1.68 (s, 3H), 1.63-0.76 (m, 23H), 1.34 (s, 3H), 1.04 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H),

- 29 045041

0.85 (s, 6H), 0.83 (s, 3H); ES-MS: m/z 672.49 (M+H)+.

Intermediate 17. Preparation of 2-(2-methoxyethoxy)acetic acid:

Step 1. Synthesis of ethyl 2-(2-methoxyethoxy)acetate:

2-Methoxyethan-1-ol (7.0 g, 91.996 mmol, 1 ,0 eq.). The reaction mixture was stirred at 0°C for about 30 minutes. Ethyl bromoacetate (19.97 g, 119.59 mmol, 1.3 eq.) and potassium iodide (3.054 g, 18.399 mmol, 0.2 eq.) were added to the reaction mixture and stirred at room temperature for about 2 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was cooled to 0°C, quenched with saturated sodium chloride solution and extracted with DCM (3x350 ml). The combined organic layer was washed with water (150 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (10 g, 67% yield) as an oil, which was used as such in the next step without further purification.

Stage 2. Synthesis of 2-(2-methoxyethoxy)acetic acid:

An aqueous 2.5N solution was added to a stirred solution of ethyl 2-(2-methoxyethoxy) acetate (step 1, 10 g, 61.656 mmol, 1.0 eq.) in methanol (100 ml) and THF (100 ml) at 0°C KOH (184.96 ml, 462.42 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, the pH was adjusted to 2.0 with 1N HCl and evaporated under reduced pressure. The resulting solid was stirred with DCM (4x150 ml), filtered and the filtrate was evaporated under reduced pressure to give the title compound (5.0 g, 60.97% yield) as an oil. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.19 (s, 2H), 3.77-3.74 (m, 2H), 3.64-3.59 (m, 2H), 3.42 (s, 3H); ES-MS: m/z 135.17 (M+H) ⁺ .

Intermediate 18. Preparation of 4-((12R,13R)-14-hydroxy-12-methyl-2,5,8,11tetraoxatetradecane-13-yl)thiomorpholine 1,1-dioxide:

Step 1. Synthesis of 2-(2-(2-methoxyethoxy)ethoxy)ethylmethanesulfonate:

Triethylamine ( 8.4 mL, 60.09 mmol, 3.0 eq.), followed by methanesulfonyl chloride (2.01 mL, 26.04 mmol, 1.3 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. Water was added to the reaction mixture and extracted with DCM (2x30 ml). The combined organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (4.3 g) as a liquid, which was used in the next step without further purification. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.40-4.36 (m, 2H), 3.78-3.75 (m, 2H), 3.70-3.61 (m, 6H), 3.56-3.52 (m, 2H), 3.37 (s, 3H), 3.07 (s, 3H); ES-MS: m/z 243.37 (M+H)+.

Step 2. Synthesis of 4-((2R,3R)-1,3-dihydroxybutan-2-yl)thiomorpholine-1,1-dioxide:

To a stirred solution of methyl (2S,3R)-2-(1,1-dioxidothiomorpholino)-3-hydroxybutanoate (intermediate 8, step 2, 15 g, 59.713 mmol, 1.0 eq.) in methanol (300 ml) at At 0°C, sodium borohydride (22.5 g, 597.13 mmol, 10.0 eq.) was added in portions. The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was neutralized with 4N HCl/1,4-dioxane (120 ml) and concentrated under reduced pressure. The residue was stirred with DCM, filtered and the filtrate was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane to give the title compound (6.5 g, 48.8% yield) as an off-white solid. 1H NMR (300 MHz,

DMSO-d ₆ ): δ ppm 4.38 (t, J=4.8 Hz, 1H), 4.26 (d, J=3.0 Hz, 1H), 4.09-4.04 (m, 1H), 3.61-3.44 (m, 2H), 3.253.16 (m, 2H) , 3.08-3.02 (m, 6H), 2.40-2.33 (m, 1H), 1.04 (d, J=6.0 Hz, 3H); ES-MS: m/z 224.16 (M+H)+.

Step 3. Synthesis of 4-((2R,3R)-1-((tert-butyldimethylsilyl)oxy)-3-hydroxybutan-2-yl)thiomorpholine1,1-dioxide:

To a stirred solution of 4-((2R,3R)-1,3-dihydroxybutan-2-yl)thiomorpholine-1,1-dioxide (step 2, 4.5 g, 20.153 mmol, 1.0 eq.) in DCM ( 90 ml) at 0°C, triethylamine (14 ml, 100.76 mmol, 5.0 eq), DMAP (0.492 g, 4.030 mmol, 0.2 eq) and TBDMSCl (7.59 g, 50.383 mmol, 0.2 eq) were added 2.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with DCM (200 ml), washed with water (200 ml) and brine (250 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of 0-60% ethyl acetate in hexane to give the title compound (4 g, 58.8% yield) as a white solid. 1H NMR (300 MHz, CDCl3): δ ppm 3.80 (dd, J=11.1, 3.0 Hz, 1H), 3.72-3.61 (m, 2H), 3.52-3.39 (m, 3H), 3.123.02 (m, 6H), 2.50-2.43 (m, 1H), 1.20 (d, J=6.0 Hz, 3H), 0.89 (s, 9H), 0.07 (s, 6H); ES-MS: m/z 338.32 (M+H) ⁺ .

Step 4. Synthesis of 4-((12R,13R)-12,16,16,17,17-pentamethyl-2,5,8,11,15-pentaoxa-16-silaoctadecan-13yl)thiomorpholine-1,1-dioxide :

To a suspension of sodium hydride (1.77 g, 74.05 mmol, 5.0 eq.) in THF (50 ml) at 0° C. was added 4((2R,3R)-1-((tert-butyldimethylsilyl)oxy) -3-hydroxybutan-2-yl)thiomorpholine-1,1-dioxide (step 3.5 g, 14.81 mmol, 1.0 eq.). The reaction mixture was stirred at 40-50°C for about 30 minutes. 2-(2-(2-methoxyethoxy)ethoxy)ethylmethanesulfonate (step 1, 4.3 g, 17.77 mmol, 1.2 eq) and potassium iodide (0.245 g, 1.48 mmol, 0 ,1 eq.) and stirred at 50-60°C overnight. The reaction mixture was cooled to 0°C, quenched with ice water and extracted with DCM (2x50 ml). The combined organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The product was purified by silica gel column chromatography using a gradient of 0-40% ethyl acetate in hexane to give the title compound (2.0 g, 27.93% yield) as a colorless liquid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.0-3.93 (m, 1H), 3.84-3.70 (m, 2H), 3.68-3.63 (m, 10H), 3.62-3.50 (m, 4H), 3.38 (s, 3H), 3.14-2.95 (m, 6H ), 2.66-2.61 (m, 1H), 1.20 (d, J=6.6 Hz, 3H), 0.89 (s, 9H), 0.07 (s, 3H), 0.05 (s, 3H); ES-MS: m/z 484.65 (M+H) ⁺ .

Step 5. Synthesis of 4-((12R,13R)-14-hydroxy-12-methyl-2,5,8,11-tetraoxatetradecane-13yl)thiomorpholine 1,1-dioxide:

To a stirred solution of 4-((12R,13R)-12,16,16,17,17-pentamethyl-2,5,8,11,15-pentaoxa-16silaoctadecan-13-yl)thiomorpholine-1,1-dioxide ( step 4, 2.0 g, 4.134 mmol, 1.0 eq.) in THF (20 mL) was added TBAF (8.26 mL, 8.269 mmol, 2.0 eq., 1.0 M in THF). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with DCM (100 ml) and washed with water (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of 0-9% methanol in dichloromethane to give the title compound (0.8 g, 52.6% yield) as a colorless liquid. ¹ H NMR (300 MHz, CDCl3): δ ppm 3.67-3.59 (m, 9H), 3.58-3.53 (m, 4H), 3.473.41 (m, 2H), 3.38 (s, 3H), 3.35-3.20 (m, 2H), 3.13-3.0 (m, 6H ), 2.63-2.56 (m, 1H), 1.20 (d, J=6.0 Hz, 3H); ESMS: m/z 370.20 (M+H) ⁺ .

Intermediate 19. Preparation of 2,5,8,11-tetraoxatridecane-13-oic acid:

ABOUT

Step 1. Synthesis of ethyl 2,5,8,11-tetraoxatridecane-13-oate:

- 31 045041

To a stirred suspension of sodium hydride (3.65 g, 152.25 mmol, 5.0 eq.) in THF (125 ml) at 0° C. was added 2-(2-(2-methoxyethoxy)ethoxy)ethan-1-ol (5.0 g, 30.45 mmol, 1.0 eq.) and tetrabutylammonium iodide (2.24 g, 6.09 mmol, 0.2 eq.). The reaction mixture was stirred at 0°C for about 30 minutes, ethyl 2-bromoacetate (6.611 g, 39.58 mmol, 1.3 eq.) was added, stirred at 0°C for about 1 hour, and stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was cooled to 0°C, quenched with ice and extracted with DCM (3x250 ml). The combined organic layer was washed with water (150 ml) and brine (150 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give the title compound (6 g, 78.74% yield) as a solid, which was used as such in the next step without further purification.

Stage 2. Synthesis of 2,5,8,11-tetraoxatridecan-13-oic acid:

O

To a stirred solution of ethyl 2,5,8,11-tetraoxatridecane-13-oate (step 1, 6.0 g, 23.97 mmol, 1.0 eq.) in methanol (60 ml) and TNF (60 ml) an aqueous solution of 2.5N KOH (71.85 mL, 179.79 mmol, 7.5 eq.) was added at 0°C. The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, water (50 ml) was added, cooled to 0°C, acidified to pH 2.0 with 1N HCl and extracted with DCM (3x250 ml). The combined organic layer was washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue (5.3 g) was used as such in the next step without further purification. 1H NMR (300 MHz, CDCh): δ ppm 4.17 (s, 2H), 3.75-3.57 (m, 12H), 3.39 (s, 3H); ES-MS: m/z 223.63 (M+H)+.

Examples

Example 1. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2-(1,1dioxidothiomorpholino)-2 -methylpropyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutan- 1-carboxylic acid:

Step 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2-((tertbutoxycarbonyl)amino)-2-methylpropyl) amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl)-( 1 R,3S)-2,2-dimethylcyclobutane -1,3-dicarboxylate:

To a stirred solution of 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro -1H-cyclopenta[a]chrysen-9-yl)-3-benzyl(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1.4 g, 5.83 mmol, 1.0 eq.) in 1,2-dichloroethane (40 ml) was added tert-butyl-(2-methyl-1-oxopropan-2-yl)carbamate (intermediate 2, 3.27 g, 17.49 mmol, 3.0 eq.) and titanium tetraisopropoxide (3.4 ml, 11.66 mmol, 2.0 eq.). The reaction mixture was stirred at room temperature for about 1 hour, and then sodium triacetoxyborohydride (3.7 g, 17.49 mmol, 3.0 eq.) was added and stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. Saturated sodium bicarbonate solution was added to the reaction mixture, the organic layer was separated and the aqueous layer was extracted with dichloromethane (2x40 ml). The combined organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 1% methanol in dichloromethane as eluent to give the title compound (4 g, 80% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.34 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.45 (m, 1H), 2.90-2.46 (m, 3H), 2.40-2.30 (m, 2H), 2.10-2.0 ( m, 2H), 1.97-1.13 (m, 32H), 1.42 (s, 9H), 1.03-0.76 (m, 22H), 0.40-0.20 (m, 4H); ESI-MS: m/z 857.75 (M+H)+.

Stage 2. Synthesis of 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2-amino-2-methylpropyl)amino)-5a,5b,8, 8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl) - 32 045041

3-Benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

O

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2((tert-butoxycarbonyl)amino)-2-methylpropyl) amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl)-( 1 R,3 S)-2,2 -dimethylcyclobutane-1,3-dicarboxylate (step 1, 4 g, 4.66 mmol, 1.0 eq.) in 1,4-dioxane (10 ml) at 0°C added 4 N HC1/1,4-dioxane (30 ml). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, made basic with saturated sodium bicarbonate solution and extracted with dichloromethane (2x40 ml). The organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to give the title compound (3 g, 85.2% yield) as an off-white solid. 1H NMR (300 MHz, CDC13): δ ppm 7.34 (m, 5H), 5.14, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.45 (m, 1H), 2.84-2.61 (m, 3H), 2.27-2.13 (m, 2H), 2.07-1.17 ( m, 34Н), 1.130.78 (m, 22Н), 0.36-0.22 (m, 4Н); ESI-MS: m/z 757.50 (M+H)+.

Step 3. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2-(1,1dioxidothiomorpholino))-2-methylpropyl) amino)-5a,5b,8,8,11 a-pentamethyl-1 -(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobutan- 1,3-dicarboxylate:

To a stirred solution 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2-amino-2methylpropyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9yl)-3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (step 2, 0.4 g, 0.52 mmol, 1.0 eq.) in 1,4dioxane (4 ml) and ethanol (4 ml) was added triethylamine (0.21 ml, 1.56 mmol, 3.0 eq.), and then divinyl sulfone (0.12 g, 1.056 mmol, 2.0 eq). The reaction mixture was heated to 85°C for about 3 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated under reduced pressure, diluted with water (100 ml) and extracted with ethyl acetate (3x30 ml). The combined organic layer was washed with water (100 ml), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a 20-40% ethyl acetate in hexane gradient to give the title compound (0.4 g, 88.8% yield) as a solid. 1H NMR (300 MHz, CDC13): δ ppm 7.35 (m, 5H), 5.14, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.49-4.40 (m, 1H), 3.05 (m, 8H), 2.86-2.73 (m, 2H), 2.71-2.60 ( m, 1H), 2.50-2.28 (m, 2H), 2.10-1.13 (m, 34H), 1.08-0.78 (m, 22H), 0.42-0.22 (m, 4H).

Stage 4. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2-(1,1dioxidothiomorpholino)-2 -methylpropyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane -1-carboxylic acid:

O

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2-(1,1dioxidothiomorpholino)-2-methylpropyl)amino) -5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro1H-cyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobutane-1,3- dicarboxylate (step 3, 0.4 g, 0.456 mmol, 1.0 eq.) in THF (4 ml) and methanol (4 ml) was added an aqueous 2.5 N solution of KOH (1.35 ml, 3.427 mmol, 7. 5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated under reduced pressure and diluted with water (5 ml). The reaction mixture was cooled to 0°C, the pH was adjusted to 5 with 1N HCl and extracted with dichloromethane (3x30 ml). The combined organic layers were washed with water (20 ml) and brine (10 ml). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 30 to 45% ethyl acetate in hexane to give

- 33 045041 of the title compound (0.15 g, 41.8% yield) as a white solid. 1H NMR (300

MHz, CDC13): δ ppm 4.46 (dd, J=10.8, 4.8 Hz, 1H), 3.10-2.92 (m, 8H), 2.86-2.74 (m, 2H), 2.65-2.53 (m, 1H),

2.39-2.28 (m, 2H), 2.10-2.0 (m, 2H), 1.98-1.05 (m, 32H), 1.03-0.78 (m, 22H), 0.40-0.20 (m, 4H). ESI-MS: m/z

785.65 (M+H) ⁺ .

Example 2. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1, 1dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutan- 1-carboxylic acid:

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1,1dioxidothiomorpholino)propyl) amino)-5a,5b,8,8,11 a-pentamethyl-1-( 1 -methylcyclopropyl)icosahydro1H-cyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobutane-1, 3-dicarboxylate:

To a stirred solution of 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl)icosahydro -1H-cyclopenta[a]chrysen-9-yl)-3-benzyl(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1, 1 g, 1.457 mmol, 1.0 eq.) in acetonitrile (20 ml) was added (S)-4-(1-chloropropan-2-yl)thiomorpholine-1,1-dioxide (intermediate 3, 0.925 g, 4.372 mmol, 3.0 eq.), potassium phosphate tribasic (1.36 g, 6.4108 mmol, 4.4 eq.) and potassium iodide (0.653 g, 3.934 mmol, 2.7 eq.). The reaction mixture was heated until condensation appeared overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was filtered through a celite pad and washed with acetonitrile (25 ml). The filtrate was evaporated under reduced pressure and the residue was purified by silica gel column chromatography using 2% methanol in dichloromethane as eluent to give the title compound (0.35 g, 27.88% yield) as an off-white solid . 1H NMR (300 MHz, CDC13): δ ppm 7.35 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.44 (dd, J=10.5, 4.8 Hz, 1H), 3.32 ( m, 1H), 3.12-3.06 (m, 8H), 2.88-2.72 (m, 2H), 2.702.52 (m, 2H), 2.50-2.48 (m, 1H), 2.18-2.0 (m, 3H), 1.92-1.17 (m, 25Н), 1.17-0.78 (m, 25Н), 0.68 (m, 1Н), 0.500.36 (m, 2Н), 0.28 (m, 1Н); ES-MS: m/z 861.79 (M+H) ⁺ .

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1, 1dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutan- 1-carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)2-(1,1-dioxidothiomorpholino)propyl) amino)-5a,5b,8,8,11 a-pentamethyl-1 -(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobutane- 1,3-dicarboxylate (step 1, 0.35 g, 0.406 mmol, 1.0 eq.) in THF (3.5 ml) and methanol (3.5 ml) at 0°C was added an aqueous 2.5N solution of KOH (1.22 ml, 3.047 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, diluted with water (10 ml), cooled to 0°C and adjusted to pH 5.0 with 1N HCl. The aqueous layer was extracted with dichloromethane (2x20 ml). The combined organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-5% methanol in DCM. The resulting compound was further purified by recrystallization from MTBE (10 mL) to give the title compound (0.1 g, 32% yield) as an off-white solid. 1H NMR (300 MHz, CDC13): δ ppm 4.46 (m, 1H), 3.30-2.90 (m, 9H), 2.88-2.72 (m, 2H), 2.68-2.50 (m, 2H), 2.28- 2.0 (m, 3H), 2.0-1.17 (m, 26H), 1.14-0.78 (m, 25H), 0.62-0.52 (m, 1H), 0.50-0.36 (m, 2H), 0.32-0.25 (m, 1H ); ES-MS: m/z 771.87 (M+H)+.

- 34 045041

Example 3. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((R)-2-(1,1dioxidothiomorpholino )-3-methylbutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2 -dimethylcyclobutane-1-carboxylic acid:

O

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((R)-2-(1,1dioxidothiomorpholino)-3- methylbutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobutane -1,3-dicarboxylate:

O

To a stirred solution of (R)-4-(1-hydroxy-3-methylbutan-2-yl)thiomorpholine 1,1 dioxide (intermediate 4, 0.241 g, 1.093 mmol, 1.25 eq.) in dichloromethane (12 ml) at At 0° C., trifluoromethanesulfonic anhydride (0.20 mL, 1.224 mmol, 1.4 eq.) was added. The reaction mixture was stirred at the same temperature for about 10 minutes. 2,6-lutidine (0.145 mL, 1.25 mmol, 1.43 eq.) was added and stirred at 0° C. for 10 minutes. 1((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl) was added to the reaction mixture )icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1, 0.6 g, 0.874 mmol, 1.0), and then triethylamine (0.175 ml 1.25 mmol, 1.43 eq.) and stirred at 0°C for about 1 hour. The cooling bath was removed and stirred at room temperature for about 4 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with dichloromethane (2x20 ml), the organic layer was washed with water, dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude compound was purified by silica gel column chromatography using 30% ethyl acetate in hexane as eluent to give the title compound (0.3 g, 38.96% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 7.35 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.45 (dd, J=11.1, 4.5 Hz, 1H), 3.22-3.06 (m, 6H), 3.02-2.94 (m, 2H), 2.88-2.58 (m, 3H), 2.52-2.18 (m, 3H), 2.10-1.90 (m, 2H), 1.88-1.10 (m, 27H), 1.06-0.78 (m, 28H), 0.40- 0.20 (m, 4H); ESMS: m/z 889.81 (M+H)+.

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((R)-2-(1,1dioxidothiomorpholino )-3-methylbutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2 -dimethylcyclobutane-1-carboxylic acid:

O

To a stirred solution 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)-2-(1,1dioxidothiomorpholino)-3 - methylbutyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1Hcyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobuane -1,3-dicarboxylate (step 1, 0.3 g, 0.337 mmol, 1.0 eq.) in methanol (3 ml) and THF (3 ml) was added an aqueous 2.5N solution of KOH (1.01 ml, 2.527 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, acidified with 1N HCl to pH 5.0 and extracted with dichloromethane (2x30 ml). The combined organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using 5% methanol in dichloromethane as eluent to give the title compound (0.100 g, 37.3% yield) as an off-white solid. 1H NMR (300 MHz, CDCI3): δ ppm 4.45 (m, 1H), 3.25-2.95 (m, 8H), 2.80-2.70 (m, 3H), 2.62-2.42 (m, 2H), 2.38-2.20 (m, 1H), 2.20-1.20 (m, 29H ), 1.18-0.78 (m, 28H), 0.42-0.20 (m, 4H); ES-MS: m/z 799.89 (M+H) ⁺ .

Example 4. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((R)-3-((tert-butyldimethylsilyl) oxy)-2-(1,1-dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1- 35 045041 methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysene- 9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1 carboxylic acid:

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((R)-3-((tert-butyldimethylsilyl)oxy)-2 -(1,1-dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl)-( 1 R ,3 S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of (R)-4-(1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2yl)thiomorpholine-1,1-dioxide (intermediate 6, 0.742 g, 2.186 mmol, 1.5 eq. ) trifluoromethanesulfonic acid anhydride (0.39 mL, 2.332 mmol, 1.6 eq.) was added to DCM (20 mL) at 0° C. The reaction mixture was stirred at 0°C for about 10 minutes, then 2,6-lutidine (0.21 ml, 2.332 mmol, 1.6 eq.) was added and stirred at 0°C for about 10 minutes. 1((1 R,3aS,5aR,5bR,7 aR,9S, 11 aR, 11 bR, 13 aR, 13bR)-3 a-amino-5a,5b,8,8,11 a-pentamethyl-1 was added sequentially -(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1.1 g, 1.457 mmol, 1.0 eq.) and triethylamine (0.32 ml, 2.332 mmol, 1.6 eq.) at 0°C and stirred at the same temperature for about 1 hour. The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with DCM (100 ml), washed with saturated sodium bicarbonate solution, water and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-15% ethyl acetate in hexane to give the title compound (1 g, 69.4% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.34 (m, 5H), 5.14, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.44 (dd, J=11.1, 4.8 Hz, 1H), 3.71-3.65 (m, 2H), 3.40-3.20 (m, 3H), 3.19-2.98 (m, 8H), 2.85-2.57 (m, 3H), 2.46-2.40 (m, 2H), 2.08-2.0 (m, 1H), 1.950.70 (m, 26H), 1.34 ( s, 3H), 1.05 (s, 3H), 0.96 (s, 3H), 0.898 (s, 6H), 0.891 (s, 6H), 0.84 (s, 9H), 0.36-0.21 (m, 4H), 0.07 (s, 3H), 0.05 (s, 3H); ESI-MS: m/z 992.16 (M+H)+.

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9SD1aRD1bRD3aRD3bR)-3a-((R)-3-((trembutyldimethylsilyl)oxy)-2-( 1, 1-dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2- dimethylcyclobutane-1carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)3-((tert-butyldimethylsilyl)oxy)- 2-(1,1-dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-(1R ,3S)-2,2-dimethylcyclobutane1,3-dicarboxylate (step 1, 1 g, 1.008 mmol, 1.0 eq.) in methanol (10 ml) and THF (10 ml) was added an aqueous 2.5N solution of KOH (3 ml, 7.56 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, pH adjusted to 5.0 with 1N HCl and extracted with DCM (2x100 ml). The combined organic layer was washed with water (100 ml) and brine (100 ml). The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0 to 6% methanol in dichloromethane to give the title compound (0.350 g, 38.5% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.53-4.45 (m, 1H), 3.76-3.62 (m, 2H), 3.40-3.23 (m, 3H), 3.20-2.93 (m, 8H), 2.80-2.52 (m, 5H), 2.02-0.78 (m , 24Н), 1.35 (s, 3Н), 1.05 (s, 3Н), 1.0 (s, 3Н), 0.98 (s, 3Н), 0.89 (s, 3Н), 0.88 (s,

- 36 045041

3H), 0.87 (s, 3H), 0.85 (s, 3H), 0.83 (s, 9H), 0.40-0.24 (m, 4H), 0.05 (s, 3H), 0.04 (s, 3H); ESI-MS: m/z 901.91 (M+H)+.

Example 5. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)-2-(1, 1dioxidothiomorpholino)-3-hydroxypronyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclononyl) and cosahydro-1 H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2 ,2-dimethylcyclobutane-1-carboxylic acid:

To a stirred solution (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)3-((tert-butyldimethylsilyl )oxy)-2-(1,1-dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-III-cyclopenta[a]chrysen-9-yl )oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid (example 4, 0.500 g, 0.554 mmol, 1.0 eq.) bTHF (15 ml) at 0°C was added TBAF (0.66 ml, 0 .6659 mmol, 1.2 eq., 1.0 M in THF). The reaction mixture was stirred at room temperature for about 4 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (100 ml), washed with water (2x100 ml) and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-10% methanol in dichloromethane to give the title compound (0.110 g, 25.2% yield) as an off-white solid. Ή NMR (300 MHz, DMSO-d6): δ ppm 4.35 (m, 1H), 3.55-3.45 (m, 1H), 3.44-3.38 (m, 1H), 3.20-3.10 (m, 2H), 3.09-2.92 (m, 6H), 2.82-2.70 (m, 3H ), 2.35-2.22 (m, 4H), 1.98-0.78 (m, 25H), 1.33 (s, 3H), 1.02 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.89 ( s, 3H), 0.83 (s, 6H), 0.82 (s, 3H), 0.35-0.20 (m, 4H); ESI-MS: m/z 787.55 (M+H) ⁺ .

Example 6. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1, 1dioxidothiomorpholino)-3-methylbutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2, 2-dimethylcyclobutane-1-carboxylic acid:

O

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1,1dioxidothiomorpholino)-3 -methylbutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2- dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of (S)-4-(1-hydroxy-3-methylbutan-2-yl)thiomorpholin-1,1 dioxide (intermediate 7, 0.290 g, 1.311 mmol, 1.5 eq.) in DCM (10 mL) trifluoromethanesulfonic acid anhydride (0.234 mL, 1.399 mmol, 1.6 eq.) was added at 0° C. The reaction mixture was stirred at 0°C for about 10 minutes. 2,6-lutidine (0.161 ml, 1.399 mmol, 1.6 eq) was added and stirred at 0°C for 10 minutes. 1((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro -1H-cyclopenta[a]chrysen-9-yl)-3-benzyl(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1, 0.6 g, 0.8745 mmol, 1 .0 eq.) and triethylamine (0.36 ml, 2.623 mmol, 3.0 eq.) at 0°C and left to stir at room temperature for about 4 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with DCM (2x20 ml). The combined organic layer was washed with water, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using 2% methanol in DCM as eluent to give the title compound (0.4 g, 51.48% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.36-7.31 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.44 (dd, J=11.1, 4.8 Hz, 1H), 3.15-3.13 (m, 4H), 3.03-2.98 ( m, 4H), 2.842.73 (m, 2H), 2.70-2.59 (m, 1H), 2.50-2.37 (m, 2H), 2.32-2.24 (m, 1H), 2.14-0.77 (m, 33H), 1.34 (s, 3H), 1.04

- 37 045041 (s, 3H), 0.96 (s, 6H), 0.91 (s, 3H), 0.87 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H), 0.39-0.20 (m, 4H); ESI-MS: m/z

889.65 (M+H) ⁺ .

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1, 1dioxidothiomorpholino)-3-methylbutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2 ,2-dimethylcyclobutane-1-carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3αS,5αR,5bR,7αR,9S,11αR,11bR,13αR,13bR)-3α-(((S)-2-(1,1dioxidothiomorpholino)-3- methylbutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(1-methylcyclopropyl)icosahydro-1Hcyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobutan- 1,3-dicarboxylate (step 1, 0.4 g, 0.449 mmol, 1.0 eq.) in methanol (4 ml) and THF (4 ml) was added an aqueous 2.5N solution of KOH (1.35 ml, 3.373 mmol , 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, pH adjusted to 5.0 with 1N HCl and extracted with DCM (2x25 ml). The combined organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using 5% methanol in DCM as eluent to give the title compound (0.120 g, 33.5% yield) as an off-white solid. 1H NMR (300 MHz, DMSO-d ₆ ): δ ppm 4.44 (m, 1H), 3.06-2.95 (m, 8H), 2.84-2.71 (m, 3H), 2.45-2.24 (m, 5H), 1.95-0.78 (m, 31H), 1.26 (s, 3H), 1.02 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.86 (s, 6H), 0.83 (s, 3H), 0.38-0.18 (m, 4H) ; ESI-MS: m/z 799.60 (M+H)+.

Example 7. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2R,3R)-2-(1 ,1dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)- 2,2-dimethylcyclobutane-1-carboxylic acid:

Stage 1. Synthesis of 1-benzyl-3-((1R,3αS,5αR,5bR,7αR,9S,11αR,11bR,13αR,13bR)-3α-(((2R,3R)-3-((tertbutyldimethylsilyl)oxy )-2 -(1,1-dioxidothiomorpholino)butyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)(III ^)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of 4-((2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1-hydroxybutan-2yl)thiomorpholine-1,1-dioxide (intermediate 8, 0.442 g, 1.311 mmol, 1.5 eq.) trifluoromethanesulfonic acid anhydride (0.234 mL, 1.399 mmol, 1.6 eq.) was added to DCM (10 mL) at 0° C. The reaction mixture was stirred at 0°C for 10 minutes. 2,6-lutidine (0.166 ml, 1.399 mmol, 1.6 eq.) was added and stirred at 0°C for about 10 minutes. 1((1R,3αS,5αR,5bR,7αR,9S,11αR,11bR,13αR,13bR)-3α-amino-5α,5b,8,8,11α-pentamethyl-1-(1-methylcyclopropyl)icosahydro -1H-cyclopenta[a]chrysen-9-yl)3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1, 0.6 g, 0.8745 mmol, 1 .0 eq.) and triethylamine (0.36 ml, 2.623 mmol, 3.0 eq.) at 0°C and stirred at the same temperature for about 1 hour. The temperature of the reaction mixture was brought to room temperature and stirred for about 4 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with DCM (2x30 ml). The combined organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using 20% ethyl acetate in hexane as eluent to give the title compound (0.4 g, 45.5% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 7.36-7.33 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.44 (dd, J=11.1, 4.5 Hz, 1H), 4.0-3.97 (m, 1H), 3.46-3.35 ( m, 2H), 3.28-3.20 (m, 2H), 2.98 (m, 4H),

- 38 045041

2.84-2.72 (m, 2H), 2.70-2.60 (m, 1H), 2.60-2.50 (m, 2H), 2.42 (m, 2H), 2.15-0.78 (m, 28H), 1.34 (s, 3H), 1.04 (s, 3H), 0.96 (s, 6H), 0.91 (s, 3H), 0.87 (brs, 12H), 0.85 (s, 3H), 0.84 (s, 3H), 0.38-0.20 (m, 4H) , 0.06 (s, 3H),

0.05 (s, 3H); ESI-MS: m/z 1005.85 (M+H)+.

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2R,3R)-3-(( tertbutyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)butyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysene-9- yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2R,3R)-3-((tert-butyldimethylsilyl)oxy )-2-(1,1-dioxidothiomorpholino)butyl)amino)-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl)ucosahydro-1H-cyclonenta[a]chrysene-9-ul)(1R, 3S)-2,2-Dimethylcyclobutane-1,3-dicarboxylate (step 1, 0.4 g, 0.3977 mmol, 1.0 eq.) in methanol (5 ml) and THF (5 ml) was added aqueous 2, 5N KOH solution (1.19 mL, 2.983 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, pH adjusted to 5.0 with 1N HCl and extracted with DCM (2x20 ml). The combined organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using 3% methanol in DCM as eluent to give the title compound (0.3 g, 82.41% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.56-4.50 (m, 1H), 3.99-3.96 (m, 1H), 3.43-3.29 (m, 4H), 3.17-3.03 (m, 2H), 3.0-2.82 (m, 3H), 2.78-2.63 (m , 2H), 2.60-2.47 (m, 3H), 2.02-0.78 (m, 29H), 1.33 (s, 3H), 1.03 (s, 3H), 0.99 (s, 3H), 0.94 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.87 (s, 9H), 0.85 (s, 3H), 0.40-0.22 (m, 4H), 0.06 (s, 3H) , 0.04 (s, 3H); ES-MS: m/z 915.60 (M+H) ⁺ .

Stage 3. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2-( 1,1dioxidothiomorpholino)-3-hydroxybutylamino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2 ,2-dimethylcyclobutane-1-carboxylic acid:

To a stirred solution (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2R,3R)-3-((tert -butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)butyl)amino)-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl )oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid (step 2, 0.3 g, 0.327 mmol, 1.0 eq.) Tetra-n-butylammonium fluoride was added to THF (6 ml) at 0°C (10 mL, 10.0 mmol, 31.0 eq., 1.0 M in THF). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with water (20 ml) and extracted with DCM (2x20 ml). The combined organic layer was washed with water, dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The crude compound was purified by silica gel column chromatography using 5% methanol in DCM as eluent to give the title compound (65 mg, 24.8% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.48-4.45 (m, 1H), 3.75 (m, 1H), 3.33 (m, 2H), 3.20-3.05 (m, 6H), 2.82-2.74 (m, 2H), 2.63-2.53 (m, 5H), 2.10-2.0 (m, 2H), 1.97-0.78 (m, 26H), 1.33 (s, 3H), 1.07 (s, 6H), 0.98 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.86 (s, 6H), 0.42-0.20 (m, 4H); ES-MS: m/z 802.2 (M+H) ⁺ .

Example 8. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1, 1dioxidothiomorpholino)-3-hydroxynronyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2 ,2-dimethylcyclobutane-1-carboxylic acid:

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-3-((tertbutyldimethylsilyl)oxy)- 2 -(1,1-dioxidothiomorpholino)nponyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1- 39 045041 methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl )-( 1 R,3 S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of (S)-4-(1-((tert-butyldimethylsilyl)oxy)-3-hydroxypropan-2yl)thiomorpholine-1,1-dioxide (intermediate 9, 0.844 g, 2.623 mmol, 1.5 eq. ) trifluoromethane sulfonic anhydride (0.47 mL, 2.798 mmol, 1.6 eq.) was added to DCM (24 mL) at 0° C. The reaction mixture was stirred at 0°C for about 10 minutes. 2,6-lutidine (0.32 mL, 2.798 mmol, 1.6 eq.) was added and stirred at 0° C. for about 10 minutes. 1((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro -1H-cyclopenta[a]chrysen-9-yl)-3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1, 1.2 g, 1.749 mmol, 1, 0 eq.) and triethylamine (0.39 ml, 2.798 mmol, 1.6 eq.) at 0°C, stirred at the same temperature for about 1 hour. The reaction mixture was brought to room temperature and stirred overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with DCM (150 ml), washed with saturated sodium bicarbonate solution, water and brine. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-15% ethyl acetate in hexane to give the title compound (1 g, 57.8% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.36-7.33 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.44 (dd, J=11.1, 4.8 Hz, 1H), 3.71-3.67 (m, 2H), 3.30-3.22 ( m, 2H), 3.18-3.06 (m, 2H), 3.05-2.90 (m, 5H), 2.83-2.72 (m, 3H), 2.70-2.59 (m, 2H), 2.46-2.32 (m, 2H), 2.10-1.20 (m, 26H), 1.0-0.78 (m, 31H), 0.39-0.23 (m, 4H), 0.05 (s, 6H); ES-MS: m/z 992.09 (M+H) ⁺ .

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-3-((tert-butyldimethylsilyl )oxy)-2-(1,1-dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl) hydroxy)carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)3-((tert-butyldimethylsilyl)oxy)- 2-(1,1-dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11a-pentamethyl-1 -(1 -methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl)-( 1 R,3 S)-2,2-dimethylcyclobutane1,3-dicarboxylate (step 1, 1.0 g, 1.008 mmol, 1.0 eq.) in methanol (12 ml) and THF (12 ml) was added aqueous 2, 5N KOH solution (3.0 mL, 7.564 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, pH adjusted to 5.0 with 1N HCl and extracted with DCM (2x100 ml). The combined organic layer was washed with water (100 ml) and brine (100 ml). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using 0-6% methanol in DCM as eluent to give the title compound (0.4 g, 44.4% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.45 (m, 1H), 3.69-3.65 (m, 2H), 3.30 (m, 2H), 3.10-2.96 (m, 7H), 2.80-2.72 (m, 3H), 2.60-2.50 (m, 2H), 2.10-0.78 (m, 38H), 1.36 (s, 3H), 0.91 (s, 3H), 0.88 (s, 9H), 0.86 (s, 6H), 0.35-0.27 (m, 4H), 0.05 (s, 6H); ES-MS: m/z 902.18 (M+H)+.

Stage 3. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1, 1-dioxidothiomorpholino)-3-hydroxypropyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(1-methylcyclopropyl)icosahydro1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2 ,2-dimethylcyclobutane-1-carboxylic acid:

- 40 045041

To a stirred solution (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-3-((tertbutyldimethylsilyl) oxy)-2-(1,1-dioxidothiomorpholino)propyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysene-9- yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid (step 2, 0.4 g, 0.444 mmol, 1.0 eq.) bTHF (10 ml) TBAF (0.532 ml, 0.532 mmol, 1.2 eq., 1.0 M in THF). The reaction mixture was stirred at room temperature for about 4 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, diluted with water and extracted with DCM. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-10% methanol in DCM to give the title compound (90 mg, 25.7% yield) as an off-white solid. ¹ H NMR (300 MHz, DMSO-d ₆ ): δ ppm 4.40-4.32 (m, 1H), 3.62-3.58 (m, 1H), 3.50-3.45 (m, 1H), 3.10-2.92 (m, 8H), 2.82-2.70 (m, 3H), 2.38-2.23 (m , 5H), 1.92-0.78 (m, 24H), 1.26 (s, 3H), 0.99 (s, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.83 ( s, 6H), 0.81 (s, 3H), 0.33-0.22 (m, 4H); ESI-MS: m/z 788.27 (M+H)+.

Example 9. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1, 1dioxidothiomorpholino)-3-fluoropropyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)- 2,2-dimethylcyclobutane-1-carboxylic acid:

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1,1dioxidothiomorpholino)-3 -fluoropropyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-(1R,3S)-2, 2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of (S)-4-(1-fluoro-3-hydroxypropan-2-yl)thiomorpholine-1,1-dioxide (intermediate 10, 0.922 g, 4.372 mmol, 3.0 eq bDCM (10 mL) at Trifluoromethanesulfonic acid anhydride (0.78 mL, 4.664 mmol, 3.2 eq.) was added at 0° C. The reaction mixture was stirred at 0° C. for about 10 minutes. 2,6-lutidine (0.54 mL, 4.664 mmol, 3 ,2 eq.), stirred at 0°C for about 10 minutes.1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b was added successively 8,8,11a-πpentamethyl1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3-benzyl-(1S,3R)-2,2-dimethylcyclobutane1,3-dicarboxylate (intermediate 1, 1.0 g, 1.457 mmol, 1.0 eq.) and triethylamine (0.65 ml, 4.664 mmol, 3.2 eq.) at 0°C, stirred at the same temperature for about 1 hour. The reaction mixture was stirred at room temperature overnight. TLC indicated that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with DCM (100 ml), washed with saturated sodium bicarbonate solution (100 ml) and brine (100 ml). The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-20% ethyl acetate in hexane to give the title compound (0.300 g, 23.4% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.36-7.33 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.67 (d, J=4.5 Hz, 1H), 4.51 (d, J=4.8 Hz, 1H), 4.44 (dd , J=11.1, 4.5 Hz, 1H), 3.28-3.14 (m, 4H), 3.11-3.07 (m, 1H), 3.05-2.98 (m, 4H), 2.85-2.73 (m, 3H), 2.69-2.62 (m, 1H), 2.53-2.47 (m, 3H), 2.08-0.78 (m, 24H), 1.34 (s, 3H), 1.0 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H ), 0.91 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H), 0.40-0.20 (m, 4H); ES-MS: m/z 880.01 (M+H) ⁺ .

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1, 1dioxidothiomorpholino)-3-fluoropropyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)- 2,2-dimethylcyclobutane-1-carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(1,1dioxidothiomorpholino)-3- fluoropyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclonponyl)icosahydro-1Hcyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobutane-1 ,3-dicarboxylate (step 1, 0.300 g, 0.341 mmol,

- 41 045041

1.0 eq.) in methanol (3 mL) and THF (3 mL) was added an aqueous 2.5N solution of KOH (1 mL, 2.559 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, pH adjusted to 4.0 with 1N HCl and extracted with DCM (2x100 ml). The combined organic layer was washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane. The resulting compound was further purified by treatment with DCM:hexane (1:9 ratio), the solid was filtered and dried in vacuo to give the title compound (0.090 g, 33.4% yield) as a white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.67 (d, J=4.5 Hz, 1H), 4.51 (d, J=4.5 Hz, 1H), 4.46 (dd, J=11.1, 4.2 Hz, 1H), 3.23-3.0 (m, 9H), 2.84-2.75 (m, 3H), 2.64-2.56 (m, 4H), 2.090.78 (m, 24H), 1.37 (s, 3H), 1.07 (s, 3H), 1.01 (s, 3H), 0.96 (s, 3H ), 0.91 (s, 3H), 0.87 (s, 6H), 0.85 (s, 3H), 0.380.29 (m, 4H); ES-MS: m/z 790.02 (M+H)+.

Example 10. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)-2-(1, 1dioxidothiomorpholino)-3-fluoropropyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1Hcyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2- dimethylcyclobutane-1-carboxylic acid:

black YES y^s=o o

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((R)-2-(1,1dioxidothiomorpholino)-3- fluoropropyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1Hcyclopenta[a]chrysen-9-yl)-(1R,3 S)-2,2-dimekylcyclobutan- 1,3-dicarboxylate:

To a stirred solution of (R)-4-(1-fluoro-3-hydroxypropan-2-yl)thiomorpholine-1,1-dioxide (intermediate 11, 0.645 g, 3.061 mmol, 3.0 eq.) in DCM(14 ml) trifluoromethanesulfonic acid anhydride (0.548 ml, 3.265 mmol, 3.2 eq.) was added at 0°C. After stirring for 10 minutes at 0° C., 2,6-lutidine (0.38 mL, 3.265 mmol, 3.2 eq.) was added and stirred at the same temperature for about 10 minutes. 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl-1(1-methylcyclopropyl) was added to the reaction mixture )icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3dicarboxylate (intermediate 1, 0.700 g, 1.020 mmol, 1.0 eq .) and triethylamine (0.45 ml, 3.265 mmol, 3.2 eq.) and stirred at 0°C for about 1 hour. The reaction mixture was allowed to stir at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with DCM (100 ml), washed with saturated sodium bicarbonate (100 ml), water and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-20% ethyl acetate in hexane to give the title compound (0.200 g, 22.2% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.36-7.35 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.64-4.62 (m, 1H), 4.48-4.36 (m, 2H), 3.27-3.22 (m, 2H), 3.183.02 (m, 7H), 2.85-2.72 (m, 3H), 2.59-2.50 (m, 1H), 2.42-2.32 (m, 1H), 2.10-0.78 (m, 26H), 1.34 (s, 3H ), 1.04 (s, 3H), 0.97 (s, 6H), 0.91 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H), 0.40-0.20 (m, 4H); ES-MS: m/z 880.01 (M+H) ⁺ .

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3α-(((R)-2-(1, 1-diokCidothiomorpholino)-3-fluoropropyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl) -2,2-dimethylcyclobutane-1-carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)-2-(1,1dioxidothiomorpholino)-3- fluoropropyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl) and cosahydro-1Hcyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobutane-1 ,3-dicarboxylate (step 1, 0.200 g, 0.227 mmol, 1.0 eq.) in methanol (2 ml) and THF (2 ml) was added an aqueous 2.5N solution of KOH (0.68 ml, 1.706 mmol, 7. 5 eq.). Reak

- 42 045041 tion mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, pH adjusted to 4.0 with 1N HCl and extracted with DCM (2x100 ml). The combined organic layer was washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane. The resulting compound was further purified by DCM:hexane (1:9 ratio) and the resulting precipitates were filtered and dried in vacuo to give the title compound (0.080 g, 44.6% yield) as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.64-4.40 (m, 3H), 3.302.98 (m, 9H), 2.80-2.50 (m, 5H), 2.20-0.78 (m, 26H), 1.35 (s, 3H), 1.05 (s, 3H), 1.01 (s, 3H), 0.92 (s, 9H), 0.88 (s, 3H), 0.87 (s, 3H), 0.40-0.20 (m, 4H); ES-MS: m/z 790.02 (M+H)+.

Example 11. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(4( isonronulsulfonyl)niperazun-1-yl)ronyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl )-2,2-dimethylcyclobutane-1-carboxylic acid:

O

Step 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(4(isopropylsulfonyl)piperazine) -1 -yl)propyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1 -methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl)-( 1 R, 3 S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of (S)-2-(4-(isopropylsulfonyl)piperazin-1-yl)propan-1-ol (intermediate 12, 0.547 g, 2.186 mmol, 1.5 eq.) bDCM (30 mL) at 0 °C, trifluoromethanesulfonic acid anhydride (0.41 mL, 2.478 mmol, 1.7 eq.) was added. After stirring for 10 minutes at 0°C, 2,6-lutidine (0.28 mL, 2.478 mmol, 1.7 eq.) was added and stirred at 0°C for about 10 minutes. 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl) was added to the reaction mixture Icosahydro-1H-cyclopenta[a]chrysen-9-yl)3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1, 1.0 g, 1.457 mmol, 1.0 eq.) dissolved in DCM (10 ml), and then triethylamine (0.6 ml, 4.372 mmol, 3.0 eq.) and stirred at 0°C for about 1 hour. The reaction mixture was allowed to stir at room temperature for about 4 hours. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with water (150 ml) and extracted with DCM (3x200 ml). The combined organic layer was washed with water (100 ml) and brine (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using 2% methanol in dichloromethane as eluent. Fractions containing the expected product were combined and concentrated under reduced pressure to give the title compound (0.350 g, 26.3% yield) as a yellow semi-solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.35 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.44 (dd, J=11.1, 4.8 Hz, 1H), 3.48-3.31 (m, 4H), 3.23-3.18 (m, 1H), 2.92-2.43 (m, 10H), 2.10-0.78 (m, 35H), 1.34 (s, 3H), 1.10 (s, 3H), 1.0 (s, 3H), 0.96 (s, 3H), 0.93 (s, 3H), 0.87 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H), 0.40-0.20 (m, 4H); ESMS: m/z 919.03 (M+H) ⁺ .

Stage 2. Synthesis of (1R,3S)-3-((((1R,3αS,5αR,5bR,7αR,9S,11αR,11bR,13αR,13bR)-3a-(((S)-2-(4- (isonronulsulfonyl)niperazin-1-ul)nronul)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl) -2,2-dimethylcyclobutane-1-carboxylic acid:

O

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((S)-2-(4(isonronylsulfonyl)niperazine-1 -yl)nronyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-(1R,3S)-2 ,2-dimethylcyclobutane-1,3-dicarboxylate (step 1, 0.350 g, 0.381 mmol, 1.0 eq.) in methanol (3.5 ml) and THF (3.5 ml) at 0°C added aqueous 2, 5N KOH solution (1.14 ml, 2.858 mmol,

- 43 045041

7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, pH adjusted to 4.0 with 1N HCl and extracted with DCM (3x150 ml). The combined organic layers were washed with water (150 ml), dried over sodium sulfate, filtered and evaporated under reduced pressure. The crude compound was purified by silica gel column chromatography using 6% methanol in dichloromethane as eluent. Fractions containing the expected product were combined and concentrated under reduced pressure. The resulting compound was dissolved in DCM (2 ml), treated with hexane (10 ml), stirred at room temperature for about 15 minutes.

The resulting precipitate was collected by filtration and dried in vacuo to give the title compound (70 mg, 22.2% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.47 (m, 1H), 3.50-3.30 (m, 4H), 3.25-3.15 (m, 1H), 2.93 (m, 1H), 2.83-2.73 (m, 2H), 2.682.50 (m, 7H), 2.10-0.78 (m, 35Н), 1.34 (s, 3Н), 1.17 (s, 3Н), 1.07 (s, 3Н), 1.03 (s, 3Н), 0.93 (s, 3Н), 0.90 (s, 3Н) , 0.86 (s, 6H), 0.52-0.22 (m, 4H); ES-MS: m/z 829.01 (M+H)+.

Example 12. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3S)-2-( 1,1dioxidothiomorpholino)-3-fluorobutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1Hcyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2 ,2-dimethylcyclobutane-1-carboxylic acid:

Step 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2R,3S)-2-(1,1-dioxygothiomorpholino )-3-fluorobutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-(1R,3S)- 2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of 4-(2R,3S)-3-fluoro-1-hydroxybutan-2-yl)thiomorpholine-1,1-dioxide (intermediate 13, 0.590 g, 2.623 mmol, 2.0 eq.) in DCM ( 20 ml) at 0°C, 2,6lutidine (0.45 ml, 3.933 mmol, 3.0 eq.) was added. The reaction mixture was stirred at 0°C for about 10 minutes, then trifluoromethanesulfonic acid anhydride (0.567 ml, 3.277 mmol, 2.5 eq.) was added and stirred at the same temperature for about 15 minutes. 1((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl) was added to the reaction mixture )icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3dicarboxylate (intermediate 1, 0.9 g, 1.311 mmol, 1, 0 eq.) and triethylamine (0.55 ml, 3.933 mmol, 3.0 eq.) and stirred at 0°C for about 4 hours. The reaction mixture was allowed to stir at room temperature overnight. Saturated sodium bicarbonate solution was added to the reaction mixture and extracted with DCM (2x30 ml). The combined organic layer was washed with water, dried over sodium sulfate and concentrated under reduced pressure.

The crude compound was purified by silica gel column chromatography using 20% ethyl acetate in hexane as eluent to give the title compound (0.3 g, 25.64% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 7.36-7.34 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.44 (dd, J=11.1, 4.5 Hz, 1H), 4.50-4.48 (m, 0.5H), 4.33-4.31 (m, 0.5H), 3.22-3.13 (m, 2H), 3.10-3.0 (m, 7H), 2.85-2.58 (m, 4H), 2.55-2.43 (m, 1H), 2.08-0.78 (m, 29H ), 1.34 (s, 3H), 0.99 (s, 3H), 0.96 (s, 6H), 0.90 (s, 3H), 0.85 (s, 6H), 0.84 (s, 3H), 0.40-0.20 (m, 4H); ES-MS: m/z 893.91 (M+H) ⁺ .

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3S)-2-( 1,1dioxidothiomorpholino)-3-fluorobutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1Hcyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2 ,2-dimethylcyclobutane-1-carboxylic acid:

- 44 045041

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3S)-2-(1,1dioxidothiomorpholino)- 3-fluorbutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1Hcyclopenha[a]chrysen-9-yl)-(1R,3S)-2,2- dimethylcyclobutane-1,3-dicarboxylate (step 1, 0.3 g, 0.335 mmol, 1.0 eq.) in methanol (3 ml) and THF (3 ml) was added an aqueous 2.5N solution of KOH (1.0 ml, 2.518 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated, cooled to 0°C, pH adjusted to 5.0 with 1N HCl and extracted with DCM (2x25 ml). The combined organic layer was washed with water, dried over sodium sulfate and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using 2% methanol in dichloromethane as eluent to give the title compound (0.13 g, 48.32% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.49-4.32 (m, 2H), 3.21-3.04 (m, 9H), 2.85-2.70 (m, 3H), 2.64-2.48 (m, 2H), 2.10-0.78 (m, 29H), 1.33 (s, 3H ), 1.0 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.86 (s, 9H), 0.85 (s, 3H), 0.40-0.22 (m, 4H); ES-MS: m/z 803.82 (M+H)+.

Example 13. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2S,3S)-2-( 1,1dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(1-methylcyclopropyl)icosahydro1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2 ,2-dimethylcyclobutane-1-carboxylic acid:

I'm n TTL

-VA ^ ^he o

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2S,3S)-3-((tert-butyldimethylsilyl)oxy )-2-(1,1-dioxidothiomorpholino)butyl)amino)-5a,5th,8,8,11 a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)- (1R,3S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of 4-((2S,3S)-3-((tert-butyldimethylsilyl)oxy)-1-hydroxybutan-2yl)thiomorpholine (intermediate 14, 0.787 g, 2.33 mmol, 2.0 eq.) in DCM (16 ml) at 0° C., trifluoromethanesulfonic acid anhydride (0.31 ml, 1.86 mmol, 1.6 eq.) was added. The reaction mixture was stirred at 0°C for about 10 minutes. 2,6-lutidine (0.21 mL, 1.86 mmol, 1.6 eq.) was then added to the reaction mixture and stirred for about 10 minutes. 1((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl) was added to the reaction mixture )icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1, 0.800 g, 1.166 mmol, 1, 0 eq.), followed by triethylamine (0.48 ml, 3.498 mmol, 3.0 eq.) and stirred at 0°C for about 1 hour. The reaction mixture was brought to room temperature and stirred overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was quenched with aqueous sodium bicarbonate and extracted with DCM (3x100 ml). The combined organic layer was washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a 20-30% ethyl acetate in hexane gradient to give the title compound (0.400 g, 34% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.36-7.30 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.45 (dd, J=11.1, 4.5 Hz, 1H), 3.95-3.92 (m, 1H), 3.44-3.39 ( m, 2H), 3.21-3.11 (m, 2H), 3.03-2.93 (m, 5H), 2.84-2.73 (m, 2H), 2.69-2.62 (m, 1H), 2.59-2.53 (m, 2H), 2.47-2.35 (m, 2H), 2.08-0.78 (m, 30H), 1.34 (s, 3H), 1.0 (s, 3H), 0.96 (s, 3H), 0.96 (s, 3H), 0.91 (s, 3H), 0.87 (s, 9H), 0.85 (s, 3H), 0.84 (s, 3H), 0.36-0.23 (m, 4H), 0.05 (s, 3H), 0.04 (s, 3H); ES-MS: m/z 1005.97 (M+H) ⁺ .

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2S,3S)-3-( (tertbutyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)butyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1 methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysene-9 -yloxy)carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid:

- 45 045041

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2S,3S)-3-((tert-butyldimethylsilyl)oxy )-2-(1,1-dioxidothiomorpholino)butyl)amino)-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-( 1 R,3S)2,2-dimethylcyclobutane-1,3-dicarboxylate (step 1, 0.400 g, 0.397 mmol, 1.0 eq.) in methanol (4 ml) and THF (4 ml) added an aqueous 1.5N solution KOH (1.9 mL, 2.98 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature for about 5 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, acidified with 1N HCl to pH 4.0 and extracted with DCM (3x50 ml). The combined organic layer was washed with water (50 ml), dried over sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-6% methanol in dichloromethane to give the title compound (0.200 g, 55% yield) as a solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.49-4.44 (m, 1H), 4.0-3.92 (m, 1H), 3.40-3.22 (m, 4H), 3.04-2.90 (m, 5H), 2.80-2.70 (m, 3H), 2.60-2.50 (m , 3H), 2.32 (m, 1H), 2.10-0.78 (m, 36H), 1.33 (s, 3H), 1.06 (s, 3H), 1.04 (s, 3H), 0.97 (s, 3H), 0.95 ( s, 3H), 0.91 (s, 3H), 0.89 (s, 6H), 0.40-0.20 (m, 4H), 0.04 (s, 3H), 0.03 (s, 3H); ES-MS: m/z 915.74 (M+H)+.

Stage 3. Synthesis of (1R,3S)-3-((((1R,3aS,5αR,5bR,7αR,9S,11αR,11bR,13αR,13bR)-3a-(((2S,3S)-2-( 1,1-dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy) carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid:

To a stirred solution (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2S,3S)-3-((tert -butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)butyl)amino)-5a,5b,8,8,11apentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl )oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid (step 2, 0.200 g, 0.218 mmol, 1.0 eq.) bTHF (2 ml) at 0° C. was added TBAF (0.654 ml, 0.654 mmol, 3.0 eq., 1.0 M in THF). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was not consumed, TBAF (1.526 ml, 1.526 mmol, 7.0 eq.) was added again at 0°C, the temperature was returned to room temperature and stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with water (50 ml) and extracted with DCM (3x30 ml). The combined organic layers were washed with water (50 ml), dried over sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane. The resulting compound was further purified by treatment with ethyl acetate (1 ml) and hexane (10 ml), stirred at room temperature for about 10 minutes, filtered and dried in vacuo to obtain the title compound (0.080 g, 45.7% yield) as a solid. . 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.46 (dd, J=11.7, 4.8 Hz, 1H), 3.80 (m, 1H), 3.42-3.32 (m, 2H), 3.18-3.0 (m, 7H), 2.80-2.50 (m, 7H), 2.08- 0.78 (m, 27Н), 1.36 (s, 3Н), 1.06 (s, 3Н), 1.03 (s, 3Н), 0.98 (s, 3Н), 0.91 (s, 3Н), 0.88 (s, 3Н), 0.85 (s, 6H), 0.35-0.20 (m, 4H); ES-MS: m/z 801.73 (M+H)+.

Example 14. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-3hydroxy-2 -(4-(isopropylsulfonyl)piperazin-1-yl)butyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl )hydroxy)carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid:

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5αR,5bR,7αR,9S,11αR,11bR,13αR,13bR)-3a-(((2R,3R)-3-((tertbutyldimethylsilyl)oxy )-2-(4-(isopropylsulfonyl)piperazin-1 -yl)butyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysene-9 -yl)-(1R,3S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

- 46 045041

To a stirred solution of (2R,3R)-3-((tert-butyldimethylsilyl)oxy)-2-(4-(isopropylsulfonyl)piperazin-1-yl)butan-1-ol (intermediate 15, 3.21 g, 8 .16 mmol, 1.6 eq.) trifluoromethanesulfonic acid anhydride (1.45 mL, 8.67 mmol, 1.7 eq.) was added to DCM (50 mL) at 0°C. The reaction mixture was stirred at 0°C for about 10 minutes. 2,6-lutidine (0.65 mL, 8.67 mmol, 1.7 eq.) was added and stirred at 0° C. for 15 minutes. 1((1R,3aS,5αR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl) was added to the reaction mixture )icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1, 3.5 g, 5.10 mmol, 1.0 eq.) and triethylamine (2.13 ml, 15.3 mmol, 3.0 eq.) and stirred at 0°C for about 1 hour. The reaction mixture was brought to room temperature and stirred overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with water (100 ml), made basic with aqueous sodium bicarbonate and extracted with DCM (3x100 ml). The combined organic layer was washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-2% methanol in dichloromethane to give the title compound (2.0 g, 36.9% yield) as a solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 7.35 (m, 5H), 5.15, 5.09 (ABq, JAB=12.6 Hz, 2H), 4.44 (dd, J=11.1, 4.5 Hz, 1H), 4.01-3.98 (m, 1H), 3.30-3.15 (m, 5Н), 2.90-2.32 (m, 10Н), 2.08-1.48 (m, 13Н), 1.471.13 (m, 24Н), 1.07-0.78 (m, 31Н), 0.38-0.18 (m, 4Н), 0.04 ( s, 3H), 0.03 (s, 3H); ES-MS: m/z 1063.68 (M+H)+.

Stage 2. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7αR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-3-hydroxy2-(4- (isopropylsulfonyl)piperazin-1 -yl)butyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1 -methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl)- (1R,3S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2R,3R)-3-((tert-butyldimethylsilyl)oxy )-2-(4-(isonropylsulfonyl)niperαzin-1-yl)butyl)amino)5α,5b,8,8,11a-pentamethyl-1-(1-methylcyclonronyl)icosahydro-1H-cyclonenta[a]chrysene-9 -yl)-(1R,3S)-2,2dimethylcyclobutane-1,3-dicarboxylate (step 1, 2.0 g, 1.88 mmol, 1.0 eq.) bTHF (20 ml) TBAF was added at 0°C (7.52 mL, 7.52 mmol, 4.0 eq., 1.0 M in THF). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with water (100 ml) and extracted with DCM (3x100 ml). The combined organic layer was washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-4% methanol in dichloromethane to give the title compound (0.500 g, 28% yield) as a solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.36-7.31 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.44 (dd, J=10.8, 4.5 Hz, 1H), 3.67-3.56 (m, 1H), 3.42-3.33 ( m, 4H), 3.25-3.13 (m, 1H), 2.95-2.57 (m, 9H), 2.43-1.90 (m, 6H), 1.88-1.18 (m, 32H), 1.030.78 (m, 22H), 0.25-0.24 (m, 4H); ES-MS: m/z 948.93 (M+H) ⁺ .

Stage 3. Synthesis of (1R,3S)-3-((((1R,3αS,5αR,5bR,7αR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-3-hydroxy -2-(4(isopronylsulfonyl)niperazin-1-yl)butyl)amino)-5α,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysene-9- yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2R,3R)-3-hydroxy-2-(4- (isopropylsulfonyl)piperazin-1-yl)butyl)amino)-5a,5b,8,8,11a-pentamethyl1 -(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-( 1 R ,3 S)-2,2-dimethylcyclobutane-1,3dicarboxylate (step 2, 0.500 g, 0.527 mmol, 1.0 eq.) bTHF (5 ml) and methanol (5 ml) at 0°C added aqueous 1.5N KOH solution (2.63 mL, 3.954 mmol, 7.5 eq.). The temperature of the reaction mixture was brought to

- 47 045041 room temperature and stirred overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated under reduced pressure. The residue was diluted with water (20 ml), cooled to 0°C, acidified with 1N HCl to pH 4.0 and extracted with DCM (3x50 ml). The combined organic layer was washed with water (50 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-10% methanol in dichloromethane. The resulting compound was treated with DCM (5 ml) and hexane (40 ml), stirred at room temperature for about 15 minutes and the solvent was decanted. DCM (5 ml) and hexane (20 ml) were added again, stirred at room temperature for about 15 minutes, the resulting precipitate was filtered and dried in vacuum for 2 hours to obtain the title compound (0.180 g, 40% yield) as a solid substances. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.46 (dd, J=11.7, 4.8 Hz, 1H), 3.75 (m, 1H), 3.35 (m, 4H), 3.233.14 (m, 1H), 2.92-2.88 (m, 2H), 2.79-2.74 ( m, 4H), 2.61-2.51 (m, 4H), 2.10-1.2 (m, 35H), 1.10-0.78 (m, 24H), 0.34-0.25 (m, 4H); ES-MS: m/z 858.82 (M+H)+.

Example 15. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2(1 ,1-dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-9 -yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid:

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-3-((tertbutyldimethylsilyl)oxy )-2 -(1,1-dioxidothiomorpholinobutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(prop1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-9- yl)-(1R,3S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of 4-((2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1-hydroxybutan-2yl)thiomorpholine-1,1-dioxide (intermediate 8, 4.14 g, 12.27 mmol , 1.5 eq.) in DCM (80 ml) at 0°C was added trifluoromethanesulfonic acid anhydride (2.33 ml, 13.9 mmol, 1.7 eq.) and stirred for about minutes. 2,6-lutidine (1.6 mL, 13.9 mmol, 1.7 eq.) was added to the reaction mixture and stirred at 0° C. for about 15 minutes. 1-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino5a,5b,8,8,11 a-pentamethyl-1-(prop-1) was added to the reaction mixture -en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 16, 5, 5 g, 8.18 mmol, 1.0 eq.) and triethylamine (3.42 ml, 24.54 mmol, 3.0 eq.), stirred at 0°C for about 1 hour and left stirring at room temperature for nights. TLC showed that the starting material was consumed and the desired product was observed. Aqueous sodium bicarbonate was added to the reaction mixture and extracted with DCM (3x100 ml). The combined organic layer was washed with water (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane to give the title compound (4.0 g, 49.3% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 7.36-7.30 (m, 5H), 5.14, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.69 (s, 1H), 4.58 (s, 1H), 4.43 (dd, J=11.7, 4.5 Hz, 1H) , 4.03-3.98 (m, 1H), 3.47-3.37 (m, 2H), 3.30-3.18 (m, 2H), 3.10-2.94 (m, 5H), 2.852.40 (m, 7H), 2.07-1.98 ( m, 2H), 1.93-0.75 (m, 25H), 1.68 (s, 3H), 1.33 (s, 3H), 1.05 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.88 (s, 9H), 0.85 (s, 3H), 0.84 (s, 3H), 0.83 (s, 3H), 0.07 (s, 6H); ES-MS: m/z 991.93 (M+H) ⁺ .

Stage 2. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2-(1,1dioxidothiomorpholino) -3-hydroxybutyl)amino)-5α,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-1Hcyclopenta[a]chrysen-9-yl)-(1R, 3 S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2R,3R)-3-((tert-butyldimethylsilyl)oxy )-2-(1,1-dioxidothiomorpholino)butyl)amino)-5a,5b,8,8,11 apentamethyl-1 -(prop-1-en-2-yl)icosahydro-1 H-cyclopenta[a]chrysene -9-yl)-(1R,3S)-2,2-dimethylcyclobu

- 48 045041 tan-1,3-dicarboxylate (step 1, 4.0 g, 4.034 mmol, 1.0 eq.) bTHF (60 ml) at 0° C. add TBAF (20.17 ml, 20.17 mmol, 5.0 eq., 1.0 M in THF). The reaction mixture was brought to room temperature and stirred overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with water (50 ml) and extracted with DCM (3x50 ml). The combined organic layer was washed with water (2x50 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-5% methanol in DCM to give the title compound (2.0 g, 56.6% yield) as a solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 7.35 (m, 5H), 5.14, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.69 (s, 1H), 4.60 (s, 1H), 4.45-4.40 (m, 1H), 3.72 (m, 1H) , 3.45-3.35 (m, 2H), 3.13-3.03 (m, 7H), 2.85-2.73 (m, 2H), 2.70-2.58 (m, 2H), 2.57-2.42 (m, 3H), 2.07-1.98 ( m, 2H), 1.90-0.75 (m, 25H), 1.68 (s, 3H), 1.34 (s, 3H), 1.05 (s, 3H), 0.96 (s, 6H), 0.85 (s, 3H), 0.84 (s, 3H), 0.83 (s, 3H); ES-MS: m/z 877.84 (M+H)+.

Stage 3. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2-( 1,1dioxidothiomorpholino)-3-hydroxybutyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-9 -yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2R,3R)-2-(1,1-dioxidothiomorpholino) -3-hydroxybutyl)αmino)-5a,5b,8,8,11 a-pentamethyl-1 -(prop-1en-2-yl)icosahydro-1H-cyclopentα[a]chrysen-9-yl)-(1R, 3S)-2,2-Dimethylcyclobutane-1,3-dicarboxylateα (step 2, 2.0 g, 2.279 mmol, 1.0 eq.) in THF (12 ml) and methanol (12 ml) at 0°C was added aqueous 1.5N KOH solution (12 ml, 17.09 mmol, 7.5 eq.). The temperature of the reaction mixture was brought to room temperature and stirred for about 4 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated under reduced pressure, diluted with water (20 ml), acidified with 1N HCl to pH 4.0 and extracted with DCM (3x50 ml). The combined organic layer was washed with water (20 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 48% methanol in DCM. The resulting compound was further purified by treatment with chloroform (5 ml) and hexane (20 ml), stirred for about 10 minutes and decanted. This procedure was repeated twice, finally filtered and dried in vacuo to obtain the title compound (0.800 g, 44.6% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.69 (s, 1H), 4.60 (s, 1H), 4.50-4.45 (m, 1H), 3.83-3.74 (m, 1H), 3.40-3.07 (m, 9H), 2.80-2.49 (m, 7H), 2.07-0.77 (m, 27H), 1.68 (s, 3H), 1.36 (s, 3H), 1.10 (s, 3H), 1.06 (s, 3H ), 0.97 (s, 3H), 0.89 (s, 3H), 0.85 (s, 6H); ES-MS: m/z 787.60 (M+H) ⁺ .

Example 16. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)-2-carboxy-2 -(1,1dioxidokyomorpholino)ethyl)αmino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)- 2,2-dimethylcyclobutane-1-carboxylic acid:

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)-2-(1,1d,ioxidothiomorpholino) -3-methoxy-3-oxopropyl)αmino)-5a,5b,8,8,11 a-pentamethyl-1 -(1-methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl)-( 1 R,3 S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution of methyl(R)-2-(1,1-dioxidothiomorpholino)-3-hydroxypropanoate (intermediate 5, 0.34 g, 1.457 mmol, 1.25 eq.) in dichloromethane (16 ml) at 0°C trifluoromethanesulfonic acid anhydride (0.27 mL, 1.632 mmol, 1.4 eq.) was added. The reaction mixture was stirred at 0°C for 10 minutes, then 2,6-lutidine (0.19 ml, 1.667 mmol, 1.43 eq.) was added and stirred at the same temperature for 10 minutes. 1 was added to the reaction mixture

- 49 045041 ((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro -1H-cyclopenta[a]chrysen-9-yl)-3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1, 0.8 g, 1.166 mmol, 1, 0 eq.), followed by triethylamine (0.23 ml, 1.667 mmol, 1.43 eq.) and stirred at 0°C for about 1 hour. The temperature of the reaction mixture was brought to room temperature and stirred for about 4 hours. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was made basic with saturated sodium bicarbonate solution and extracted with dichloromethane (3x50 ml). The combined organic layer was washed with water (20 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 03% methanol in dichloromethane to give the title compound (0.680 g, 64.7% yield) as a solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 7.35 (m, 5H), 5.15, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.44 (dd, J=11.1, 4.5 Hz 1H), 3.70 (s, 3H), 3.42-3.35 (m, 1H), 3.35-3.25 (m, 1H), 3.15-2.94 (m, 7H), 2.852.57 (m, 5H), 2.08-1.0 (m, 28H), 1.0-0.78 (m, 22H), 0.38-0.20 (m , 4H).

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)-2-(1, 1-dioxidothiomorpholino)-3-methoxy-3-oxopropyl)αmino)-5α,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro1H-cyclopenta[a]chrysen-9-yl)oxy) carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid:

To a suspension of Pd/C (500 mg) in ethyl acetate (7 ml) and methanol (7 ml) was added 1-benzyl-3((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)- 3a-(((R)-2-(1,1-dioxidothiomorpholino)-3-methoxy-3oxopropyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobutane-1,3-dicarboxylate (step 1, 0.35 g, 0.386 mmol, 1.0 eq.) and formate ammonium (0.121 g, 1.933 mmol, 5.0 eq.). The reaction mixture was stirred at room temperature for about 1 hour. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was filtered through a celite pad and washed with 5% methanol in DCM (100 ml). The filtrate was evaporated under reduced pressure and the residue was purified by silica gel column chromatography using a 5 to 10% gradient of methanol in DCM with one drop of acetic acid. The resulting solid was treated with ethyl acetate (2 ml) and hexane (8 ml) and stirred at room temperature for 20 minutes. The resulting precipitate was collected by filtration and dried under vacuum to give the title compound (40 mg, 12.69% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.47-4.45 (m, 1H), 3.70 (s, 3H), 3.42-3.35 (m, 1H), 3.28 (m, 1H), 3.15-3.02 (m, 4H), 2.98 (m, 3H), 2.85- 2.75 (m, 2H), 2.65-2.52 (m, 3H), 2.10-1.18 (m, 25H), 1.10-0.78 (m, 25H), 0.40-0.20 (m, 4H); ES-MS: m/z 815.88 (M+H)+.

Stage 3. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((R)-2-carboxy-2( 1,1-dioxidothiomorpholino)ethyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2, 2-dimethylcyclobutane-1-carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)-2-(1,1d,ioxidothiomorpholino)- 3-methoxy-3-oxopropyl)αmino)-5a,5b,8,8,11 a-pentamethyl-1 -(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)-(1R,3S )-2,2-dimethylcyclobutane-1,3-dicarboxylate (0.3 g, 0.33 mmol, 1.0 eq.) in methanol (3 ml) and THF (3 ml) added an aqueous solution of 2.5N KOH solution (1.0 ml, 2.48 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, diluted with water (10 ml), cooled to 0°C, acidified with 1N HCl to pH 2.0 and extracted with DCM (3x25 ml). The combined organic layer was washed with water (20 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using 10% methanol in DCM as eluent using one drop of acetic acid. The resulting compound was further purified by treatment with ethyl acetate (20 ml), heated until condensation appeared for about 30 minutes. The reaction mixture was cooled to room temperature, the solid was filtered, washed with ethyl acetate (5 ml) and dried in vacuo to give the title compound (50 mg, 18.8% yield) as a white solid. 1H NMR (300 MHz, DMSO-d ₆ ): δ ppm 4.38-4.32 (m, 1H), 3.08-2.93 (m, 8H), 2.82-2.68 (m, 4H), 2.37-2.23 (m, 2H), 2.0-0.78 (m, 26H), 1.27 (s, 3H ), 1.05 (s, 3H), 0.95 (s, 3H), 0.91 (s, 6H), 0.85 (s, 3H), 0.84 (s, 3H), 0.83 (s, 3H), 0.4-0.18 (m, 4H); ESI-MS: m/z 823.55

- 50 045041 (M+Na)+.

Example 17. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2(1 ,1-dioxidothiomorpholino)-3 -(2-(2-methoxyethoxy)acetoxy)butyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 (1-methylcyclopropyl)icosahydro-1H-cyclopenta[a] chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutan-1-carboxylic acid:

To the stirred solution (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2R,3R)-2-( 1, 1-dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl) -2,2-dimethylcyclobutane-1-carboxylic acid (example 8, 0.400 g, 0.499 mmol, 1.0 eq.) in DCM (10 ml) was added DMAP (0.030 g, 0.24 mmol, 0.5 eq.) , EDC.HCl (0.430 g, 2.24 mmol, 4.5 eq.) and 2-(2-methoxyethoxy)acetic acid (intermediate 17, 0.167 g, 1.24 mmol, 2.5 eq., step 2 ). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with water (10 ml) and extracted with DCM (3x20 ml). The combined organic layer was washed with water (20 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-4% methanol in dichloromethane. The resulting compound was further purified by treatment with chloroform (2 ml) and hexane (8 ml), stirred at room temperature for about 30 minutes, filtered and dried in vacuo to obtain the title compound (0.100 g, 21.8% yield) as a solid. . ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 5.15-5.09 (m, 1H), 4.48 (m, 1H), 4.16 (s, 2H), 3.73-3.71 (m, 2H), 3.62-3.58 (m, 2H), 3.38 (s, 3H), 3.27- 3.19 (m, 4H), 3.0-2.95 (m, 5H), 2.85-2.73 (m, 2H), 2.65-2.55 (m, 1H), 2.50-2.35 (m, 2H), 2.08-2.0 (m, 2H ), 1.90-0.78 (m, 27Н), 1.36 (s, 3Н), 1.09 (s, 3Н), 1.06 (s, 3Н), 0.98 (s, 3Н), 0.90 (s, 3Н), 0.90 (s, 3H), 0.87 (s, 3H), 0.87 (s, 3H), 0.38-0.25 (m, 4H); ES-MS: m/z 917.95 (M+H) ⁺ .

Example 18. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)13-(1 ,1-dioxidothiomorpholino)-12-methyl-2,5,8,11-tetraoxatetradecane-14-yl)amino)-5a,5b,8,8,11a-pentamethyl-1(1-methylcyclopropyl)icosahydro-1H-cyclopenta [a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutan-1-carboxylic acid:

Stage 1. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)-13-(1,1dioxidothiomorpholino) -12-methyl-2,5,8,11-tetraoxatetradecane-14-yl)αmino)-5a,5b,8,8,11a-pentamethyl1 -(1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysene- 9-yl)-(1 R,3 S)-2,2-dimethylcyclobutane-1,3dicar boxylate:

To a stirred solution of 4-((12R,13R)-14-hydroxy-12-methyl-2,5,8,11-tetraoxαtetradecαn-13yl)thiomorpholine-1,1-dioxide (intermediate 18, 0.775 g, 2.099 mmol, 1.6 eq.) trifluoromethanesulfonic acid anhydride (0.39 mL, 2.361 mmol, 1.8 eq.) was added to DCM (20 mL) at 0° C. After stirring for 10 minutes at 0°C, 2,6-lutidine (0.3 ml, 2.623 mmol, 2.0 eq.) was added and stirred at the same temperature for about 10 minutes. 1((1 R,3aS,5aR,5bR,7aR,9S, 11 aR, 11bR, 13aR, 13bR)-3a-amino-5a,5b,8,8,11 a-pentamethyl-1 -( 1-methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)3-benzyl-(1S,3R)-2,2-dimethylcyclobutane-1,3-dicarboxylate (intermediate 1, 0.900 g, 1.311 mmol, 1.0 eq.) and triethylamine (0.5 ml, 3.935 mmol, 3.0 eq.) and stirred at 0°C for about 1 hour. The reaction mixture was allowed to stir at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with DCM (100 ml), washed with saturated sodium bicarbonate solution (100 ml), water and brine. The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane, with

- 51 045041 obtaining the title compound (1.0 g, 73.5% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 7.35 (m, 5H), 5.15, 5.09 (ABq, JAB=12.6 Hz, 2H), 4.44 (dd, J=10.5, 4.2 Hz, 1H), 3.67-3.65 (m, 4H), 3.64-3.61 (m, 6H), 3.60-3.58 (m, 1H), 3.57-3.52 (m, 3H), 3.45-3.40 (m, 1H), 3.38 (s, 3H), 3.36-3.27 (m, 1H), 3.23-3.10 ( m, 1H), 3.09-3.06 (m, 1H), 3.05-2.92 (m, 6H), 2.83-2.64 (m, 3H), 2.60-2.54 (m, 3H), 2.10-0.78 (m, 27H), 1.34 (s, 3H), 1.03 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.86 (s, 3H), 0.85 (s, 3H), 0.40-0.19 (m, 4H); ES-MS: m/z 1038.95 (M+H)+.

Stage 2. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)-13-( 1,1dioxidothiomorpholino)-12-methyl-2,5,8,11-tetraoxatetradecane-14-yl)amino)-5a,5b,8,8,11a-pentamethyl1-(1-methylcyclopropyl)icosahydro-1H-cyclopenta[ a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutan-1-carboxylic acid:

△ /Р n/A IJAU °о

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7αR,9S,11αR,11bR,13αR,13bR)-3a-(((12R,13R)-13(1,1-dioxidothiomorpholino) -12-methyl-2,5,8,11-tetraoxatetradecane-14-yl)amino)-5 a,5b,8,8,11 a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1 H-cyclopenta[a] chrysen-9-yl)(1R,3S)-2,2-dimethylcyclobutane-1,3-dicarboxylate (step 1, 1.0 g, 0.963 mmol, 1.0 eq.) in THF (10 ml) and methanol (10 mL) at 0° C., aqueous 2.5N KOH solution (2.89 mL, 7.229 mmol, 7.5 eq.) was added. The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was evaporated under reduced pressure, cooled to 0°C, acidified with 1N HCl to pH 4.0 and extracted with DCM (2x100 ml). The combined organic layer was washed with water (100 ml), dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of 0-7% methanol in DCM to give the title compound (0.050 g, 5.47% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.464.46 (dd, J=11.4, 4.8 Hz, 1H), 3.65-3.50 (m, 14H), 3.42-3.32 (m, 5H), 3.183.10 (m, 2H), 3.05-2.95 (m, 6H), 2.82-2.72 (m, 3H), 2.70-2.50 (m, 3H), 2.08-0.76 (m, 27H), 1.36 (s, 3H), 1.07 (s, 3H), 1.02 (s, 3H), 0.96 ( s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.86 (s, 3H), 0.86 (s, 3H), 0.40-0.20 (m, 4H); ESMS: m/z 947.99 (M+H) ⁺ .

Example 19. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2(1 ,1-dioxidethiomorpholino)-3-(2-(2-methoxyethoxy)acetoxy)butyl)amino)-5a,5b,8,8,11a-pentamethyl-1(prop-1-en-2-yl)icosahydro-1H -cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid:

To the stirred solution (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2R,3R)-2-( 1, 1-dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(prop-1 en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl )oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid (example 15, 0.650 g, 0.825 mmol, 1.0 eq.) in DCM (13 ml) was added 2-(2methoxyethoxy)acetic acid (intermediate 17 , 0.276 g, 2.06 mmol, 2.5 eq.), DMAP (0.050 g, 0.41 mmol, 0.5 eq.) and EDC.HCl (0.711 g, 3.71 mmol, 4.5 eq. ). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with water (50 ml) and extracted with DCM (3x50 ml). The combined organic layer was washed with water (50 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-6% methanol in dichloromethane. The resulting compound was further purified by treatment with ethyl acetate (5 ml) and hexane (50 ml), stirred at room temperature for about 30 minutes, filtered and dried in vacuo to obtain the title compound (0.480 g, 64% yield) as a white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 5.14-5.09 (m, 1H), 4.68 (s, 1H), 4.59 (s, 1H), 4.53-4.48 (m, 1H), 4.17 (s, 2H), 3.75-3.68 (m, 2H), 3.623. 57 (m, 2H), 3.38 (s, 3H), 3.33-2.85 (m, 9H), 2.81-2.40 (m, 5H), 2.05-0.77 (m, 26H), 1.68 (s, 3H), 1.38 ( s, 3H), 1.24 (d, J=6.6 Hz, 3H), 1.12 (s, 3H), 1.06 (s, 3H), 0.97 (s, 3H), 0.90 (s, 3H), 0.86 (s, 3H ), 0.85 (s, 3H); ES-MS: m/z 904.04 (M+H) ⁺ .

Example 20. Preparation of 4-(((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2-(1,1dioxidothiomorpholino)-3- Hydroxybutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)-2,2 -dimethyl-4-oxobutanoic acid:

- 52 045041

Stage 1. Synthesis of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((tert-butoxycarbonyl)amino)5a,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-1 H-cyclopenta[a]chrysen-9-ylacetate:

To a stirred solution tert-butyl((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9hydroxy-5a,5b,8,8,11 a-pentamethyl-1 -(prop-1 -en-2-yl)icosahydro-3aH-cyclopenta[a]chrysen-3a-yl)carbamate (prepared as described in WO 2017/149518A2, 12 g, 22.734 mmol, 1.0 eq.) bTHF (120 ml) at 0°C, triethylamine (9.5 ml, 68.203 mmol, 3.0 eq), DMAP (0.333 g, 2.728 mmol, 0.12 eq) and acetic anhydride (3 ml, 31.828 mmol, 1.4 eq) were added .). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with ethyl acetate (200 ml) and washed with water (2x200 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was treated with n-hexane (100 mL), stirred at room temperature for about 1 hour, filtered and dried in vacuo to give the title compound (12 g, 93% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.71 (s, 1H), 4.60 (s, 1H), 4.47 (dd, J=9.9, 4.5 Hz, 1H), 4.34 (s, 1H), 2.58-2.30 (m, 3H), 2.05 (s, 3H) , 2.02-0.77 (m, 22H), 1.68 (s, 3H), 1.44 (s, 9H), 1.01 (s, 3H), 0.95 (s, 3H), 0.85 (s, 3H), 0.84 (s, 3H ), 0.83 (s, 3H); ES-MS: m/z 570.39 (m+n) ⁺ .

Step 2. Synthesis of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino-5a,5b,8,8,11a-pentamethyl1 -(prop-1-ene-2 -yl)icosahydro-1 H-cyclopenta[a]chrysen-9-ylacetate:

To a stirred solution (1 R,3aS,5aR,5bR,7aR,9S, 11aR,11bR, 13aR,13bR)-3a-((tert-butoxycarbonyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-1 H-cyclopenta[a]chrysene-9-ylacetate (step 1, 12 g, 21.057 mmol, 1.0 eq.) in 1,4-dioxane (24 ml) 4N HCl in 1,4dioxane (24 ml) was added. The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was concentrated under reduced pressure and made basic with saturated sodium bicarbonate solution. The resulting solid was filtered and washed with water. The solid was dissolved in DCM, dried over sodium sulfate, filtered and concentrated under reduced pressure. The solid was further purified by trituration with n-hexane (100 ml), filtered, washed with hexane and dried in vacuo to give the title compound (7 g, 70.7% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.71 (s, 1H), 4.59 (s, 1H), 4.47 (dd, J=9.0, 5.4 Hz, 1H), 2.57-2.48 (m, 1H), 2.04 (s, 3H), 1.68 (s, 3H) , 1.66-0.77 (m, 24Н), 1.04 (s, 3Н), 0.95 (s, 3Н), 0.85 (s, 3Н), 0.84 (s, 3Н), 0.83 (s, 3Н); ES-MS: m/z 470.44 (M+H)+.

Step 3. Synthesis of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-3-((tert-butyldimethylsilyl)oxy)-2-( 1,1-dioxidothiomorpholino)butyl)amino)-5a,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2yl)icosahydro-1H-cyclopenta[a]chrysene-9-ylacetate:

To the stirred solution

4-((2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1-hydroxybutan-2yl)thiomorpholine-1,1-dioxide (intermediate 8, 8.5 g, 25.326 mmol, 1.7 eq.) 2,6-lutidine (3.8 mL, 32.775 mmol, 2.2 eq.) was added to DCM (70 mL) at 0°C. After 10 minutes of stirring at 0°C, add

- 53 045041 trifluoromethanesulfonic acid anhydride (5 ml, 29.795 mmol, 2.0 eq.) and stirred at 0°C for about 20 minutes. (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-amino5a,5b,8,8,11a-pentamethyl-1-(prop-1-ene-2) was added to the reaction mixture -yl)icosahydro-1H-cyclopenta[a]chrysene-9-ylacetate (step 2.7 g, 14.897 mmol, 1.0 eq.) and triethylamine (7.27 ml, 52.142 mmol, 3.5 eq.) and stirred at 0° for about 1 hour. The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with DCM (200 ml), washed with saturated sodium carbonate solution (100 ml) and water (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of 010% ethyl acetate in hexane. The resulting compound was further purified by treatment with n-hexane, filtered and dried in vacuo to give the title compound (7.0 g, 59% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.68 (s, 1H), 4.58 (s, 1H), 4.494.44 (m, 1H), 4.08-4.0 (m, 1H), 3.44-3.39 (m, 2H), 3.28-3.22 (m, 2H), 3.06-3.01 (m, 4H), 2.60-2.40 (m, 4H), 2.04 (s, 3H), 1.93-0.76 (m, 27H), 1.68 (s, 3H), 1.04 (s, 3H), 0.94 ( s, 3H), 0.87 (s, 9H), 0.85 (s, 3H), 0.84 (s, 3H), 0.83 (s, 3H), 0.07 (s, 6H); ES-MS: m/z 789.67 (M+H)+.

Step 4. Synthesis of 4-((2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1-(((1R,3aS,5aR,5bR,7aR,9S, 11 aR, 11 bR, 13 aR, 13bR)-9-hydroxy-5 a,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-3 aH-cyclopenta[a]chrysen-3a-yl)amino )butan-2-yl)thiomorpholine-1,1-dioxide:

To the stirred solution (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-3-((tert-butyldimethylsilyl)oxy)-2 -(1,1 -dioxidothiomorpholino)butyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1 (prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-9-ylacetate (step 3 , 7.0 g, 8.868 mmol, 1.0 eq.) in methanol (70 mL) and THF (70 mL), a 1N aqueous solution of KOH (53.2 mL, 53.212 mmol, 6.0 eq.) was added. The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was concentrated under reduced pressure, DCM (400 ml) was added and washed with water (200 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of 0-30% ethyl acetate in hexane to give the title compound (6.0 g, 91% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.69 (s, 1H), 4.58 (s, 1H), 4.04-3.99 (m, 1H), 3.44-3.39 (m, 2H), 3.28-3.16 (m, 3H), 3.07-2.96 (m, 4H), 2.61-2.40 (m, 4H), 1.97-0.66 (m, 27H), 1.68 (s, 3H), 1.04 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.87 (s, 9H), 0.82 (s, 3H), 0.75 (s, 3H), 0.07 (s, 6H); ES-MS: m/z 747.74 (M+H)+.

Step 5. Synthesis of 1-(tert-butyl)-4-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-((2R,3R)-3((tert -butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)butylamino)-5a,5b,8,8,11a-pentamethyl1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene -9-yl)-2,2-dimethylsuccinate:

To a stirred solution 4-((2R,3R)-3-((tert-butyldimethylsilyl)oxy)-1-(((1R,3aS,5aR,5bR, 7aR,9S,11aR,11bR,13aR,13bR)-9 -hydroxy-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-3aNcyclopenta[a]chrysen-3a-yl)amino)butan-2-yl)thiomorpholin- 1,1-dioxide (step 4, 1 g, 1.338 mmol, 1.0 eq.) in DCM (20 ml), add EDC.HCl (0.897 g, 4.683 mmol, 3.5 eq.), DMAP (0.081 g , 0.669 mmol, 0.5 eq.) and 4-(tert-butoxy)-3,3-dimethyl-4-oxobutanoic acid (prepared as described in WO 2013/020245, 0.676 g, 3.345 mmol, 2.5 eq. .). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with DCM (100 ml) and washed with water (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of 0-30% ethyl acetate in hexane to give the title compound (1 g, 80.2% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.68 (s, 1H), 4.58 (s, 1H), 4.48 (dd, J=10.5, 6.0 Hz, 1H), 4.02-3.99 (m, 1H), 3.47-3.39 (m, 2H), 3.27-3.22 ( m, 2H), 3.0 (m, 4H), 2.62-2.40 (m, 4H), 2.53 (s, 2H), 1.97-0.75 (m, 33H), 1.68 (s, 3H), 1.43 (s, 9H) , 1.04 (s, 3H), 0.94 (s, 3H), 0.87 (s, 9H), 0.84 (s, 6H),

- 54 045041

0.82 (s, 3H), 0.07 (s, 6H); ES-MS: m/z 932.06 (M+H) ⁺ .

Step 6. Synthesis of 1-(tert-butyl)-4-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2-( 1,1dioxidothiomorpholino)-3-hydroxybutyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-9 -yl)-2,2-dimethylsuccinate:

To a stirred solution of 1-(tert-butyl)-4-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2R,3R)-3-((tert -butyldimethylsilyl)oxy)-2-(1,1-dioxidothiomorpholino)butyl)amino)-5 a,5b,8,8,11 apentamethyl-1 -(prop-1-en-2-yl)icosahydro-1 H- cyclopenta[a]chrysen-9-yl)-2,2-dimethylsuccinate (step 1, g, 1.073 mmol, 1.0 eq.) bTHF (10 ml) added TBAF (4.29 ml, 4.294 mmol, 4.0 eq., 1.0 M in THF). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with ethyl acetate (100 ml) and washed with water (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography using a gradient of 0-4% methanol in dichloromethane to give the title compound (0.800 g, 91.2% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm 4.68 (s, 1H), 4.59 (s, 1H), 4.48 (dd, J=10.2, 6.0 Hz, 1H), 3.73-3.68 (m, 1H), 3.42-3.37 (m, 2H), 3.18-3.05 ( m, 6H), 2.65-2.45 (m, 6H), 1.96-0.76 (m, 24H), 1.68 (s, 3H), 1.43 (s, 9H), 1.26 (d, J=6.0 Hz, 3H), 1.23 (s, 3H), 1.22 (s, 3H), 1.04 (s, 3H), 0.94 (s, 3H), 0.84 (s, 6H), 0.82 (s, 3H); ESMS: m/z 818.12 (M+H) ⁺ .

Stage 7. Synthesis of 4-(((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2-(1,1dioxidothiomorpholino)-3 - hydroxybutyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1 -(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)-2 ,2-dimethyl-4-oxobutanoic acid:

To a stirred solution of 1-(tert-butyl)-4-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a(((2R,3R)-2-( 1, 1 -dioxidothiomorpholino)-3 -hydroxybutyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1 -(prop-1 en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-9- yl)-2,2-dimethylsuccinate (step 2, 0.800 g, 0.978 mmol, 1.0 eq.) in 1,4-doxane (2 ml) was added 4N HCl in 1,4-dioxane (2 ml). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was concentrated under reduced pressure. The crude compound was dissolved in DCM (100 ml) and washed with water (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane. The resulting compound was further purified by recrystallization from DCM:hexane (1:5, 10 ml). The mixture was cooled, filtered, washed with hexane and dried in vacuo to give the title compound (0.500 g, 67% yield) as an off-white solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 4.69 (s, 1H), 4.59 (s, 1H), 4.49-4.44 (m, 1H), 3.73 (m, 1H), 3.423.37 (m, 2H), 3.20-3.05 (m, 6H), 2.70- 2.48 (m, 6H), 1.95-0.75 (m, 33H), 1.67 (s, 3H), 1.05 (s, 3H), 0.96 (s, 3H), 0.84 (s, 3H), 0.83 (s, 3H) , 0.80 (s, 3H); ES-MS: m/z 762.0 (M+H)+.

Example 21. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((15R,16R)-16-( 1,1dioxidothiomorpholino)-15-methyl-13-oxo-2,5,8,11,14-pentaoxaheptadecan-17-yl)amino)-5 a,5b,8,8,11 a-pentamethyl-1-(prop -1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid:

To the stirred solution (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a (((2R,3R)-2-( 1, 1 -dioxidothiomorpholino)-3 -hydroxybutyl)amino)-5 a,5b,8,8,11 a-pentamethyl-1 -(prop-1 en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-9- yl)oxy)carbonyl-2,2-dimethylcyclobutane-1-carboxylic acid (example 15, 0.350 g, 0.444 mmol, 1.0 eq.) in DCM (5 ml) was added EDC.HCl (0.383 g,

- 55 045041

2.000 mmol, 4.5 eq.), DMAP (0.027 g, 0.222 mmol, 0.5 eq.) and 2,5,8,11-tetraoxatridecano-13-oic acid (0.247 g, 1.111 mmol, 2.5 eq. .). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was diluted with DCM (100 ml), washed with 1N HCl (50 ml) and water (100 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane. The resulting compound was further purified by treatment with 1:8 (MTBE:hexane; 18 mL), filtered, washed with n-hexane and dried in vacuo to give the title compound (60 mg, 13.6% yield) as an off-white solid substances. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 5.13-5.08 (m, 1H), 4.68 (s, 1H), 4.59 (s, 1H), 4.53-4.48 (m, 1H), 4.23-4.17 (m, 2H), 3.78-3.63 (m, 10H), 3.57-3.54 (m, 2H), 3.38 (s, 3H), 3.30-2.86 (m, 8H), 2.82-2.73 (m, 2H), 2.63-2.32 (m, 6H), 2.10-0.78 (m, 27H ), 1.68 (s, 3H), 1.38 (s, 3H), 1.11 (s, 3H), 1.06 (s, 3H), 0.97 (s, 3H), 0.89 (s, 3H), 0.86 (s, 3H) , 0.85 (s, 3H).

Example 22. Preparation of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)13-(1 ,1-dioxidothiomorpholino)-12-methyl-2,5,8,11-tetraoxatetradecane-14-yl)amino)-5a,5b,8,8,11a-pentamethyl-1 -(prop-1-en-2- yl)icosahydro-1 H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid:

Stage 1. Synthesis of (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)-13-(1,1dioxidothiomorpholino)-12-methyl-2, 5,8,11-tetraoxatetradecan-14-yl)amino)-5a,5b,8,8,11a-pentamethyl1 -(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysene-9 -yl acetate:

O

To a stirred solution of 4-((12R,13R)-14-hydroxy-12-methyl-2,5,8,11-tetraoxatetradecane-13yl)thiomorpholine-1,1-dioxide (intermediate 18, 0.849 g, 2.298 g) , mmol, 1.8 equiv.) 2,6-lutidine (0.32 ml, 2.79 mmol, 2.2 equiv.) was added to DCM (20 ml) at 0°C. The reaction mixture was stirred at the same temperature for about 10 minutes, triflate anhydride (0.43 ml, 2.54 mmol, 2.0 eq.) was added and stirred at 0°C for about 15 minutes. (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3aamino-5a,5b,8,8,11a-pentamethyl-1-(prop-1-ene-2) was added to the reaction mixture -yl)icosagylro-1H-cyclopenta[a]chrysene-9-ylacetate (example 20, step 2, 0.600 g, 1.277 mmol, 1.0 eq.) and triethylamine (0.56 ml, 4.06 mmol, 3. 5 eq.), stirred at 0°C for 1 hour and stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was detected. The reaction mixture was made basic with aqueous sodium bicarbonate and extracted with DCM (3x50 ml). The combined organic layer was washed with water (30 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. Another 500 mg portion with the same experimental procedure was combined and purified by silica gel column chromatography using a 40-50% ethyl acetate in hexane gradient to give the title compound (1.0 g, 95.33% yield) as solid matter. 1H NMR (300 MHz, CDC13): δ ppm 4.69 (s, 1H), 4.59 (s, 1H), 4.49-4.44 (m, 1H), 3.66-3.53 (m, 13H), 3.45-3.35 (m, 2H), 3.38 (s, 3H), 3.07- 2.97 (m, 6H), 2.04 (s, 3H), 1.90-0.77 (m, 31H), 1.68 (s, 3H), 1.05 (s, 3H), 0.95 (s, 3H), 0.85 (s, 3H) , 0.84 (s, 3H), 0.83 (s, 3H).

Stage 2. Synthesis of 4-((12R,13R)-14-(((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9-hydroxy5a,5b,8,8,11a -pentamethyl-1-(prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysen-3a-yl)amino)-12-methyl-2,5,8,11-tetraoxatetradecane-13-yl )thiomorpholine-1,1-dioxide:

To a stirred solution (1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)-13(1,1-dioxidothiomorpholino)-12-methyl-2, 5,8,11 -tetraoxatetradecan-14-yl)amino)-5 a,5b,8,8,11 a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a] chrysene-9-ylacetate (step 1, 1.0 g, 1.217 mmol, 1.0 eq.) in methanol (10 ml) and THF (10 ml) was added an aqueous solution of 2.5N KOH (3.57 ml, 9. 13 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated

- 56 045041 under reduced pressure, diluted with water (40 ml), neutralized with 1N HCl solution and extracted with DCM (3x50 ml). The combined organic layer was washed with water (30 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-3% methanol in dichloromethane to give the title compound (0.500 g, 52.74% yield) as an off-white solid. 1H NMR (300 MHz, CDCl ₃ ): δ ppm (s, 1H), 4.58 (s, 1H), 3.66-3.53 (m, 14H), 3.38 (s, 3H), 3.303.0 (m, 8H), 2.58-2.56 (m, 1H), 2.41 (m , 1H), 2.0-0.75 (m, 44H), 1.67 (s, 3H).

Step 3. Synthesis of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)-13-(1,1dioxidothiomorpholino) -12-methyl-2,5,8,11-tetraoxatetradecan-14-yl)amino)-5a,5b,8,8,11a-pentamethyl1 -(prop-1-en-2-yl)icosahydro-1 H- cyclopenta[a]chrysen-9-yl)-(1R,3 S)-2,2-dimethylcyclobutane-1,3-dicarboxylate:

To a stirred solution 4-((12R,13R)-14-(((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-9hydroxy-5a,5b,8,8,11a- pentamethyl-1-(prop-1-en-2-yl)icosahydro-3aH-cyclopenta[a]chrysen-3a-yl)amino)-12-methyl-2,5,8,11-tetraoxatetradecane-13-yl) thiomorpholine-1,1-dioxide (step 2, 0.500 g, 0.641 mmol, 1.0 eq.) in DCM (20 ml) was added (1S,3R)-3-((benzyloxy)carbonyl)-2,2-dimethylcyclobutane -1carboxylic acid (prepared according to the procedure described in WO 2011/007230 A2, 0.505 g, 1.925 mmol, 3.0 eq.), then EDC.HCl (0.733 g, 3.846 mmol, 6.0 eq.) and DMAP (0.070 g, 0.577 mmol, 0.9 eq.). The reaction mixture was stirred at room temperature overnight. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was diluted with water (30 ml) and extracted with DCM (3x30 ml). The combined organic layer was washed with water (30 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using a gradient of 0-3% methanol in dichloromethane to give the title compound (0.060 g, 22.8% yield) as a solid. ¹ H NMR (300 MHz, CDCl ₃ ): δ ppm 7.34 (m, 5H), 5.14, 5.09 (ABq, JAB=12.3 Hz, 2H), 4.69 (s, 1H), 4.58 (s, 1H), 4.46-4.40 (m, 1H), 4.06-4.0 (m, 1H), 3.78-3.52 (m, 13H), 3.37 (s, 5H), 3.07-2.97 (m, 6H), 2.84-2.52 (m, 5H), 2.42-2.38 (m, 1H), 2.08-0.77 ( m, 31H), 1.68 (s, 3H), 1.33 (s, 3H), 1.04 (s, 3H), 0.96 (s, 3H), 0.95 (s, 3H), 0.84 (s, 3H), 0.83 (s , 3H).

Stage 4. Synthesis of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)-13-( 1,1dioxidothiomorpholino)-12-methyl-2,5,8,11-tetraoxatetradecane-14-yl)amino)-5a,5b,8,8,11a-pentamethyl1-(prop-1-en-2-yl)icosahydro -1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid:

To a stirred solution of 1-benzyl-3-((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)-13(1,1-dioxidothiomorpholino) -12-methyl-2,5,8,11-tetraoxatetradecane-14-yl)amino)-5a,5b,8,8,11 a-pentamethyl-1 (prop-1-en-2-yl)icosahydro-1 N-cyclopenta[a]chrysen-9-yl)-(1R,3S)-2,2-dimethylcyclobutane-1,3-dicarboxylate (step 3, 0.200 g, 0.195 mmol, 1.0 eq.) bTHF (2 ml) and methanol (2 ml) was added an aqueous solution of 1.5N KOH (0.97 ml, 1.46 mmol, 7.5 eq.). The reaction mixture was stirred at room temperature for 2 days. TLC showed that the starting material was consumed and the desired product was observed. The reaction mixture was evaporated under reduced pressure, diluted with water (10 ml), acidified with 1N HCl to pH 4 to 5 and extracted with DCM (3x50 ml). The combined organic layer was washed with water (30 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude compound was purified by silica gel column chromatography using a gradient of 0-5% methanol in dichloromethane. The resulting compound was treated with ethyl acetate (2 ml) and hexane (8 ml), stirred at room temperature for 20 minutes, the solvent was decanted and this procedure was repeated twice, finally filtered, washed with hexane (2 ml) and dried in vacuum to obtain the above title compound (0.060 g, 32.96% yield) as a solid. 1H NMR (300 MHz, CDCI3): δ ppm 4.68 (s, 1H), 4.59 (s, 1H), 4.54 (m, 1H), 3.80-3.52 (m, 12H), 3.37 (s, 3H), 3.27-2.32 (m, 16H), 2.03-0.77 ( m, 25Н), 1.68 (s, 3Н), 1.38 (s, 3Н), 1.18 (s, 3Н), 1.06 (s, 3Н), 0.98 (s, 3Н), 0.92 (s, 3Н), 0.87 (s , 6H), 0.83 (s, 3H); ES-MS: m/z 933.94 (M+H)+.

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Biological activity

The compounds described herein are tested for their antiviral activity in accordance with procedures known to one of ordinary skill in the art. For example, the following protocols can be used to test connections. These protocols are illustrative and do not limit the scope of the present invention.

Example 23. Evaluation of the antiviral activity of compounds against HIV-1 strain 92HT599 in MT2 cells.

MT2 cells were infected with HIV-1 strain 92HT599 (10 CPD50/30,000 cells). Infected cells were seeded in a 96-well plate at a concentration of ~30,000 cells per well. The test compound was added to the microplate in a defined format with a final concentration of DMSO (vehicle) of no more than 1%. Incubation was carried out in a CO2 incubator for ~96 hours for viral infection. At the end of the incubation period, an aliquot was removed from each well for p24 assessment. Quantitative determination of p24 is an indicator of the antiviral activity of the compound. The percentage of inhibition was calculated relative to control values (medium control values). p24 was assessed using the Advance biosciences kit according to the procedure detailed by the supplier.

In a serological binding assay, A refers to an IC50 value less than 10 nM, B refers to an IC50 value in the range of 10.01-20 nM, and C refers to an IC50 value greater than 20 nM. IC50 values (nM) are given in table. 1 below.

Table 1

Sample N IC50 (nM) 1 WITH 2 A 3 WITH 4 IN 5 A 6 A 7 A 8 A 9 A 10 A

Sample N IC50 (nM) eleven A 12 WITH 13 A 14 A 15 A 16 IN 17 A 18 A 19 A 20 A

Example 24. Evaluation of the antiviral activity of compounds against p-NL4-3/WT and V7A strains in MT4 cells.

MT4 cells were transfected with HIV-1 plasmid (pNL4-3-WT&V7A) (cells were incubated with the required amount of HIV-1 CPD50() for 1.5 h at 37°C). After infection, infected cells were seeded in a 96-well plate at a concentration of 3x104 cells per well. The test compound was added to the test plate in a specified format with a final DMSO concentration of no more than 1%. Incubation was carried out in a CO2 incubator for 4 days for viral infection. At the end of the incubation period, an aliquot was removed from each well for p24 assessment. p24 assessment was performed using the Advance biosciences kit according to the procedure detailed by the supplier.

For the pNL4-3 WT assay, selected compounds exhibit an IC50, where Ά refers to an IC50 value less than 10 nM, B refers to an IC50 value in the range of 10.01-50 nM, and C refers to an IC50 value greater than 50 nM.

For the pNL4-3 V7Ά assay, selected compounds exhibit an IC50, where Ά refers to an IC50 value less than 1-50 nM, B refers to an IC50 value in the range of 50.01-100 nM, and C refers to an IC50 value greater than 100 nM. IC50 values (nM) are given in table. 2.

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Claims (11)

Links. Antiviral methods and protocols (Eds: D Kinchington and R. F. Schinazi) Humana Press Inc., 2000. HIV protocols (Eds: N. L. Michael and J. H. Kim) Humana Press Inc, 1999. DAIDS Virology manual from HIV laboratories, Publication NIH-97-3838, 1997. HIV-1 p24 antigen capture assay, enzyme immunoassay for detection of Human immunodeficiency Virus Type 1 (HIV-1) p24 in tissue culture media - Advanced bio science laboratories, Inc kit procedure. Although the present invention has been described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Therefore, it should be understood that numerous modifications may be made to the illustrative embodiments, and that other variations may be developed without departing from the spirit and scope of the present invention as described above. All publications and patent applications cited in this patent application are hereby incorporated by reference to the same extent as if each individual publication or patent application had been specifically and individually cited for inclusion herein by reference. CLAIM 1. A compound of formula (I) wherein R ₁ is selected from or R ₂ is selected from C1-C6 alkyl, optionally substituted C ₂ -C ₆ alkenyl or optionally substituted C ₃ -C ₈ cycloalkyl; wherein the optional substituent is C1-C6 alkyl; R ₃ represents hydrogen; R4 is selected from optionally substituted C ₁ -C ₆ alkyl or -C(O)ORa; wherein C ₁ -C ₆ alkyl is ethyl and the optional substituent is selected from halogen, hydroxy, alkoxy or -OC(O)CH ₂ alkoxy; where alkoxy refers to a straight or branched hydrocarbon chain with an oxygen radical, consisting of carbon and hydrogen atoms, containing saturation or unsaturation, having from one to eight carbon atoms, which is attached through an oxygen atom to the rest of the molecule by a single bond; and one of the hydrogen atoms in the alkoxy group may be further replaced by one or more alkoxy groups to further increase the length of the alkoxy chain with a group selected from methyloxy, ethyloxy, isopropoxy, n-propyloxy, t-butyloxy, 1-methylethyloxy 1-dimethylethyloxy (isopropyloxy), n-butyloxy, n-pentyloxy, 1, (t-butyloxy), - 59 045041 and a ring represents Ra represents hydrogen or C1-C6 alkyl; or pharmaceutically acceptable salts, pharmaceutically acceptable stereoisomers thereof, or combinations thereof. 2. A compound according to claim 1, which is a compound of formula (IA) R4 is selected from optionally substituted C ₁ -C ₆ alkyl or -C(O)OR _a ; wherein C ₁ -C ₆ alkyl is ethyl and the optional substituent is selected from halogen, hydroxy, alkoxy or -OC(O)CH ₂ alkoxy; where alkoxy refers to a straight or branched hydrocarbon chain with an oxygen radical, consisting of carbon and hydrogen atoms, containing saturation or unsaturation, having from one to eight carbon atoms, which is attached through an oxygen atom to the rest of the molecule by a single bond; and one of the hydrogen atoms in the alkoxy group may be further replaced by one or more alkoxy groups to further increase the length of the alkoxy chain with a group selected from methyloxy, ethyloxy, isopropoxy, n-propyloxy, t-butyloxy, 1-methylethyloxy (isopropyloxy), n-butyloxy, n -pentyloxy, 1,1-dimethylethyloxy (t-butyloxy), ΠΛΑΤ PLAY GT O Q ring ' represents ⁰ or ^o > and Ra represents hydrogen or C1-C6 alkyl; or pharmaceutically acceptable salts, pharmaceutically acceptable stereoisomers thereof, or combinations thereof. 3. A compound according to claim 1, which is a compound of formula (IB) where R ₃ represents hydrogen; R4 is selected from optionally substituted C ₁ -C ₆ alkyl or -C(O)ORa; wherein C1-C6 alkyl is ethyl and the optional substituent is selected from halogen, hydroxy, alkoxy or -OC(O)CH and alkoxy; where alkoxy refers to a straight or branched hydrocarbon chain with an oxygen radical, consisting of carbon and hydrogen atoms, containing saturation or unsaturation, having from one to eight carbon atoms, which is attached through an oxygen atom to the rest of the molecule by a single bond; and one of the hydrogen atoms in the alkoxy group may be further replaced by one or more alkoxy groups to further increase the length of the alkoxy chain with a group selected from methyloxy, ethyloxy, isopropoxy, n-propyloxy, t-butyloxy, 1-methylethyloxy (isopropyloxy), n-butyloxy, n -pentyloxy, 1,1-dimethylethyloxy (t-butyloxy), '/o—°—0-, _or and Ra represents hydrogen or C1-C6 alkyl; or pharmaceutically acceptable salts, pharmaceutically acceptable stereoisomers thereof, or combinations thereof. - 60 045041 4. A compound selected from the group consisting of (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R, 3R)-2-(1,1dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy )carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid, (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(( (2R,3S)-2-(1,1dioxidothiomorpholino)-3-fluorobutyl)amino)-5a,5b,8,8,11 a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysene-9 -yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid, (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)- 3a-(((2S,3S)-2-(1,1dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a] chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid, (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR, 13bR)-3a-(((2R,3R)-3-hydroxy-2-(4(isopropylsulfonyl)piperazin-1 -yl)butyl)amino)-5a,5b,8,8,11a-pentamethyl-1-( 1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1carboxylic acid, (1R,3S)-3-((((1R,3aS,5aR,5bR, 7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2-(1,1dioxidothiomorpholino)-3-hydroxybutyl)amino)-5a,5b,8,8,11a-pentamethyl- 1-(prop-1-en-2yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid, (1R,3S)-3-( (((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((R)-2-carboxy-2-(1,1dioxidothiomorpholino)ethyl)amino)-5a, 5b,8,8,11a-pentamethyl-1-(1-methylcyclopropyl)icosahydro-1Hcyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid, (1R,3S) -3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((2R,3R)-2-(1,1dioxidothiomorpholino)-3-(2 -(2-methoxyethoxy)acetoxy)butyl)amino)-5a,5b,8,8,11a-pentamethyl-1-(1methylcyclopropyl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2 ,2-dimethylcyclobutane-1carboxylic acid, (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R) -13-(1,1dioxidothiomorpholino)-12-methyl-2,5,8,11-tetraoxatetradecane-14-yl)amino)-5a,5b,8,8,11a-pentamethyl1-(1-methylcyclopropyl)icosahydro-1H -cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2dimethylcyclobutane-1-carboxylic acid, (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR ,11bR,13aR,13bR)-3a-(((2R,3R)-2-(1,1dioxidothiomorpholino)-3-(2-(2-methoxyethoxy)acetoxy)butyl)amino)-5a,5b,8,8 ,11a-pentamethyl-1(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutan-1carboxylic acid, 4-(((1R,3αS,5αR,5bR,7αR,9S,11αR,11bR,13αR,13bR)-3α-(((2R,3R)-2-(1,1-duoxudotuomorpholino)-3hydroxybutyl)amino) -5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2-yl)icosahydro-1H-cyclopenta[a]chrysen9-yl)oxy)-2,2-dimethyl-4-oxobutanoic acids, (1R,3S)-3-((((1R,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((15R,16R)-16-(1, 1-dioxidσgiomorpholino)-15-methyl-13-oxo-2,5,8,11,14-pentaoxaheptadecan-17-yl)amino)-5a,5b,8,8,11a-pentamethyl-1(prop-1- en-2-yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy)carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid, and (1R,3S)-3-((((1R ,3aS,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a-(((12R,13R)-13-(1,1-dioxideσhyomorpholino)-12-methyl-2,5,8, 11-tetraoxatetradecan-14-yl)amino)-5a,5b,8,8,11a-pentamethyl-1-(prop-1-en-2yl)icosahydro-1H-cyclopenta[a]chrysen-9-yl)oxy) carbonyl)-2,2-dimethylcyclobutane-1-carboxylic acid, or pharmaceutically acceptable salts, pharmaceutically acceptable stereoisomers, or combinations thereof. 5. A pharmaceutical composition containing a compound according to any one of claims 1 to 4 and at least one pharmaceutically acceptable excipient. 6. The pharmaceutical composition according to claim 5, wherein the pharmaceutically acceptable excipient is a carrier or diluent. 7. A method of preventing, ameliorating, or treating a virus-mediated disease, disorder, or syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 4. 8. The method according to claim 7, characterized in that the virus-mediated disease, disorder or syndrome is HIV infection, HBV infection, HCV infection, retroviral infection genetically associated with AIDS, respiratory disorders (including respiratory distress syndrome in adults (ADSV)), an inflammatory disease, or a combination of these. 9. A method of treating HIV in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 4. - 61 045041 10. A method of preventing, ameliorating or treating a virus-mediated disease, disorder or syndrome in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to claims 5 and 6 containing a therapeutically effective amount of a compound. 11. The method according to claim 10, characterized in that the disease, disorder or syndrome caused by the virus is HIV infection, HBV infection, HCV infection, retroviral infection genetically associated with AIDS, respiratory disorders (including respiratory distress syndrome in adults (ARDS), an inflammatory disease, or a combination thereof. Kommersant Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2