Irreversible 17 [beta]-HSD1 inhibitors

Abstract

Compounds of Formula (I), pharmaceutical compositions or kits thereof are disclosed. Wherein A1 is C (O) or CHRz1, Rz1 is OH, amino, imino, halogen, (C1-C5) alkoxy, OC (O) (C1-C5) alkyl, and OSO2-amino; a2 is CH2 or O; r1 is hydrogen, a (C1-C5) alkyl group optionally substituted by one or more Z substituents, a (C1-C5) alkoxy group optionally substituted by one or more Z substituents, a (C2-C5) alkenyl group optionally substituted by one or more Z substituents, a (C2-C5) alkynyl group optionally substituted by one or more Z substituents; (C3-C8) cycloalkyl, aryl or heteroaryl groups; z is halogen, OH, (C1 to C5) alkyl, (C1 to C5) haloalkyl, (C1 to C5) alkoxy, (C1 to C5) haloalkoxy, or amino; r2 is a heterocyclic aromatic ring having 5, 6 or 7 members and having at least one N, S and / or O; r3 is a (C1-C5) alkyl group, which is substituted by one or more Br, Br76; i, I123, I124 or I131 substitutions; m is an integer selected from 0 to 2; n is an integer selected from 0 to 2; and p is an integer selected from 0 to 5. The compounds of formula I are inhibitors of 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD1) for use in the treatment or prevention of steroid hormone dependent diseases such as breast cancer, lung cancer, prostate cancer, ovarian cancer, uterine cancer, endometrial cancer and endometrial hyperplasia. (I).

Description

[0001] Cross-references to related applications

[0002] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 519,779, entitled “Irreversible 17β-HSD1 Inhibitor,” filed August 15, 2023, with the United States Patent and Trademark Office, the contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the inhibition of 17β-HSD1. In particular, the invention provides compounds of formula (I) and compositions comprising thereof, which provide effective, non-estrogenic, and irreversible inhibition of 17β-HSD1. The invention further provides therapeutic methods and uses based on these novel compounds and compositions. Background Technology

[0004] In premenopausal women, estradiol (E2) is primarily produced in the ovaries. E2 reaches target tissues via the endocrine pathway, where it exerts its effects through interaction with estrogen receptor (ER)α. Postmenopausal, plasma E2 levels decrease to about one-tenth of premenopausal levels. E2 is then primarily produced in peripheral tissues (e.g., breast tissue, endometrium, adipose tissue, skin) from inactive precursors. These reactions occur in peripheral tissues (where active estrogen exerts its effects) with the participation of various steroid-producing enzymes (hydroxysteroid dehydrogenase, aromatase). Due to this endocrine mechanism of E2 formation, E2 concentrations in peripheral tissues (especially in estrogen-dependent diseases) are higher than in healthy tissues. In particular, the growth of many cancer cell lines is stimulated by locally increased E2 concentrations. Furthermore, the occurrence and progression of diseases such as endometriosis, leiomyomas, adenomyosis, menorrhagia, uterine bleeding, and dysmenorrhea depend on significantly elevated E2 levels in the corresponding lesion tissues. Endometriosis is an estrogen-dependent disease affecting approximately 5-10% of women of reproductive age, and 35-50% of women experiencing abdominal pain and / or infertility show signs of endometriosis. The disease is defined as histologically confirmed ectopic endometrial glands and stromal tissue. This relapsing, chronic condition causes pain of varying intensities and natures and, if it occurs in a particular form, can lead to infertility. Three macroscopic states are distinguished: peritoneal endometriosis, deep retroperitoneal endometriosis (including adenomyosis), and ovarian cystic endometriosis.

[0005] 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) converts estrone (E1) to E2, the most potent natural ligand for ERα. This enzyme also catalyzes the reduction of dehydroepiandrosterone (DHEA) to 5-androstenedione-3β,17β-diol (A5-diol), a weaker estrogen, but particularly important in postmenopause. Therefore, 17β-HSD1 inhibitors are attractive therapeutic agents for controlling estrogen-dependent diseases such as breast cancer and endometriosis.

[0006] Over the past three decades, tremendous efforts have been made to design effective inhibitors of this key steroid-producing enzyme, but only recently have lead candidates with very good inhibitory activity been reported. The presence of residual estrogenic activity associated with steroid inhibitors (typically built around an estradiol backbone) represents a major obstacle to their development.

[0007] 16β-(m-carbamoylbenzyl)estradiol (CC-156) has been reported as a potent 17β-HSD1 inhibitor. Despite its good inhibitory efficacy, it has been found to significantly reduce its therapeutic potential by stimulating MCF-7 and T-47D estrogen-sensitive breast cancer cell lines in vitro.

[0008] Many efforts have been made to overcome unwanted residual estrogen activity without negatively impacting the inhibitory effect.

[0009] Among the new series of compounds developed in this field, the E2 derivative 3-{[(16β,17β)-3-(2-bromoethyl)-17-hydroxyestradiol-1,3,5(10)-trien-16-yl]methyl}benzamide (hereinafter also referred to as “PBRM”) has been reported as the first non-estrogenic irreversible steroidal 17β-HSD1 inhibitor (Trottier A. et al., “Insight into the modeof action and selectivity of PBRM, a covalent steroidal inhibitor of 17β-hydroxysteroid dehydrogenase type 1”, Biochemical Pharmacology, 2017, 144, 149–161):

[0010]

[0011] Identifying the covalent binding of PBRM to 17β-HSD1 confirmed its irreversibility (Maltais R. et al., “Discovery of a non-estrogenic irreversible inhibitor of 17β-hydroxysteroiddehydrogenase type 1 from 3-substitted-16β-(m-carbamoylbenzyl)-estradiolderivatives”, 2014, J. Med. Chem., 55(7), 204-222; Li T. et al., “Combined biophysical chemistry reveals a new covalent inhibitor with low-reactivityalkyl halide”, 2018, J. Phys. Chem. Lett. 9, 5275-5280). From a therapeutic perspective, the irreversible nature of this interaction is particularly advantageous for enhancing efficacy and prolonging the effect through this mechanism.

[0012] However, despite efforts, there is still a need for non-estrogenous irreversible 17β-HSD1 inhibitors with improved properties. Summary of the Invention

[0013] The inventors surprisingly discovered that when the PBRM compound was modified by replacing the original 16β-methylbenzamide moiety with the amide moiety of another formula (i):

[0014]

[0015] The resulting compound not only retained the irreversibility and non-estrogenicity of the original PBRM, but also achieved significant improvements in the inhibitory effect on 17β-HSD1 and metabolic stability.

[0016] on the one hand, Figure 1The results showed that when one of the compounds of the present invention (PBRM-II) was administered to the ER+ breast cancer cell line T-47D, cell growth was significantly reduced compared to the control. This indicates that the compounds of the present invention are effective in treating estrogen-dependent diseases associated with high levels of estrogen, and importantly, they do not “add” estrogenity to the environment. This further indicates the safety of the compounds of the present invention when they contain the amide moiety of formula (i). Conversely, for example, CC-156 (a reversible 17β-HSD1 inhibitor), when administered to cells, induces estrogenity and leads to increased cell growth of tumor cells, even at low concentrations (see [link to relevant documentation]). Figure 1 ).

[0017] from Figure 1 It can also be seen that, compared with PBRM, the compounds of the present invention not only inhibit the target more effectively, but this higher potency translates into more effective treatment of the disease at the lowest doses (0.1 and 1 μM).

[0018] On the other hand, Table 1 below shows that when PBRM is modified by replacing the methylbenzamide at the 15β position with the amide moiety of formula (i) and R2 is a thiazolyl ring, IC 50 The value decreased to 1 / 26. Furthermore, Figure 2 This indicates that the stability of the compound has increased by more than 30%.

[0019] In summary, the compounds provided by this invention represent a significant advancement in the field of irreversible 17β-HSD1 inhibitors in terms of inhibitory efficacy, efficiency, and safety.

[0020] Therefore, in a first aspect, the present invention provides compounds of formula (I), their stereoisomers, or salts:

[0021]

[0022] in:

[0023] A1 is selected from C(O) and CHR z1 ;

[0024] A2 is selected from CH2 and O;

[0025] R1 is selected from: hydrogen, (C1-C5) alkyl optionally substituted with one or more Z-substituents, (C1-C5) alkoxy optionally substituted with one or more Z-substituents, (C2-C5) alkenyl optionally substituted with one or more Z-substituents, (C2-C5) alkynyl optionally substituted with one or more Z-substituents; (C3-C8) cycloalkyl; aryl; and heteroaryl;

[0026] R2 is a heterocyclic aromatic ring having 5, 6, or 7 members selected from: CR z2 N, S, and O, provided that at least one member is N, S, or O;

[0027] R3 is a (C1-C5) alkyl group, which is reacted with one or more Br, Br 76 ;I、I 123 I 124 or I 131 replace;

[0028] R z1 Selected from OH, NR x1 R x2 =N-OR x3 Halogens, (C1-C5)alkoxy groups, OC(O) (C1-C5)alkyl groups, and OSO2NR x4 NR x5 ;

[0029] R z2 Selected from hydrogen, OH, or (C1-C) groups optionally substituted with one or more Z substituents. 10 ) alkyl, and (C1-C) substituted with one or more Z substituents optionally 10 )alkoxy;

[0030] Z is selected from halogen, OH, (C1-C5)alkyl, (C1-C5)haloalkyl, (C1-C5)alkoxy, (C1-C5)haloalkoxy, and NR. x1 R x2 ;

[0031] R x1 R x2 R x4 and R x5 The same or different, and selected from: hydrogen, (C1-C5)alkyl, (C1-C5)haloalkyl, (C1-C5)alkoxy and (C1-C5)haloalkoxy;

[0032] R x3 Selected from H and (C1-C5) alkyl groups;

[0033] m represents an integer value selected from 0 to 2;

[0034] n represents an integer value selected from 0 to 2;

[0035] p represents an integer value selected from 0 to 5;

[0036] in:

[0037] Aryl groups contain 5 or 6 CRs c The aromatic ring system of the members, where R cSelected from H, halogen, cyano, nitro, (C1-C5)alkyl, (C1-C5)haloalkyl, -O-(C1-C5)alkyl and -O-(C1-C5)haloalkyl; and

[0038] Heteroaryl is an aromatic ring system comprising 5 or 6 members selected from: CR d , O, N, NH and S; where R d Selected from H, halogen, cyano, nitro, (C1-C5)alkyl, (C1-C5)haloalkyl, -O-(C1-C5)alkyl, and -O-(C1-C5)haloalkyl; and

[0039] Compound of formula (I) has the ability to covalently (i.e. irreversibly) bind to 17β-hydroxysteroid dehydrogenase type 1.

[0040] The present invention also provides a method for synthesizing the compounds of the present invention.

[0041] Therefore, in a second aspect, the present invention provides a method for preparing a compound of formula (I) as defined in the first aspect of the present invention, the method comprising the steps provided in scheme 1 or 2 below:

[0042] Option 1: Obtain C15 analogues

[0043]

[0044] Alk = (C1-C5) alkyl; Alk-X = (C1-C5) alkyl substituted with one X, where X is selected from Br, Br 76 ;I、I 123 I 124 or I 131 .

[0045] Option 2: Obtain C16 analogues

[0046]

[0047] Alk = (C1-C5) alkyl; Alk-OH = (C1-C5) alkyl substituted with one OH substituent; Alk-X = (C1-C5) alkyl substituted with one X, where X is selected from Br, Br 76 ;I、I 123 I 124 or I 131 .

[0048] The amide coupling step between the carboxylic acid compound of formula (II) and the amine of formula (III) can be carried out using amide coupling agents known in the art. Illustrative and non-limiting examples of amide coupling agents are independently selected from TBTU, TCTU, HATU, T3P, or COMU. The coupling reaction is carried out in the presence of DMF or any other suitable aprotic solvent and a base (such as DIPEA).

[0049] The halogenation step (bromination or iodination) of the OH derivative of formula (IV) can be carried out using known reagents and conditions. Illustrative and non-limiting conditions, namely PPh3 and CBr4, are provided below.

[0050] The reduction step of C17 ketones can also be carried out using any suitable reducing agent and conditions. The reducing agent specifically reduces the ketone and converts it to an alcohol. In some embodiments, the specific water-soluble agent is a non-toxic and / or green agent. Non-limiting examples of specific water-soluble reducing agents include sodium borohydride (NaBH4); sodium cyanoborohydride (NaCNBH3); catalysts such as nickel (Ni), platinum (Pt), or palladium (Pd, in the presence of hydrogen (H2)); ammoniaborane (H3NBH3), borane dimethylamine complex [(CH3)2NH·BH3]; borane tert-butylamine complex [(CH3)3CNH2·BH3]; or borane-pyrimidine complexes.

[0051] In a third aspect, the present invention provides a pharmaceutical composition comprising a compound as defined in the first aspect of the invention and one or more pharmaceutically acceptable excipients or carriers.

[0052] In a fourth aspect, the present invention provides a kit comprising:

[0053] (i) the compound of formula (I) as defined in the first aspect of the present invention, and

[0054] (ii) Detectable markers.

[0055] In a fifth aspect, the present invention provides compounds as defined in the first aspect of the invention for use in therapeutics or diagnostics.

[0056] The compounds of this invention can be used therapeutically, particularly for the treatment or prevention of steroid hormone-dependent diseases or conditions in animals, especially mammals, and humans that require inhibition of the 17β-HSD1 enzyme. Specifically, compounds of formula (I) represent inhibitors of the 17β-HSD1 enzyme that have pharmacological properties for the treatment and / or prevention of malignant steroid-dependent diseases or conditions (such as breast cancer, lung cancer, prostate cancer, ovarian cancer, uterine cancer, endometrial cancer, and endometrial hyperplasia) and also for the treatment and / or prevention of benign steroid-dependent diseases or conditions (such as endometriosis, uterine fibroids, uterine leiomyomas, adenomyosis, dysmenorrhea, menorrhagia, uterine bleeding, prostatodynia, benign prostatic hyperplasia, urinary dysfunction, polycystic ovary syndrome, or lower urinary tract syndrome). Other estrogen-dependent diseases that can be treated and / or prevented with effective amounts of the compounds of this invention include multiple sclerosis, obesity, rheumatoid arthritis, colon cancer, tissue trauma, skin wrinkles, and cataracts.

[0057] Therefore, in a sixth aspect, the present invention provides a method for inhibiting the 17β-HSD1 enzyme in a subject, the method comprising the step of administering to the subject in need a therapeutically effective amount of a compound of formula (I) as defined in the first aspect of the present invention or a pharmaceutical composition as defined in the third aspect of the present invention. This aspect can be described as the use of a compound of formula (I) as defined in the first aspect of the present invention or a pharmaceutical composition as defined in the third aspect of the present invention for treating and / or preventing disease by inhibiting the 17β-HSD1 enzyme. This aspect can also be alternatively described as the use of a compound of formula (I) as defined in the first aspect of the present invention or a pharmaceutical composition as defined in the third aspect of the present invention in the preparation of a medicament for treating and / or preventing disease by inhibiting the 17β-HSD1 enzyme.

[0058] In a seventh aspect, the present invention provides a method for treating and / or preventing estrogen-dependent diseases, the method comprising the step of administering to a subject in need a therapeutically effective amount of a compound of formula (I) as defined in the first aspect of the present invention or a pharmaceutical composition as defined in the third aspect of the present invention. This aspect may alternatively be described as the use of a compound of formula (I) as defined in the first aspect of the present invention or a pharmaceutical composition as defined in the third aspect of the present invention for treating and / or preventing estrogen-dependent diseases. This aspect may alternatively be described as the use of a compound of formula (I) as defined in the first aspect of the present invention or a pharmaceutical composition as defined in the third aspect of the present invention in the preparation of a medicament for treating and / or preventing estrogen-dependent diseases. Attached Figure Description

[0059] Figure 1 Effects of 17β-HSD1 inhibitors on estrogen-dependent T-47D cell proliferation.

[0060] Figure 2Metabolic stability of 17β-HSD1 inhibitors CC-156 (comparative target), PBRM (comparative target), and PBRM-II (the present invention) in human liver microsomes.

[0061] Figure 3 The irreversibility of the compound PBRM-II of this invention.

[0062] Figure 4 : Concentration of PBRM-II in mice when orally administered (30 mg / kg, in DMSO:sunflower oil / 8:92).

[0063] Figure 5A Remaining PBRM-II percentage (%PR) vs. time (min).

[0064] Figure 5B : Remaining percentage of PBRM-II (%PR) Ln vs time (min) and half-life of 4 μM PBRM-II in human liver microsomes (HLM).

[0065] Figure 5C Remaining PBRM percentage (%PR) vs. time (min).

[0066] Figure 5D Ln vs time (min) of remaining PBRM percentage (%PR) and half-life of 4 μM PBRM-II in human liver microsomes (HLM). Detailed Implementation

[0067] Terms not specifically defined herein shall be given the meanings that a person skilled in the art may assign based on the disclosure and context. However, as used in the specification, unless otherwise stated, the following terms have specified meanings and follow the conventions.

[0068] Throughout this specification and its accompanying terms, the word "comprising" and its variations such as "containing" and "including" shall be understood as inclusive. That is, these words are intended to convey that, where the context permits, other elements or integers not specifically listed may be included. The word "comprising" also includes the term "consisting of".

[0069] For the purposes of this invention, any range given includes both the lower and upper endpoints of that range.

[0070] In a first aspect, the present invention provides an irreversible 17β-HSD1 inhibitor.

[0071] In the context of this invention, a compound is a 17β-HSD1 inhibitor when it reduces the activity of an enzyme. In one embodiment, the compound inhibits at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the enzyme activity. Well-known methods in the art exist for determining inhibitory activity. Illustrative examples are provided below, based on (a) determining labeled E1 and E2 after contacting the estrogen-sensitive breast cancer cell line T-47D with [14C]-E1 + cold E1 and the test compound, and (b) calculating conversion % and inhibition % as follows: %conversion = 100 [14C]-E2 / ([14C]-E1 + [14C]-E2), %inhibition = 100 (%conversion without inhibitor − %conversion with inhibitor) / %conversion without inhibitor. The concentrations of E1 and E2 (labeled and unlabeled) can be determined using any suitable technique. Any standard method or tool, such as GraphPad Prism 6 software, can be used to determine the concentration (IC50) that inhibits 50% of the E1 to E2 conversion. 50 ).

[0072] In the context of this invention, irreversibility arises because the compound of formula (I) can irreversibly covalently bind to 17β-hydroxysteroid dehydrogenase type 1, thereby blocking the enzyme's ability to convert estrone (E1) to estradiol (E2). Well-known methods in the art exist for determining whether a compound covalently binds to this enzyme (Zang et al. Cell Chem Biol., 2019, 26 (11), 1486-1500). A wash assay (i.e., cells first exposed to an inhibitor, then washed away, and then enzyme activity measured) is a valuable method in the validation process. The conversion of estrone to estradiol in T-47D cells with and without wash was compared, with or without the presence of an inhibitor. The sustained effect of the covalently bound inhibitor undergoing the wash assay in blocking the conversion of estrone to estradiol is attributed to the irreversible nature of its target binding. Specific conditions and reagents are given in the examples provided below.

[0073] In the context of this invention, the term "alkyl" refers to a straight-chain or branched hydrocarbon chain group that does not contain unsaturated bonds and is connected to the rest of the molecule by single bonds. Typical alkyl groups have 1 to 5 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, and n-pentyl.

[0074] In the context of this invention, the term "alkenyl" refers to a straight-chain or branched hydrocarbon chain group containing at least two carbon atoms and at least one C=C double bond, which is connected to the rest of the molecule by single bonds. Typical alkenyl groups have 2 to about 10, 2 to about 8, or 2 to about 6 carbon atoms. In one particular embodiment, the alkenyl group is vinyl, 1-methyl-vinyl, 1-propenyl, 2-propenyl, or butenyl.

[0075] In the context of this invention, the term "alkynyl" refers to a straight-chain or branched hydrocarbon chain group containing one or more C≡C triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, 2-propynyl, and 2-butynyl.

[0076] In the context of this invention, the term "cycloalkyl" refers to a cyclic alkyl group, wherein "alkyl" is as defined above. Illustrative and non-limiting examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl.

[0077] In the context of this invention, the term "alkoxy" refers to -O-alkyl, wherein "alkyl" is as defined above. Illustrative, non-limiting examples of hydroxyalkyl are methoxy, ethoxy, or tert-butoxy, etc.

[0078] In the context of this invention, the term "haloalkyl" refers to a straight-chain or branched hydrocarbon chain group that does not contain unsaturated bonds, wherein one or more hydrogen atoms are substituted with a halogen. Illustrative and non-limiting examples of haloalkyl groups are chloromethyl, trifluoromethyl, 1-chloro-2-fluoroethyl, etc.

[0079] In the context of this invention, the term "haloalkoxy" refers to -O-haloalkyl, wherein "haloalkyl" is as defined above.

[0080] In the context of this invention, the term "halogen" refers to bromine, chlorine, iodine, or fluorine.

[0081] In the context of this invention, the term "nitro" refers to NO2.

[0082] In the context of this invention, the term "cyano" refers to HC≡N.

[0083] In the context of this invention, the term "salt" must be understood to mean any form of compound used according to the invention, wherein the compound is in ionic form or charged and coupled with a counter ion (cation or anion), or is in solution. This definition also includes quaternary ammonium salts and complexes of active molecules with other molecules and ions, particularly complexes formed through ionic interactions. This definition specifically includes physiologically acceptable salts; the term must be understood to be equivalent to "pharmacologically acceptable salt" or "pharmaceutical-acceptable salt".

[0084] In the context of this invention, the term "pharmaceutically acceptable salt" means any salt that is physiologically tolerable when used appropriately for treatment, application, or use, particularly in humans and / or mammals (generally implying its non-toxicity, especially due to counterions). These physiologically acceptable salts can form with cations or bases, and in the context of this invention, should be understood as salts formed from at least one compound used according to the invention (typically an acid (deprotonated), such as an anion) and at least one physiologically tolerable (particularly for use in humans and / or mammals) cation (preferably an inorganic cation). Salts formed with alkali metals and alkaline earth metals are particularly preferred, as are salts with ammonium cations (NH4). + Salts formed with (a) or (b) sodium, (a) or (b) potassium, magnesium, or calcium. These physiologically acceptable salts can also form with anions or acids, and in the context of this invention, it should be understood as salts formed by at least one compound used according to the invention (typically protonated, e.g., on nitrogen, as a cation) and at least one physiologically tolerable (particularly when used in humans and / or mammals) anion. In the context of this invention, this definition specifically includes salts formed by physiologically tolerable acids, i.e., salts of a specific active compound with a physiologically tolerable (particularly when used in humans and / or mammals) organic or inorganic acid. Examples of this type of salt are salts formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, formic acid, acetic acid, oxalic acid, succinic acid, malic acid, tartaric acid, mandelic acid, fumaric acid, lactic acid, or citric acid.

[0085] Any compound of formula (I) mentioned herein is intended to represent that particular compound as well as certain variants or forms. In particular, the compounds mentioned herein may have an asymmetric center and thus exist in different enantiomers or diastereomers. Therefore, any given compound of formula (I) mentioned herein is intended to represent any one of a racemic mixture, one or more enantiomers, one or more diastereomers, or mixtures thereof. Similarly, stereoisomerism or geometric isomerism with respect to double bonds is also possible, and thus, in some cases, the molecule may exist as (E)-isomers or (Z)-isomers (trans and cis isomers). When a molecule contains multiple double bonds, each double bond will have its own stereoisomerism, which may be the same as or different from the stereoisomerism of the other double bonds in the molecule. Furthermore, the compounds mentioned herein may exist as transisomers. All stereoisomers of the compounds mentioned herein, including enantiomers, diastereomers, geometric isomers, and transisomers, and mixtures thereof, are considered to be within the scope of this invention.

[0086] Furthermore, any compound of formula (I) mentioned herein may exist as a tautomer. Specifically, the term tautomer refers to one of two or more structural isomers of a compound that exist in equilibrium and are readily converted from one isomer to another.

[0087] In one embodiment of the invention, the compound of the invention is one of formula (Ia) or (Ib):

[0088]

[0089]

[0090] R1 to R3, A1, A2, m, n and p are as defined in the first aspect.

[0091] In another embodiment, the compound of the present invention has formula (Ic) or (Id):

[0092]

[0093]

[0094] R1 to R3, A1, A2, m, n and p are as defined in the first aspect.

[0095] In one embodiment of the invention, the compound is a compound in which A1 is C(O). In an alternative embodiment, A1 is CR. z1 , where R z1 Selected from OH, =N-OH, =N-OCH3, NH2, F, OCH3, OCOCH3, OSO2NH2; especially R z1 It is OH. In one embodiment, A1 is selected from C(O) or CHOH.

[0096] In one embodiment of the invention, the compound of the invention is a compound in which m is not 0 and A2 = CH2. Specifically, m is 1, A2 = CH2 and n is 0.

[0097] In one alternative embodiment, the compound of the present invention is a compound in which m=0 and A2=-O-. In another embodiment, the compound of the present invention is a compound in which m=0, A2=-O- and n is not 0, particularly n=1.

[0098] In one embodiment of the present invention, the compound of the present invention is a compound wherein R1 is hydrogen.

[0099] In one embodiment of the invention, the compound of the invention is a compound in which R3 is bromoethyl, particularly 2-bromoethyl.

[0100] In one embodiment of the invention, the compound of the invention is a compound in which p is 0 when R2 represents a 5-membered heterocyclic aromatic ring.

[0101] In another embodiment, the compound of the present invention is a compound in which p is 1 when R2 represents a 6-membered heterocyclic aromatic ring.

[0102] In one embodiment of the invention, the compound of the invention is a compound in which R2 is a heterocyclic aromatic ring having 5 or 6 members. Specifically, R2 is a heterocyclic aromatic ring having one or two heteroatoms. In another specific embodiment, R2 is a heterocyclic aromatic ring having one or two heteroatoms selected from N or S. In one embodiment, R2 is a thiazolyl ring, wherein R2 is as defined above, and specifically R2 is selected from H and (C1-C5) alkyl groups. In an alternative embodiment, R2 represents a pyridyl ring, wherein R2 is as defined above, and specifically R2 is selected from H and (C1-C5) alkyl groups.

[0103] In a further embodiment, the compounds of the present invention are selected from:

[0104] 3-[3-(2-bromoethyl)-17-oxostera-1(10),2,4-trien-15β-yl]-N-(5-methyl-1,3-thiazo-2-yl)propionamide (PBRM-II);

[0105] 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-15β-yl]-N-(5-methyl-1,3-thiazolyl-2-yl)propionamide (PBRM-II-OH);

[0106] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-thiazo-2-yl)propionamide (PBRM-III);

[0107] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-thiazo-2-yl)acetamide (PBRM-IV);

[0108] 3-[3-(2-bromoethyl)-17-β-hydroxyestradiol-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-thiazolyl-2-yl)acetamide (PBRM-V);

[0109] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl]propionamide (PBRM-VII);

[0110] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl]acetamide (PBRM-VIII);

[0111] 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl]propionamide (PBRM-IX);

[0112] 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl]acetamide (PBRM-X);

[0113] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-15β-yl]-N-[(pyridin-3-yl)methyl]propionamide (PBRM-XI);

[0114] 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-15β-yl]-N-[(pyridin-3-yl)methyl]propionamide (PBRM-XII);

[0115] 2-{[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-15β-yl]oxy}-N-(5-methyl-1,3-thiazo-2-yl)acetamide (PBRM-XIII); and

[0116] 2-{[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-15β-yl]oxy}-N-(5-methyl-1,3-thiazolyl-2-yl)acetamide (PBRM-XIV);

[0117] And any of its salts or stereoisomers.

[0118] In a third aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the compound of the present invention as defined above.

[0119] The term "therapeutic effective dose" should be understood as the amount of a compound applied being sufficient to prevent the development of the targeted disease or to alleviate one or more symptoms of the targeted disease to some extent.

[0120] The precise therapeutic dose of the components and the amount of the compounds of the present invention may depend on several variables. Some of these may be: route of administration, drug release time (e.g., immediate or prolonged), administration regimen, pain severity, patient condition, etc.

[0121] The pharmaceutical composition can be prepared as a liquid, semi-solid, or solid dosage form, such as an injectable solution, drops, juice, syrup, spray, suspension, tablet, patch, capsule, dressing, suppository, ointment, cream, lotion, gel, emulsion, aerosol, or multi-particulate form, such as in the form of pills or granules, and, if necessary, compressed into tablets, encapsulated, or suspended in a liquid, or applied directly.

[0122] These compositions can be prepared using conventional means, equipment, methods or processes known in the art.

[0123] Pharmaceutically acceptable adjuvants, solvents, or excipients that can be used in such compositions are those known to those skilled in the art or commonly used in the preparation of therapeutic compositions, and may be selected, for example, excipients, fillers, solvents, diluents, surfactants, colorants, preservatives, disintegrants, flow aids, lubricants, flavoring agents, or binders.

[0124] The term "pharmaceutical acceptable" refers to pharmaceutically acceptable materials, compositions, or solvents. Each component must be pharmaceutically acceptable in the sense of compatibility with the other components of the pharmaceutical composition. It must also be suitable for contact with human tissues or organs and, in animals and especially in humans, not cause excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0125] The choice of physiologically compatible adjuvant or the amount of adjuvant used depends on the form of administration of the pharmaceutical composition, i.e., oral, subcutaneous, parenteral, intravenous, intraperitoneal, intradermal, intramuscular, intranasal, buccal, rectal, ear, or tympanic cavity. Formulations in the form of tablets, sugar-coated pills, capsules, granules, pellets, drops (especially ear drops), juices, or syrups are preferably suitable for oral administration; solutions, suspensions, readily reconstituted dry formulations, or sprays are preferably suitable for parenteral, topical, or inhalation administration. Compounds of the invention used in the pharmaceutical compositions of the present invention, if in reservoir, dissolved, or dressing form, or if appropriate, with the addition of other agents that facilitate skin penetration, are suitable for transdermal administration. Formulations that can be administered orally or transdermally may also release the corresponding compound according to the invention in a delayed manner.

[0126] For example, for oral administration in tablet or capsule form, the active pharmaceutical ingredient can be combined with an orally administered, non-toxic, pharmaceutically acceptable inert carrier such as lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, etc.; for oral administration in liquid form, the oral pharmaceutical ingredient can be combined with any orally administered, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerin, water, etc. Furthermore, suitable binders, lubricants, disintegrants, and colorants can be incorporated into the mixture when needed or necessary. Suitable binders include starch, gelatin, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth, or sodium alginate, carboxymethyl cellulose, polyethylene glycol, waxes, etc. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc.

[0127] Gelatin capsules contain active ingredients and powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, and stearic acid. Similar diluents can be used to manufacture compressed tablets. Both tablets and capsules can be manufactured as sustained-release products to provide continuous drug release over several hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant tastes and protect the tablet from atmospheric effects, or enteric-coated for selective disintegration in the gastrointestinal tract.

[0128] Liquid dosage forms for oral administration may contain colorants and flavoring agents to increase patient acceptance.

[0129] Of course, the dosage of the drug composition administered will vary depending on the intended use and known factors such as the recipient's age, health condition, and weight; the nature and severity of symptoms; concomitant treatment (if any); frequency of treatment; and desired outcome. The recipient can be any type of mammal, but humans are preferred.

[0130] The present invention also provides methods and uses for treatment and prevention based on the compounds and compositions provided by the present invention.

[0131] Throughout the instructions, the term “treatment” includes, but is not limited to, reducing or eliminating one or more symptoms of the condition; reducing the severity of the disease, stabilizing (i.e., not worsening) the condition, delaying or slowing its progression, alleviating or improving the condition, and relieving (completely or partially) the condition.

[0132] As used in this invention, the term "prevention" refers to preventing pain attacks in patients who are susceptible but have not yet developed disease symptoms.

[0133] "Estrogen-dependent conditions" refer to diseases, conditions, or tumors whose initiation and / or proliferation and / or growth are stimulated by estrogen. "Estrogen-dependent conditions can be benign or malignant."

[0134] Estrogen-dependent conditions can be benign (local proliferation without metastasis) or malignant (local proliferation with metastasis). Malignant conditions are typically cancer.

[0135] In one implementation, the subject suffers from a malignant estrogen-dependent disease or condition, such as cancer. Illustrative, non-limiting examples of cancer include breast cancer, lung cancer, prostate cancer, endometrial cancer, uterine cancer, and ovarian cancer.

[0136] In another embodiment, the subject suffers from benign estrogen-dependent diseases or conditions, such as endometriosis, uterine fibroids, uterine leiomyomas, adenomyosis, dysmenorrhea, menorrhagia, uterine bleeding, prostatodynia, benign prostatic hyperplasia, urinary dysfunction, polycystic ovary syndrome, lower urinary tract syndrome, multiple sclerosis, obesity, rheumatoid arthritis, colon cancer, tissue trauma, skin wrinkles, or cataracts.

[0137] In another embodiment of the composition, method, and use, the compound of the present invention is administered in combination with one or more other therapeutic agents suitable for treating estrogen-dependent diseases.

[0138] The term "therapeutic agent" is used to describe pharmaceutical agents other than those of the compounds of the present invention, which are used in combination with the compounds of the present invention as biologically active agents to help achieve the intended therapeutic, inhibitory, and / or preventative / controlling effects of using the compounds of the present invention. Preferred biologically active agents used herein include those having pharmacological activities similar to those of using or administering the compounds of the present invention, including, for example, anticancer agents, antiviral agents (especially anti-HIV and anti-HCV agents), antimicrobial agents, antifungal agents, nonsteroidal anti-inflammatory compounds (NSAIDs), retinoid compounds, matrix metalloproteinase inhibitors, anti-estrogens, GnRH agonists or antagonists, selective progesterone receptor modulators (SPRMs), angiogenesis inhibitors, progesterone-like compounds, aromatase inhibitors, 17β-HSD-7 inhibitors, 17β-HSD5 inhibitors, or any combination thereof.

[0139] According to the present invention, the nonsteroidal anti-inflammatory compound (NSAID) is, for example, selected from the following compounds: acetylsalicylic acid, indomethacin, sulindac, phenylbutazone, diclofenac, fentiac, ketoroxyprofen, piroxicam, tenoxicam, mecoxicam, meloxicam, sinoxicam, isobutylphenylacetic acid, ibuprofen, naproxen, ketoprofen, nabumetone, niflufenicol, and nimesulide, or pharmaceutically acceptable salts thereof. Preferred NSAIDs are diclofenac, piroxicam, tenoxicam, mecoxicam, meloxicam, isobutylphenylacetic acid, ibuprofen, naproxen, and ketoprofen, or pharmaceutically acceptable salts thereof.

[0140] According to the present invention, examples of retinoid compounds include, for example, Accutane; Adapalene; Allergan AGN-193174; Allergan AGN-193676; Allergan AGN-193836; Allergan AGN-193109; Aronex AR-623; BMS-181162; Galderma CD-437; Eisai ER-34617; Etratilate; Fenivel Aamine; Ligand LGD-1550; Lysalicalciferol; Maxia Pharmaceuticals MX-781; Mofarotin; Molecular Design MDI-101; Molecular Design MDI-301; Molecular Design MDI-403; Movel Aamine; Eisai 4-(2-[5-(4-methyl-7-ethylbenzofuran-2-yl)pyrrole])benzoic acid; Johnson & Johnson N-[4-[2-ethyl-1-(1H-imidazol-1-yl)butyl]phenyl]-2-benzothiazolamide; Solyatan; Roche SR-11262; Tocoretinoic acid; Advanced Polymer Systems all-trans retinoic acid; UAB Research Foundation UAB-8; Tazarotene; TopiCare; Taiho TAC-101; and Vesanoid.

[0141] According to the present invention, examples of matrix metalloproteinase inhibitors include those known to include:

[0142] 1-Cyclopropyl-N-hydroxy-4-[[4-[4-(trifluoromethoxy)phenoxy]phenyl]sulfonyl]-4-piperidinecarboxamide monohydrochloride;

[0143] N-hydroxy-1-(phenylmethyl)-4-[[4-[4-[4-(trifluoromethoxy)phenoxy]-1-piperidinyl]sulfonyl]-4-piperidincarboxamide monohydrochloride;

[0144] N-hydroxy-1-(pyridylmethyl)-4-[[4-[4-[4-(trifluoromethyl)phenoxy]phenyl]sulfonyl]-4-piperidinecarboxamide dihydrochloride;

[0145] N-hydroxy-2,3-dimethoxy-6-[[4-[4-(trifluoromethyl)phenoxy]-1-piperidinyl]sulfonyl]benzamide;

[0146] N-hydroxy-1-(4-pyridylmethyl)-4-[[4-[4-(trifluoromethyl)phenoxy]phenyl]sulfonyl]-4-piperidinecarboxamide dihydrochloride;

[0147] N-hydroxy-1-(3-pyridylmethyl)-4-[[4-[4-(trifluoromethyl)phenoxy]phenyl]sulfonyl]-4-piperidinecarboxamide dihydrochloride;

[0148] N-hydroxy-1-(2-pyridylmethyl)-4-[[4-[4-[4-(trifluoromethyl)phenoxy]phenyl]sulfonyl]-4-piperidinecarboxamide monohydrochloride;

[0149] British Biotech BB-2516 (Marimastastat), N4-[2,2-dimethyl-1-[(methylamino)carbonyl]-propyl]-N1,2-dihydroxy-3-(2-methylpropyl)-, [2S-[N4(R*), 2R*, 3S*]]-);

[0150] BMS 275291 was disclosed in WO97 / 19075;

[0151] Bayer Ag Bay-12-9566 (Tanostat), 4-[(4'-chloro[1,1-biphenyl]-4-yl)oxy]-2-[(phenylthio)methyl]butyric acid;

[0152] Agouron Pharmaceuticals AG-3340, N-hydroxy-2,2'-dimethyl-4-[[4-(4-pyridyloxy)phenyl]sulfonyl]-3-thiomorpholine carboxamide;

[0153] CollaGenex Pharmaceuticals CMT-3 (metastastat), 6-demethyl-6-deoxy-4-dedimethylaminotetracycline, bamastat (BB-94); and

[0154] Chiroscience D-2163, 2-[1S-([(2R,S)-acetylmercapto-S-phthalimino]pentanoyl-L-leucyl)amino-3-methylbutyl]imidazolium.

[0155] Anti-estrogens, such as selective estrogen receptor modulators (SERMs), are preferably SERMs without uterine nutritional activity. Examples of SERMs according to the invention include tamoxifen, toremifene, azoxifen, edoxifene, EM800, fulvestrant, and droloxifen.

[0156] According to the present invention, examples of GnRH (LHRH) agonists are, for example, leuprorelin, dilorelin, triptorelin, buserorelin, nafarelin, goserelin, avorelin, histidinerelin, compound PTL 03001 (5-oxo-L-prolyl-L-histyl-L-tryptophanyl-L-seryl-L-tyrosinyl-D-tryptophanyl-L-leucyl-L-arginyl-N-ethyl-L-prolylamide) (Peptech), and compound AN 207(6-[N6-[5-[2-[1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-4-[[2,3,6-trideoxy-3-(2,3-dihydro-1H-pyrrolo-1-yl).α.-L-lyso-pyranohexosyl]oxy]-2-tetraphenyl]-1,5-dioxopentyl]-D-lysine]-,(2S-cis)-)(ASTA Medica Inc.), compound AN 238 L-threonamide, N-[5-[2-[(2S,4S)-1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-7-methoxy-6,11-dioxo-4-[[2,3,6-trideoxy-3-(2,3-dihydro-1H-pyrrolo-1-yl).α.-L-lyso-pyranohexosyl]oxy]-2-tetraphenyl]-2-oxoethoxy]-1,5-dioxopentyl]-D-phenylalanyl-L-cysteyl-L-tyrosinyl-D-tryptophanyl-L-lysyl-L-valine-L-cysteyl cyclic (2→7)-disulfide (ASTAMedica Inc.) and compound SPD 424 (LHRH-hydrogel implant) (Shire Pharmaceuticals Group), or pharmaceutically acceptable salts thereof.

[0157] Preferred examples are triptorelin, leuprorelin and goserelin, or pharmaceutically acceptable salts thereof, particularly triptorelin or pharmaceutically acceptable salts thereof, such as triptorelin dihydroxynaphthyl salt.

[0158] According to the present invention, examples of GnRH (LHRH) antagonists are, for example, cetrorex, abalix, lamorix, tevirilix, ganirix, compound A75998 (acetyl-D-(2-naphthyl)alanyl-D-(4-chlorophenyl)alanyl-D-(3-pyridyl)alanyl-seryl-(N-methyl)tyrosyl-N6-(nicotinyl)-D-lysyl-leucyl-N6-(isopropyl)lysyl-alanyl-D-alanamide) and A 84861 (Tetrahydrofuran-2-(S)-ylcarbonyl-glycyl-D-(2-naphthyl)alanyl-D-(4-chloro)phenylalanyl-D-(3-pyridyl)-alanyl-L-(N-methyl)tyrosyl-D-[N6-(3-pyridylcarbonyl)]lysyl-L-leucyl-L-(N6-isopropyl)lysyl-L-alanyl-D-alanylamide) (AbbotLabs.), GnRH immunogen (Aphton Co.), compound T 98475 (3-(N-benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-2-(4-isobutyrylaminophenyl)-4-oxothiopheno[2,3-b-pyridine-5-carboxylic acid isopropyl hydrochloride) (Takeda), and compound MI 1544 (acetyl-D-tryptophanyl-D-cyclopropyl-alanyl-D-tryptophanyl-L-seryl-L-tyrosyl-D-lysyl-L-leucyl-L-arginyl-L-propyl-D-alanylamide), or a pharmaceutically acceptable salt thereof.

[0159] According to the present invention, an example of a selective progesterone receptor modulator (SPRM) is, for example, dinogest or a pharmaceutically acceptable salt thereof.

[0160] Angiogenesis inhibitors include, for example, αvβ3 integrin inhibitors, protein kinase inhibitors, angiostatin, platelet factor 4 (endostatin), VEGF inhibitors, or thalidomide.

[0161] Vascular endothelial growth factor (VEGF) inhibitors and telomerase inhibitors are well known in the art.

[0162] In addition, known VEGF inhibitors or antagonists are agents that inhibit angiogenesis by reducing the binding of VEGF to cell receptors, including but not limited to, blocking monoclonal antibodies against growth factors (e.g., rhuMAbVEGF, Ryan et al., Toxicol Pathol 1999, 27:78-86), antibodies against receptors (e.g., DC101 and its derivatives, Witte et al., Cancer Metastasis Rev 1998, 17:155-61), soluble forms of VEGF receptors (e.g., soluble Flt, Aiello et al., Proc Natl Acad Sci USA 1995, 92:10457-61), or compounds that directly antagonize the interaction between VEGF and cell surface receptors (e.g., Fairbrother et al., Biochemistry 1998, 37:17754-64).

[0163] According to the present invention, the protein kinase inhibitor is, for example, a tyrosine kinase inhibitor, particularly the compounds 3-[4-(2-carboxyethyl-3,5-dimethylpyrrolo-2-yl)methylene]-2-indolone and 3-[(2,4-dimethylpyrrolo-5-yl)methylene]-2-indolone.

[0164] Examples of αvβ3 integrin inhibitors are known:

[0165] Vitaxin antibody (Ixsys); Merck KgaA EMD-121974, cyclo[RGDF-N(Me)V-];

[0166] (10S)-10,11-dihydro-3-[3-(2-pyridylamino)propoxy]-5H-dibenzo[a,d]cycloheptene-10-acetic acid;

[0167] (2S)-7[[(1H-benzimidazol-2-ylmethyl)methylamino]carbonyl]-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-2-acetic acid;

[0168] (2S)-2,3,4,5-Tetrahydro-4-methyl-7-[[[(5-methyl-1H-imidazo[4,5-b]pyridin-2-yl]methyl]amino]carbonyl]-3-oxo-1H-1,4-benzodiazepine-2-acetic acid;

[0169] (bR)-b-[[[(3R)-2-oxo-3-[2-(5,6,7,8-tetrahydro-[1,8]-naphthidin-2-yl)ethyl]-1-1-pyrrolyl]acetyl]amino]-d-(1H-indol-3-yl)valeric acid; and

[0170] (3R)-N-[3-hydroxy-5-[(1,4,5,6-tetrahydro-5-hydroxy-2-pyrimidinyl)amino]benzoyl]-glycyl-3-(3-bromo-5-chloro-2-hydroxyphenyl)-β-alanine.

[0171] Angiostatin, endostatin, and thalidomide are well known in the art. Pharmaceutically acceptable salts of the compounds mentioned herein are well known in the art.

[0172] In one embodiment, the one or more other therapeutic agents are administered simultaneously, sequentially, or separately from the compounds or compositions of the present invention.

[0173] Other objects, advantages, or features of the invention will be apparent to those skilled in the art, either in part from the description or in part from practice of the invention. The following examples are provided by way of illustration and are not intended to limit the invention.

[0174] Example

[0175] Chemical synthesis of the compounds of this invention

[0176] I. Materials and Methods

[0177] Chemical reagents were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada). 2-(3-bromopropoxy)-tert-butyldimethylsilane was purchased from Combiblock (San Diego, California, USA).

[0178] Commonly used solvents were purchased from Fisher Scientific (Montreal, Quebec, Canada) and used as is. Anhydrous dichloromethane (DCM), tetrahydrofuran (THF), and dimethylformamide (DMF) were purchased from Sigma-Aldrich.

[0179] Thin-layer chromatography (TLC) and rapid column chromatography were performed on 0.20-mm silica gel 60 F254 plates and 230-400 mesh ASTM silica gel 60 plates, respectively (E. Merck; Darmstadt, Germany).

[0180] Infrared (IR) spectra were recorded on a Horizon MB 3000 ABB FTIR spectrometer (Quebec, Quebec, Canada), and only significant bands are reported (in cm⁻¹). −1 (Unit: )

[0181] Nuclear magnetic resonance (NMR) spectra at 300 MHz and 400 MHz 1 H) and 75MHz and 100.6MHz ( 13 C) were recorded on a Bruker Avance NEO 300 and Avance 400 digital spectrometer (Billerica, Massachusetts, USA). Chemical shifts (δ) are expressed in ppm and are expressed as chloroform (7.26 and 77.0 ppm), acetone (2.05 and 28.9 ppm), methanol (3.31 and 49.0 ppm), or dimethyl sulfoxide (2.49 and 39.5 ppm), respectively. 1 H and 13 Reference for C NMR.

[0182] High-performance liquid chromatography (HPLC) analysis for chemical purity was performed on a Shimadzu Prominence instrument (Kyoto, Japan) using a diode array detector and an Altima C18 analytical reversed-phase column (5 μm, 4.6 x 250 mm) under the conditions described (wavelength detection and solvent gradient).

[0183] Low-resolution mass spectrometry (LRMS) was performed on a Shimadzu Prominence instrument (Kyoto, Japan) equipped with a Shimadzu LCMS-2020 mass spectrometer and an APCI (atmospheric pressure chemical ionization) probe, and the results are expressed in m / z. X-ray analysis was performed using plateforme dediffraction des rayons X (Department of Chemistry, University of Montreal, Quebec, Canada).

[0184] II. Synthesis of the compound 3-[3-(2-bromoethyl)-17-oxostera-1(10),2,4-trien-15β-yl]-N-(5-methyl-1,3-thiazolyl-2-yl)propionamide (PBRM-II) of the present invention

[0185]

[0186] Scheme 3. Synthesis of PBRM-II. Reagents and conditions.

[0187] 17-Oxyestrol-1(10),2,4-trien-3-yltrifluoromethanesulfonate(1)

[0188] The compound was prepared according to the literature method (R. Maltais et al. Org. Process Res. Dev. 2019, 23, 11, 2323-2334). 1 The H NMR data are completely consistent with those reported in the literature.

[0189] 3-[2-(benzyloxy)ethyl]estrost-1(10),2,4-trien-17-one(2)

[0190] The compound was prepared according to the literature method (R. Maltais et al. Org. Process Res. Dev. 2019, 23, 11, 2323-2334). 1 The H NMR data are completely consistent with those reported in the literature.

[0191] 3-[2-(benzyloxy)ethyl]estrost-1(10),2,4,15-tetraen-17-one(3)

[0192] Under an argon atmosphere, at -78 °C, a 1.5 M lithium diisopropylamino (LDA) THF solution (8.92 mL, 13.4 mmol) was added dropwise to an anhydrous THF solution of compound 2 (2.6 g, 6.7 mmol) (130 mL). The solution was stirred for 40 minutes, followed by the sequential addition of trimethylamine (TEA) (2.74 mL, 20.1 mmol) and trimethylchlorosilane (TMSCl) (2.54 mL, 20.0 mmol). The solution was then warmed to room temperature and stirred for another 30 minutes. The resulting solution was poured into a 10% sodium bicarbonate solution and extracted with DCM. The organic layer was dried over sodium sulfate and evaporated under reduced pressure. The crude compound was diluted in a mixture of ACN / DCM (64 mL / 16 mL), Pd(OAc)2 (1.5 g, 6.7 mmol) was added, and the solution was stirred at 40 °C for 1 hour. The resulting solution was evaporated and the crude compound was purified by rapid chromatography using EtOAc / hexane (2:8) to give 1.4 g (54%) of compound 3.

[0193] 1 H NMR (CDCl3): 1.11 (s, 3H), 1.47-2.54 (m, 8H), 2.89 (t, 2H, J = 7.0Hz), 2.95 (m, 2H), 3.69 (t, 2H, J = 7.1 Hz), 4.54 (s, 2H), 6.09 (d, 1H, J =5.4 Hz), 6.99 (s, 1H), 7.04 (d, 1H, J = 7.9 Hz), 7.22 (d, 1H, J = 7.9 Hz), 7.33 (m, 5H), 7.63 (d, 1H, J = 5.7 Hz). 13C NMR (Acetone-d6): 20.3, 25.2,26.5, 31.7, 35.5, 35.6, 45.3, 51.0, 55.9, 71.2, 72.2, 124.8, 126.4, 127.4(3C), 128.2 (2C), 129.4, 131.3, 136.0, 136.6, 137.5, 139.1, 158.1, 211.2;LRMS for C 27 H 31 O2 [M + H] + 387.2 m / z.

[0194] 3-[2-(benzyloxy)ethyl]-15β-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)estradiol-1 (10), 2,4-trien-17-one (4)

[0195] Magnesium powder (0.1–0.3 mm) (282 mg, 11.8 mmol) and iodide crystals were placed in a 100 mL round-bottom flask and dried in a flame under an argon atmosphere. Then, an anhydrous THF solution (40 mL) of 2-(3-bromopropoxy)-tert-butyldimethylsilane (2.29 g, 9.04 mmol) was added dropwise at room temperature (over 20 minutes). The solution was then stirred at room temperature for 90 minutes. The resulting turbid gray solution was allowed to stand for 5 minutes until a black solid deposit formed at the bottom of the flask. The solution was then transferred through a sleeve to a flame-dried 200 mL round-bottom flask without transferring the black solid deposit. The solution was cooled to -40 °C, and then CuI (984 mg, 5.18 mmol) was added rapidly in a single batch. The solution was stirred vigorously at this temperature for 15 minutes, and the color of the solution gradually changed from light gray to light grayish-purple. Then, an anhydrous THF solution of compound 3 (1.0 g, 2.59 mmol) in 40 mL was added dropwise at -40 °C (1 drop per second for 70 minutes), followed by stirring at -40 °C for another 15 minutes. Next, glacial acetic acid (1.0 mL) was added dropwise at -40 °C, and the mixture was stirred at this temperature for 30 minutes. The resulting solution was poured into a 10% ammonium chloride solution and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give 2.3 g of crude material. The crude compound was purified by rapid chromatography using EtOAc / hexane (5:95 to 10:90) to give 1.02 g (70%) of compound 4.

[0196] 1H NMR (Acetone-d6): 0.09 (s, 6H), 0.93 (s, 9H), 1.05 (s, 3H), 1.42-2.38 (m, 15H), 2.83-2.93 (m, 5H), 3.66-3.74 (m, 4H), 4.53 (s, 2H), 6.99 (s,1H), 7.03 (d, 1H, J = 8.0 Hz), 7.21 (d, 1H, J = 8.0 Hz), 7.23-7.35 (m, 5H). 13 C NMR (Acetone-d6): -6.0 (2C), 17.2, 17.9, 25.3, 25.4 (3C), 26.6, 27.0,29.1, 32.6, 33.9, 34.1, 35.6, 36.0, 42.0, 44.9, 46.7, LRMS for C 36 H 53 O3Si [M + H] + 562.4 m / z.

[0197] 15β-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-3-(2-hydroxyethyl)estrost-1(10), 2,4-Trien-17-one (5)

[0198] Carbon-supported palladium hydroxide (20% wt, 600 mg) was added to a MeOH / DCM mixture solution of compound 4 (6.0 g, 10.7 mmol) at room temperature under an argon atmosphere. The round-bottom flask was purged three times with hydrogen and stirred for 1 hour at room temperature under a hydrogen atmosphere. The resulting solution was filtered through diatomaceous earth, washed with MeOH, and evaporated under reduced pressure to give 5.0 g of crude compound 5. The compound was evaporated twice with DCM to remove residual trace amounts of MeOH. The crude compound was purified by rapid chromatography using EtOAc / hexane (4:6) to give 3.5 g (70%) of compound 5.

[0199] 1H NMR (Acetone-d6): 0.08 (s, 6H), 0.92 (s, 9H), 1.05 (s, 3H), 1.43-2.43 (m, 16H), 2.73-2.94 (m, 5H), 3.66-3.77 (m, 4H), 6.97 (s, 1H), 7.01 (d,1H, J = 8.0 Hz), 7.21 (d, 1H, J = 8.0 Hz). 13 C NMR (Acetone-d6): -6.0 (2C),17.2, 17.9, 25.3, 25.4 (3C), 26.7, 27.0, 29.1, 32.6, 33.9, 34.1, 36.0, 39.0,42.0, 44.9, 46.7, 52.7, 62.3, 63.1, 124.8, 126.3, 129.4, 136.1, 136.8, 137.8,~220.0; LRMS for C 29 H 47 O3Si [M + H] + 471.3 m / z.

[0200] 3-(2-Bromoethyl)-15β-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)estrost-1(10),2, 4-Trien-17-one (6)

[0201] At 0 °C, imidazole (4.98 g, 73.1 mmol) and triphenylphosphine (7.8 g, 29.7 mmol) were added to a DCM (300 mL) solution of crude compound 5 (3.45 g, 7.32 mmol). Then, at 0 °C, a DCM (30 mL) solution of carbon tetrabromide (9.8 g, 29.7 mmol) was slowly added (for 2 min) to this solution. The solution was stirred at 0 °C for 5 min, then allowed to return to room temperature and stirred for another 45 min. The resulting solution was poured into a sodium bicarbonate solution (10%), extracted with DCM, filtered through a phase separator syringe, and evaporated under reduced pressure. Rapid chromatographic purification using EtOAc / hexane (1:9) yielded 2.93 g (75%) of bromide compound 6.

[0202] 1H NMR (Acetone-d6): 0.08 (s, 6H), 0.93 (s, 9H), 1.05 (s, 3H), 1.44-2.44 (m, 16H), 2.79-2.96 (m, 2H), 3.11 (t, 2H, J = 7.5 Hz), 3.66 (t, 2H, J =7.5 Hz), 3.64-3.74 (m, 2H), 7.03 (s, 1H), 7.06 (d, 1H, J = 8.0 Hz), 7.26 (d,1H, J = 8.0 Hz). 13 C NMR (Acetone-d6): -6.0 (2C), 17.2, 17.9, 25.2, 25.4 (3C), 26.6, 27.0, 29.1, 32.6, 33.3, 33.9, 34.1, 35.9, 38.7, 42.0, 44.9, 46.7, 52.7,62.3, 125.1, 126.0, 129.1, 136.4, 136.5, 138.6, ~220.0; LRMS for C 29 H 46 BrO2Si[M + H] + 533.2 and 535.1 m / z.

[0203] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-15β-yl]propionic acid (7)

[0204] Jones' reagent (3.0 mL) was added to a 300 mL solution of compound 6 (2.93 g, 5.49 mmol) in acetone at 0 °C, and the reaction mixture was stirred at this temperature for 30 min. The resulting solution was quenched with isopropanol (5 mL), stirred for 15 min, then poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give 2.4 g of crude compound, which was used directly as is in the next step.

[0205] 1H NMR (Acetone-d6): 1.07 (s, 3H), 1.39-2.46 (m, 17H), 2.92 (m, 2H), 3.11 (t, 2H, J = 7.5 Hz), 3.66 (t, 2H, J = 7.5 Hz), 7.03 (s, 1H), 7.06 (d,1H, J = 8.0 Hz), 7.26 (d, 1H, J = 7.9 Hz), 10.6 (broad s, 1H). 13 C NMR(Acetone-d6): 17.1, 25.2, 25.8, 26.5, 29.0, 32.8, 33.4, 33.7, 34.1, 35.8,38.7, 41.4, 45.0, 46.6, 52.6, 125.1, 126.0, 129.1, 136.4, 136.6, 138.6,173.7, ~220.0; LRMS for C 23 H 30 BrO3 [M + H] + 433.1 and 435.1 m / z.

[0206] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-15β-yl]-N-(5-methyl-1,3-thia) (PbM-II)-2-azolyl propionamide

[0207] To an anhydrous DMF (5 mL) solution of compound 7 (900 mg, 2.08 mmol), dimethylamino-morpholino-carbomony hexafluorophosphate (COMU) (978 mg, 2.28 mmol) was added. The mixture was stirred for 5 min, followed by the addition of 2-amino-5-methylthiazole (474 ​​mg, 4.15 mmol) and diisopropylethylamine (DIPEA) (723 µL, 4.6 mmol). The resulting solution was stirred at room temperature for 30 min. The solution was then poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using EtOAc / hexane (1:1 to 7:3) to give 835 mg (76%) of PBRM-II.

[0208] 1H NMR (CDCl3): 1.06 (s, 3H), 1.42-2.65 (m, 16H), 2.42 (s, 3H), 2.93(m, 2H), 3.11 (t, 2H, J = 7.7 Hz), 3.56 (t, 2H, J = 7.7 Hz), 6.96 (s, 1H),7.00 (d , 1H, J = 8.0 Hz), 7.05 (d, 1H, J = 1.2 Hz), 7.24 (d, 1H, J = 8.3 Hz), 11.0 (s, 1H); 13 C NMR (CDCl3): 11.6, 17.8, 25.3, 26.1, 26.7, 29.2, 33.0, 33.8,33.9, 35.5, 35.7, 39.0, 42.0, 45.0, 47.1, 52.8, 125.3, 126.0, 127.8, 129.2,133.0, 136.5, 136.7, 138.5, 158.2, 170.1, 220.2; LRMS for C 27 H 34 BrN2O2S [M + H] + 529.2 and 531.2 m / z. HPLC purity = 98.7%.

[0209] III. Synthesis of other compounds of the present invention

[0210] Chemical synthesis of PBRM-II-OH, PBRM-XI and PBRM-XII

[0211]

[0212] Scheme 4. Synthesis of PBRM-II-OH, PBRM-XI, and PBRM-XII. Reagents and conditions.

[0213] 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-15β-yl]-N-(5-methyl-1,3-thiazolinone) (PbRM-II-OH)-2-yl-propionamide

[0214] Sodium borohydride (11 mg, 0.028 mmol) was added to a solution of PBRM-II (50 mg, 0.094 mmol) in MeOH (3 mL). The resulting solution was stirred at 0 °C for 1 hour, then poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using EtOAc / hexane (8:2 to 10:0) to give 40 mg (80%) PBRM-II-OH.

[0215] 1 H NMR (CDCl3): 0.90 (s, 3H), 1.20-2.62 (m, 17H), 2.85 m, 2H), 3.10(t, 2H, J = 7.7 Hz), 3.55 (t, 2H, J = 7.7 Hz), 3.73 (t, 1H, J = 8.6 Hz), 6.93 (s, 1H), 6.98 (d, 1H, J = 8.0 Hz), 7.05 (d, 1H, J = 0.8 Hz), 7.23 (d,1H, J = 8.0 Hz), 12.1 (s, 1H); 13 C NMR (CDCl3): 11.6, 14.6, 25.4, 27.6, 27.7,29.3, 33.0, 35.0, 35.1, 35.7, 38.0, 38.6, 39.0, 43.0, 45.0, 52.0, 81.7,125.2, 125.9, 127.4, 129.1, 133.0, 136.3, 137.1, 139.2, 158.3, 170.8; LRMSfor C 27 H 36 BrN2O2S [M + H] + 531.0 and 533.0 m / z. HPLC purity = 99.4%.

[0216] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-15β-yl]-N-[(pyridin-3-yl)methyl] [By]propionamide (PBRM-XI)

[0217] To an anhydrous DMF (2 mL) solution of compound 7 (50 mg, 0.094 mmol), 45 mg (0.105 mmol) of (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholino-carbomony hexafluorophosphate (COMU) was added. The mixture was stirred for 5 min, followed by the addition of 474 mg (4.15 mmol) of 1-(pyridin-3-yl)methylamine and 55 µL (0.32 mmol) of diisopropylethylamine (DIPEA). The resulting solution was stirred at room temperature for 30 min. The solution was then poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using DCM / MeOH (95:5 to 9:1) to give 55 mg (95%) of PBRM-XI.

[0218] 1H NMR (Acetone-d6): 1.04 (s, 3H), 1.30-2.48 (m, 18H), 2.89 (m, 2H), 3.11 (t, 2H, J = 7.5 Hz), 3.66 (t, 2H, J = 7.5 Hz), 4.44 (d, 2H, J = 6.0 Hz),7.02 (s, 1H), 7.05 (d, 1H, J = 8.0 Hz), 7.25 (d, 1H, J = 8.0 Hz), 7.30 (m,1H), 7.72 (d, 2H, J = 7.8 Hz), 8.46 (d, 1H, J = 4.0 Hz), 8.55 (s, 1H); 13 C NMR(Acetone-d6): 17.2, 25.2, 26.4, 26.6, 29.0, 33.4, 33.8, 34.1, 35.2, 35.8,38.7, 40.3, 41.6, 44.9, 46.7, 52.7, 123.3, 125.1, 126.0, 129.2, 135.0 (2C),136.4, 136.6, 138.6, 148.3, 149.2, 172.1, ~220.0; LRMS for C 29 H 36 BrN2O2 [M + H] + 523.2 and 525.2 m / z. HPLC purity = 99.2%.

[0219] 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-15β-yl]-N-[(pyridin-3-yl)methyl] [Bacillus propionamide (PBRM-XII)]

[0220] Sodium borohydride (4 mg, 0.105 mmol) was added to a solution of PBRM-XI (20 mg, 0.032 mmol) in MeOH (1 mL). The resulting solution was stirred at 0 °C for 1 hour, then poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using DCM / MeOH (94:6) to give 8 mg (40%) of PBRM-XII.

[0221] 1H NMR (MeOH-d4): 0.88 (s, 3H), 1.23-2.33 (m, 18H), 2.83 (m, 2H), 3.06(t, 2H, J = 7.5 Hz), 3.56 (t, 2H, J = 7.5 Hz), 3.64 (t, 1H, J = 8.6 Hz), 4.40(s, 1H), 4.60 (br s, 1H), 6.93 (s, 1H), 6.96 (d, 1H, J = 8.0 Hz), 7.20 (d,1H, J = 7.9 Hz), 7.41 (dd, 1H, J1 = 4.9 Hz, J2 = 7.8 Hz), 7.79 (d, 1H, J = 7.9Hz), 8.43 (br s, 1H), 8.49 (br s, 1H); 13 C NMR (MeOH-d4): 15.3, 26.7, 28.8,30.0, 30.3, 33.9, 36.1 (2C), 37.2, 38.5, 40.0, 40.1, 41.6, 44.1, 46.3, 53.2,82.5, 125.3, 126.1, 126.9, 130.1, 136.9, 137.6, 137.7, 138.0, 140.4, 148.8,149.5, 176.2; LRMS for C 29 H 38 BrN2O2 [M + H] + 525.1 and 527.1 m / z. HPLC purity = 98.2%.

[0222] Chemical synthesis of PBRM-III to PBRM-X

[0223]

[0224] Algorithm 5. Synthesis of PBRM-III to PBRM-X from intermediate compound 2. Reagents and conditions. PBRM-VI was not obtained from PBRM-IV.

[0225] 3-[2-(benzyloxy)ethyl]-16-(hydroxymethylene)-estrost-1(10),2,4-trien-17-one(8)

[0226] To an ice-cooled solution of compound 2 (500 mg, 1.29 mmol) in toluene (7 mL), NaH (60% mineral oil, 731 mg, 12.9 mmol) was added. Ethyl formate (1 mL, 12.9 mmol) was added to the suspension, and the mixture was stirred overnight at room temperature. The solution was quenched with 10% HCl and extracted with EtOAc. The organic phase was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The oily residue was purified by rapid chromatography using EtOAc / hexane (1:4) to give 424 mg (79%) of syrupy compound 8.

[0227] 1 H NMR (DMSO-d6): 0.79 (s, 3H), 1.33-2.60 (m, 11H), 2.73-2.80 (m, 4H), 3.58 (t, 2H, J = 6.9 Hz), 4.45 (s, 2H), 6.91 (s, 1H), 6.96 (d, 1H, J = 8.0Hz), 7.16 (d, 1H, J = 8.0 Hz), 7.25-7.34 (m, 5H), 7.38 (s, 1H), 10.59 (bs,1H); 13 C NMR (DMSO-d6): 14.3, 24.0, 25.4, 26.2, 28.8, 31.4, 35.0, 37.2, 43.8,47.7, 48.4, 70.6, 71.7, 113.2, 124.9, 126.1, 127.2, 127.3, 127.4, 128.2 (2C),129.1, 135.9, 136.0, 137.4, 138.5, 150.1, 208.6.

[0228] (E)-Benzyl 3-[3-(2-(benzyloxy)ethyl)-17-oxostero-1(10),2,4-trien-16-ylidene]propionic acid Ester (9)

[0229] Benzyl (triphenylphosphine) acetate (640 mg, 1.56 mmol) was added to a THF (5 mL) solution of compound 8 (540 mg, 1.3 mmol). The mixture was stirred overnight at room temperature. After evaporation, the crude compound was purified by rapid chromatography using EtOAc / hexane (1:9) to give syrupy benzyl ester 9 (570 mg, 80%), and a mixture of E (83%) and Z (17%) isomers.

[0230] 1H NMR (CDCl3): 0.96 (s, 3H), 1.40-2.55 (m, 11H), 2.62-2.75 (m, 1H), 2.92-2.97 (m, 4H), 3.32 (d, 2H, J = 7.3 Hz), 3.75 (t, 2H, J = 7.2 Hz), 4.59(s, 2H), 5.22 (s, 2H), 6.84 (t, 1H, J = 7.4 Hz), 7.04 (s, 1H), 7.09 (d, 1H, J= 8.0 Hz), 7.28 (d, 1H, J = 8.0 Hz), 7.34-7.42 (m, 10H); 13 C NMR (CDCl3):14.2, 25.6, 26.3, 26.6, 29.2, 31.4, 35.0, 35.7, 37.6, 44.1, 47.6, 48.3, 66.7,71.1, 72.8, 125.2, 126.3, 127.2, 127.3, 127.4, 127.5, 128.0, 128.1, 128.2,128.3, 128.4, 129.4, 135.4, 136.1, 136.2, 136.3, 137.1, 137.4, 138.3, 139.9,169.7, 207.6; LRMS for C 37 H 40 O4 [M + H] + 549.3 m / z.

[0231] 3-[3-(2-hydroxyethyl)-17-oxostero-1(10),2,4-trien-16β-yl]propionic acid(10)

[0232] Under an argon atmosphere, carbon-supported palladium hydroxide (20% by weight, 60 mg) was added to a 1:1 EtOAc / MeOH mixture (10 mL) solution of compound 9 (300 mg, 0.53 mmol). The flask was purged with hydrogen three times and stirred at room temperature for 1 hour. The suspension was then filtered through diatomaceous earth, washed with MeOH, and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using MeOH / DCM (1:99 to 7:93) to give 161 mg (82%) of acid 10.

[0233] 1H NMR (CDCl3): 0.87 (s, 3H), 1.37-2.48 (m, 14H), 2.51 (t, 2H, J = 7.2Hz), 2.81 (t, 2H, J = 6.6 Hz), 2.88-2.91 (m, 2H), 3.85 (t, 2H, J = 6.6 Hz), 5.88 (bs, 1H), 6.97 (s, 1H), 7.01 (d, 1H, J = 8.0 Hz), 7.23 (d, 1H, J = 8.0Hz); 13 C NMR (CDCl3): 14.0, 25.5, 26.6, 27.3, 28.4, 29.3, 31.8, 32.3 (2C), 37.6, 44.3, 48.1, 48.4, 48.8, 63.5, 125.4, 126.4, 129.6, 135.8, 136.5, 137.8,178.2, ~219.0; LRMS for C 23 H 30 O4 [M + H] + 371.2 m / z.

[0234] Benzyl 3-[3-(2-(benzyloxy)ethyl)-17-oxostero-1(10),2,4-trien-16β-yl]acetate (11)

[0235] Under argon atmosphere, LDA (1.8 M in THF, 2.8 mL, 5 mmol) was added to an ice-cooled solution of compound 2 (1.5 g, 3.86 mmol) in dry THF (20 mL). The resulting deep yellow solution was stirred for 1 hour, and then the ice-cooling bath was replaced with a dry ice-acetone bath. After 10 minutes, a solution of dry HMPA (0.77 mL, 5 mmol) and benzyl bromoacetate (0.79 mL, 5 mmol) in dry THF (5 mL) was added to the mixture. The cooling bath was then removed, and the mixture was stirred for 3 hours, followed by quenching with a saturated NH4Cl aqueous solution. After extraction with EtOAc, the combined organic phases were washed with water and brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude product was purified by rapid chromatography (EtOAc / hexane 5:95) to give 0.77 g of recovered ketone (30%), followed by 1.4 g (42%) of 16-alkylated product, which was a mixture of two 16α / 16β diastereomers in a ratio of 90:10. Data for the major 16α-diastereomer are reported only. 1H NMR (CDCl3): 0.97 (s, 3H), 1.43-2.43 (m, 13H), 2.85-2.90 (m,4H), 2.93-3.03 (m, 1H), 3.71 (t, 2H, J = 7.3 Hz), 4.54 (s, 2H), 5.15 (s, 2H),6.96 (s, 1H), 7.01 (d, 1H, J = 8.0 Hz), 7.20 (d, 1H, J = 8.0 Hz), 7.28-7.38(m, 10H); 13 C NMR (CDCl3): 14.5, 25.5, 26.3, 27.7, 29.3, 31.5, 35.5, 35.7,38.1, 40.9, 44.2, 47.7, 48.2, 66.5, 71.2, 72.9, 125.3, 126.3, 127.5, 127.6(2C), 128.2 (2C), 128.3 (3C), 128.5 (2C), 129.5, 135.7, 136.3 (2C), 137.5,138.4, 172.2, ~219.0. Under argon atmosphere, commercially available LDA (1.8 M in THF, 0.6 mL, 14 mmol) was slowly added to an ice-cooled dry THF (20 mL) solution of the above-mentioned 16-alkylated steroid (769 mg, 1.4 mmol). After stirring for 1 hour, the pale yellow solution gradually turned brown and was cooled to -78 °C. A THF (5 mL) solution of dry MeOH (0.6 mL, 14 mmol) was slowly added, and the resulting mixture was stirred at -78 °C for 1 hour, followed by quenching with a saturated NH4Cl aqueous solution. After extraction with EtOAc, the combined organic phases were washed with water and brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude product was purified by rapid chromatography using EtOAc / hexane (5:95) to give 16β-alkylated compound 11 (301 mg, 39%) containing 12% of the 16α-diastereomer.

[0236] 1H NMR (CDCl3): 0.86 (s, 3H), 1.16-2.67 (m, 13H), 2.86-2.93 (m, 4H), 2.96 (dd, 1H, J1 = 4.0 and J2 = 16.0 Hz), 3.69 (t, 2H, J = 7.3 Hz), 4.54 (s,2H), 5.16 (s, 2H), 6.97 (s, 1H), 7.03 (d, 1H, J = 8.0 Hz), 7.23 (d, 1H, J =8.0 Hz), 7.25-7.38 (m, 10H); 13 C NMR (CDCl3): 14.2, 25.5, 26.6, 28.7, 29.2,31.8, 35.7, 36.4, 37.6, 44.3, 45.6, 48.3, 49.1, 66.5, 71.2, 72.8, 125.2,126.3, LRMS for C 36 H 40 O4 [M + H] + 537.2 m / z.

[0237] 3-[3-(2-hydroxyethyl)-17-oxostero-1(10),2,4-trien-16β-yl]acetic acid(12)

[0238] Under an argon atmosphere, carbon-supported palladium hydroxide (20% by weight, 30 mg) was added to a 1:1 EtOAc / MeOH mixture (10 mL). The flask was purged with hydrogen three times and stirred at room temperature for 1 hour. The suspension was then filtered through diatomaceous earth, washed with MeOH, and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using MeOH / DCM (1:99 to 4:96) to give 108 mg (54%) of acid 12.

[0239] 1H NMR (CDCl3): 0.89 (s, 3H), 1.40-2.65 (m, 13H), 2.82 (t, 2H, J = 6.5Hz), 2.88-2.99 (m, 3H), 3.86 (t, 2H, J = 6.5 Hz), 6.98 (s, 1H), 7.03 (d, 1H, J = 8.0 Hz), 7.24 (d, 1H, J = 8.0 Hz); 13 C NMR (CDCl3): 14.3, 25.6, 26.7,28.8, 29.3, 31.9, 36.1, 37.6, 38.6, 44.4, 45.5, 48.5, 49.2, 63.7, 125.5,126.5, 129.7, 135.9, 136.7, 137.8, 176.8, ~219.0; LRMS for C 22 H 28 O4 [M + H] + 357.2 m / z.

[0240] 3-[3-(2-hydroxyethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-thia] (13) Azol-2-yl)propionamide

[0241] HATU (364 mg, 0.96 mmol) was added to a dry DMF (1 mL) solution of compound 10 (255 mg, 0.7 mmol). After stirring at room temperature for 10 min, 5-methyl-1,3-thiazole (111 mg, 0.96 mmol) was added, followed by DIPEA (366 μL, 2.1 mmol). The resulting yellow mixture was stirred overnight at room temperature. After dilution with water and extraction with EtOAc, the organic phase was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified by rapid chromatography using acetone / hexane (3:7) to give 195 mg (60%) of foamy amide 13.

[0242] 1H NMR (CDCl3): 0.90 (s, 3H), 1.38-2.38 (m, 14H), 2.41 (d, 3H, J = 1.1Hz), 2.71 (t, 2H, J = 7.1 Hz), 2.82 (t, 2H, J = 6.5 Hz), 2.87-2.92 (m, 2H),3.86 (t, 2H, J = 6.5 Hz), 6.97 (s, 1H), 7.00 (d, 1H, J = 8.0 Hz), 7.12 (d,1H, J = 1.2 Hz), 7.23 (d, 1H, J = 8.0 Hz); 13 C NMR (MeOD): 11.3, 14.5, 26.9,27.9, 28.8, 29.4, 30.4, 32.1, 34.8, 39.3, 39.8, 45.7, 49.7, 49.8, 50.1, 64.4,126.2, 127.4, 128.6, 130.5, 135.4, 137.4 (2C), 138.8, 158.4, 172.8, ~219.0;LRMS for C 27 H 35 N₂O₃S [M + H] + 467.2 m / z.

[0243] [3-[3-(2-hydroxyethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-yl]-N-(5-methyl-1,3-yl) Thiazol-2-yl)acetamide (14)

[0244] Amide 14 was prepared as described in the preparation of compound 13. Thus, starting from acid 12 (61 mg, 0.17 mmol), chromatography was performed using MeOH / DCM (0.5:99.5), yielding 55 mg (71%) of foamy amide 14.

[0245] 1¹H NMR (CDCl₃) (a mixture of two conformational isomers, 57:43): 0.62 and 0.90 (2s, 3H), 1.39–2.53 (m, 12H), 2.40 and 2.41 (2d, 3H, J = 1.0 Hz), 2.63–2.73 (m, 1H), 2.68–2.93 (m, 4H), 3.01–3.19 (m, 1H), 3.81–3.87 (m, 2H), 6.95 and 6.96 (2s, 1H), 6.98–7.03 (m, 1H), 7.09 and 7.14 (2d, 1H, J = 1.1 Hz), 7.22 (d, 1H, J = 8.0 Hz); 13 C10 NMR (CDCl3) (a mixture of two conformational isomers, 57:43): 11.5, 11.6, 14.4, 14.6, 25.6, 26.4, 26.7, 27.4, 28.9, 29.3, 29.4, 31.9, 33.4, 33.6, 37.6, 38.2, 38.6, 38.7, 42.7, 43.5, 44.4, 45.9, 48.5, 49.2, 49.3, 50.7, 63.6, 125.5, 125.6, 126.3, 126.4, 127.5, 127.9, 129.6, 129.7, 133.6, 133.8, 135.8, 136.0,136.6, 136.7, 137.8, 138.1, 156.1, 157.8, 169.2, 172.5, ~219.0; LRMS forC 26 H 33 N₂O₃S [M + H] + 453.2 m / z.

[0246] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-thia) (PBRM-III) 2-Azolium propionamide

[0247] PBRM-III was prepared as described in the preparation of compound 6. Thus, the bromination of amide 13 (195 mg, 0.42 mmol) was followed by chromatography using acetone / DCM (5:95) to give 57 mg (26%) of PBRM-III as a solid.

[0248] 1H NMR (Acetone-d6): 0.91 (s, 3H), 1.39-2.45 (m, 14H), 2.38 (s, 3H), 2.69 (t, 2H, J = 7.2 Hz), 2.80-2.92 (m, 2H), 3.10 (t, 2H, J = 7.4 Hz), 3.66(t, 2H, J = 7.4 Hz), 7.00 (s, 1H), 7.06 (d, 1H, J = 7.8 Hz), 7.07 (s, 1H),7.26 (d, 1H, J = 7.8 Hz); 13 C NMR (Acetone-d6): 10.4, 13.5, 25.5, 26.6, 27.7,28.2, 29.2, 32.0, 33.3, 33.7, 37.8, 38.7, 44.4, 48.1, 48.2, 48.7, 125.4,126.0, 126.5, 129.2, 134.6, 136.3, 136.5, 138.3, 156.4, 170.2, ~219.0; LRMSfor C 27 H 34 BrN2O2S [M + H] + 529.1 and 531.5 m / z. HPLC purity = 94.8%.

[0249] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-thia) (PbM-IV) 2-Azol-Acetamide

[0250] PBRM-IV was prepared as described in the preparation of compound 6. Thus, the bromination of amide 14 (50 mg, 0.11 mmol) was followed by chromatography using acetone / DCM (5:95) to give 25 mg (32%) of PBRM-IV as a white solid.

[0251] 1 H NMR (CDCl3): 0.63 (s, 3H), 1.12-2.46 (m, 11H), 2.42 (s, 3H), 2.63-2.73 (m, 1H), 2.83-2.93 (m, 2H), 3.04-3.16 (m, 3H), 3.55 (t, 2H, J = 7.6 Hz), 5.93 (bs, 1H), 6.93 (s, 1H), 6.99 (d, 1H, J = 7.7 Hz), 7.09 (s, 1H), 7.23 (d, 1H, J = 7.9 Hz);13 C NMR (CDCl3): 11.5, 14.6, 26.4, 27.4, 29.4, 33.0, 33.4,33.6, 38.6, 38.7, 39.0, 42.7, 43.6, 49.3, 50.7, 125.6, 126.0, 128.0, 129.2,133.8, 136.3, 136.8, 138.6, 156.1, 172.5, ~219.0; LRMS for C 26 H 32 BrN2O2S [M +H] + 515.1 and 517.1 m / z. HPLC purity = 99.9%.

[0252] 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-thiazolinone) (2-Azol-2-yl)acetamide (PBRM-V)

[0253] PBRM-V was prepared as described in the preparation of PBRM-XII. Therefore, PBRM-III (50 mg, 0.09 mmol) was reduced with NaBH4 and recrystallized (MeOH) to give 10.3 mg (20%) of white solid PBRM-V.

[0254] 1 H NMR (CDCl3): 0.80 (s, 3H), 1.02-2.38 (m, 12H), 2.41 (s, 3H), 2.53-2.63 (m, 2H), 2.83-2.87 (m, 2H), 3.10 (t, 2H, J = 7.8 Hz), 3.55 (t, 2H, J =7.6 Hz), 3.81 (d, 1H, J = 9.2 Hz), 6.93 (s, 1H), 6.99 (d, 1H, J = 8.0 Hz), 7.08 (s, 1H), 7.25 (d, 1H, J = 8.0 Hz); 13 C NMR (CDCl3): 11.6, 12.4, 26.0,27.4 (2C), 29.5, 32.6, 33.0, 35.6, 37.6, 38.0, 39.0, 39.5, 44.2, 44.3, 48.7,82.0, 125.6, 125.9, 126.6, 129.2, 133.6, 136.2, 136.9, 138.9, 157.4, 170.9.LRMS for C 27 H 36BrN2O2S [M + H] + 531.1 and 533.1 m / z. HPLC purity = 96.3%.

[0255] [3-[3-(2-hydroxyethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)] [Methyl]propionamide (15)

[0256] HATU (114 mg, 0.3 mmol) was added to a dry DMF (0.2 mL) solution of compound 10 (74 mg, 0.2 mmol). After stirring at room temperature for 10 min, 1-(pyridin-3-yl)methylamine (32 mg, 0.3 mmol) was added, followed by DIPEA (103 µL, 0.4 mmol). The resulting yellow mixture was stirred overnight at room temperature. After dilution with water and extraction with EtOAc, the organic phase was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified by chromatography using MeOH / DCM (4:96) to give 70 mg (70%) of foamy amide 13.

[0257] 1 H NMR (Acetone-d6): 0.85 (s, 3H), 1.32-2.30 (m, 13H), 2.35-2.42 (m,3H), 2.75 (t, 2H, J = 7.0 Hz), 2.83-2.86 (m, 2H), 3.74 (t, 2H, J = 7.0 Hz), 4.43 (d, 2H, J = 5.9 Hz), 6.95 (s, 1H), 6.99 (d, 1H, J = 8.0 Hz), 7.19 (d,1H, J = 7.9 Hz), 7.29-7.33 (m, 1H), 7.71 (d, 1H, J = 7.6 Hz), 7.82 (bt, 1H),8.46 (bs, 1H), 8.56 (bs, 1H). 13 C NMR (Acetone-d6): 13.6, 25.6, 26.6, 28.2,28.3, 29.2, 32.0, 34.2, 37.8, 39.0, 40.3, 44.4, 48.1, 48.4, 48.8, 63.1,123.4, 125.1, 126.3, 129.5, 135.1, 135.4, 136.1, 136.7, 137.4, 148.2, 149.2,172.1, ~219.0; LRMS for C 29 H 37 N₂O₃ [M + H]+ 461.2 m / z.

[0258] 3-[3-(2-hydroxyethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl] Acetamide (16)

[0259] Amide 16 was prepared as described in the preparation of compound 15. Thus, starting from acid 12 (45 mg, 0.13 mmol), chromatography using MeOH / DCM (4:96) yielded 35 mg (57%) of foamy amide 15.

[0260] 1 H NMR (CDCl3): 0.82 (s, 3H), 1.35-2.46 (m, 12H), 2.52-2.65 (m, 1H), 2.71 (dd, 1H, J1 = 5.6 and J2 = 14.6 Hz), 2.80 (t, 1H, J = 6.6 Hz), 2.84-2.92(m, 2H), 3.83 (t, 2H, J = 6.6 Hz), 4.43 (t, 2H, J = 5.3 Hz), 6.95-7.02 (m,3H), 7.20 (d, 1H, J = 8.0 Hz), 7.26 (d, 1H, J = 7.5 Hz), 7.64 (d, 1H, J = 7.6Hz), 8.46–8.50 (m, 2H); 13 C NMR (CDCl3): 14.2, 25.5, 26.6, 29.0, 29.2, 31.8,37.5, 38.3, 38.6, 41.0, 44.2, 46.1, 48.5, 48.9, 63.5, 123.5, 125.3, 126.3,129.6, 134.0, 135.6, 136.0, 136.5, 137.6, 148.6, 149.0, 171.3, ~219.0; LRMSfor C 28 H 35 N₂O₃ [M + H] + 447.2 m / z.

[0261] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl] [Bacillus propionamide (PBRM-VII)]

[0262] PBRM-VII was prepared as described in the preparation of compound 6. Thus, the bromination of amide 15 (63 mg, 0.12 mmol) was followed by chromatography using acetone / diethyl ether / DCM (4:20:80) to give 24 mg (34%) of PBRM-VII as a yellow solid.

[0263] 1 H NMR (Acetone-d6): 0.88 (s, 3H), 1.30-2.35 (m, 12H), 2.39 (t, 2H, J= 7.1 Hz), 2.83-2.90 (m, 3H), 3.10 (t, 2H, J = 7.5 Hz), 3.66 (t, 2H, J = 7.5Hz), 4.43 (d, 2H, J = 6.0 Hz), 7.01 (s, 1H), 7.05 (d, 1H, J = 8.2 Hz), 7.25(d, 1H, J = 8.0 Hz), 7.31 (dd, 1H, J = 4.5 and 7.7 Hz), 7.71 (d, 1H, J =7.8 Hz), 8.46 (d, 1H, J = 3.9 Hz), 8.55 (bs, 1H); 13 C NMR (Acetone-d6): 13.5,25.5, 26.5, 28.2, 28.4, 29.1, 32.0, 33.3, 34.1, 37.7, 38.7, 40.2, 44.4, 48.1,48.3, 48.8, 123.2, 125.4, 126.0, 129.2, 135.0, 135.3, 136.3, 136.5, 138.3,148.3, 149.2, 171.9, ~219.0; LRMS for C 29 H 36 BrN2O2 [M + H] + 523.2 and 525.2 m / z. HPLC purity = 71.1%.

[0264] 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl] Acetamide (PBRM-VIII)

[0265] PBRM-VIII was prepared as described in the preparation of compound 6. Thus, the bromination of amide 16 (34 mg, 0.07 mmol) yielded 19 mg (50%) of PBRM-VIII as a yellow solid after chromatographic analysis (acetone / ethyl ether / DCM (3:20:80)).

[0266] 1H NMR (CDCl3): 0.84 (s, 3H), 1.38-2.42 (m, 12H), 2.56-2.65 (m, 1H), 2.72 (dd, 1H, J = 5.8 and 14.6 Hz), 2.88-2.92 (m, 2H), 3.10 (t, 2H, J = 7.6Hz), 3.55 (t, 2H, J = 7.6 Hz), 4.45 (d, 2H, J = 6.0 Hz), 6.73 (t, 1H, J = 5.5Hz), 6.94 (s, 1H), 6.99 (d, 1H, J = 8.0 Hz), 7.23 (d, 2H, J = 8.1 Hz), 7.65(d, 1H, J = 7.8 Hz), 8.53 (bs, 2H); 13 C NMR (CDCl3): 14.3, 25.6, 26.7, 29.3,31.9, 32.9 (2C), 37.6, 38.4, 39.0, 41.1, 44.4, 46.2, 48.6, 49.1, 123.6,125.5, 126.0, 129.3, 135.0, 135.6, 136.4, 136.7, 138.3, 148.8, 149.2, 171.3,~219.0; LRMS for C 28 H 34 BrN2O2 [M + H] + 509.2 and 511.2 m / z. HPLC purity = 92.2%.

[0267] 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl]propionamide (PBRM-IX)

[0268] PBRM-IX was prepared as described in the preparation of PBRM-XII. Therefore, PBRM-VII (17 mg, 0.03 mmol) was reduced with NaBH4 and chromatographically subjected to MeOH / DCM (8:92) to give 13 mg (76%) of white foamy PBRM-IX.

[0269] 1H NMR (Acetone-d6): 0.81 (s, 3H), 1.01-2.41 (m, 14H), 2.80-2.90 (4Hmasked), 3.10 (t, 2H, J = 7.4 Hz), 3.65 (t, 2H, J = 7.5 Hz), 3.74 (d, 1H, J =9.5 Hz), 4.43 (d, 2H, J = 6.0 Hz), 6.98 (s, 1H), 7.04 (d, 1H, J = 8.3 Hz),7.22-7.33 (m, 2H), 7.60 (bs, 1H), 7.71 (d, 1H, J = 7.8 Hz), 8.47 (s, 1H),8.55 (s, 1H); 13 C NMR (DMSO-d6): 12.6, 25.7, 27.0, 28.1, 29.0, 31.8, 34.5,34.9, 37.4, 37.8 (2C), 38.0, 43.6 (2C), 43.8, 48.2, 80.3, 123.4, 125.2,125.9, 128.9, 135.0, 135.3, 135.8, 136.1, 138.5, 147.9, 148.7, 172.7; LRMSfor C 29 H 38 BrN2O2 [M + H] + 525.1 and 527.1 m / z. HPLC purity = 91.4%.

[0270] 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl] Acetamide (PBRM-X)

[0271] PBRM-X was prepared as described in the preparation of PBRM-XII. Therefore, PBRM-VIII (10 mg, 0.07 mmol) was reduced with NaBH4 and chromatographically subjected to MeOH / DCM (8:92) to give 3.3 mg (33%) of white foamy PBRM-X.

[0272] 1H NMR (CDCl3): 0.79 (s, 3H), 1.08-2.38 (m, 12H), 2.56-2.66 (m, 1H), 2.77 (dd, 1H, J1 = 10.6 and J2 = 14.7 Hz), 2.83-2.88 (m, 2H), 3.10 (t, 2H, J =7.7 Hz), 3.55 (t, 2H, J = 7.6 Hz), 3.88 (d, 1H, J = 9.6 Hz), 4.46 (d, 2H, J =6.0 Hz), 6.22 (t, 1H, J = 5.4 Hz), 6.92 (s, 1H), 7.01 (d, 1H, J = 8.0 Hz),7.23-7.32 (m, 2H), 7.64 (d, 1H, J = 7.9 Hz), 8.54 (bs, 2H); 13 C NMR (CDCl3):12.7, 26.0, 27.5, 29.5, 29.7, 32.8, 33.0, 37.8, 37.9, 38.6, 39.0, 41.2, 44.2,44.3, 49.2, 80.8, 123.7, 125.6, 125.9, 129.2, 135.6 (2C), 136.1, 136.9,139.0, 148.9, 149.1, 174.5; LRMS for C 28 H 36 BrN2O2 [M + H] + 511.1 and 513.1 m / z. HPLC purity = 94.8%.

[0273] Chemical synthesis of PBRM-XIII and PBRM-XIV

[0274]

[0275] Scheme 6. Synthesis of PBRM-XIII and PBRM-XIV from intermediate compound 3. Reagents and conditions.

[0276] 3-[2-(benzyloxy)ethyl]-15β-(2-hydroxyethoxy)estrost-1(10),2,4-trien-17-one(17)

[0277] Ethylene glycol (15 mL) and 1 mL of NaOH (5%) aqueous solution were added to a DCM (15 mL) solution of compound 3 (700 mg, 1.81 mmol). The solution was stirred vigorously at room temperature for 72 hours. The resulting solution was diluted with DCM and washed with water. The organic layer was dried over a cotton plug and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using EtOAc / hexane (2:8 to 10:0) to give 580 mg (72%) of compound 17.

[0278] 1 H NMR (CDCl3): 1.19 (s, 3H), 1.40-2.47 (m, 12H), 2.91 (m, 4H), 3.45(m, 1H), 3.70 (m, 5H), 4.26 (t, 1H, J = 5.4 Hz), 4.56 (s, 2H), 7.00 (s, 1H), 7.04 (d, 1H, J = 8.0 Hz), 7.23 (d, 1H, J = 8.0 Hz), 7.29-7.39 (m, 5H); 13 C NMR(CDCl3): 17.6, 25.6, 26.3, 29.2, 32.7, 34.8, 35.8, 43.2, 44.5, 47.3, 54.6,62.0, 70.8, 71.3, 73.0, 75.3, 125.2, 126.4, 127.6, 127.7 (2C), 128.4 (2C),129.6, 136.4 (2C), 137.7, 138.5, 219.3; LRMS for C 29 H 37 O4 [M + H] + 449.2 m / z.

[0279] 3-[2-(benzyloxy)ethyl]-15β-{2-[(oxacyclohexane-2-yl)oxy]ethoxy}estrost-1(10),2, 4-Trien-17-one (18)

[0280] At 0 °C, 3,4-dihydro-2H-pyran (544 mg, 6.46 mmol) and p-toluenesulfonic acid monohydrate (p-TSA) (3 mg, 0.015 mmol) were added to a DCM (15 mL) solution of compound 17 (580 mg, 1.29 mmol). The solution was stirred at 0 °C for 10 min, then allowed to warm to room temperature and stirred for 1 h. The solution was then poured into a saturated sodium bicarbonate solution and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using EtOAc / hexane (3:7, containing 1% TEA) to give 580 mg (84%) of compound 18.

[0281] 1 H NMR (Acetone-d6): 1.17 and 1.18 (2s, 3H), 1.38-2.50 (m, 16H), 2.70-2.98 (m, 5H), 3.44 (m, 1H), 3.55 (m, 2H), 3.68 (t, 2H, J = 7.0 Hz), 3.82 (m,3H), 4.32 (t, 1H, J = 5.4 Hz), 4.53 (s, 2H), 4.67 (m, 1H), 6.99 (s, 1H), 7.03(d, 1H, J = 8.0 Hz), 7.22 (d, 1H, J = 8.0 Hz), 7.33 (m, 5H); 13 C NMR (Acetone-d6): 17.0, 19.0, 25.4, 25.6, 26.0, 29.1, 30.5, 32.8, 35.0, 35.6, 42.7, 44.5,46.9, 54.2, 61.0, 66.3, 68.9, 71.2, 72.3, 75.0, 98.1, 125.0, 126.2, 127.2,127.4 (2C), 128.1 (2C), 129.4, 136.2, 136.5, 137.7, 139.1, 217.9.

[0282] 3-(2-hydroxyethyl)-15β-{2-[(oxacyclohexane-2-yl)oxy]ethoxy}estrost-1(10),2,4-tristan ...2,2,2,2,2,2,2,2,2,2,2,2,2,2,2 En-17-one (19)

[0283] Under an argon atmosphere, 50 mg of carbon-supported palladium hydroxide (20% by weight) was added to a 30 mL solution of compound 18 (500 mg, 0.94 mmol) in MeOH. The flask was purged three times with hydrogen and stirred at room temperature for 1 hour. The suspension was then filtered through diatomaceous earth, washed with MeOH, and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using EtOAc / hexane (1:1 to 7:3; containing 1% TEA) to give 180 mg (45%) of compound 19.

[0284] 1 H NMR (Acetone-d6): 1.17 and 1.18 (2s, 3H), 1.40-2.46 (m, 18H), 2.68-2.90 (m, 5H), 3.46 (m, 1H), 3.58 (m, 2H), 3.73 (m, 3H), 3.82 (m, 2H), 4.33(t, 1H, J = 5.4 Hz), 4.67 (m, 1H), 6.97 (s, 1H), 7.00 (d, 1H, J = 7.9 Hz), 7.21 (d, 1H, J = 7.9 Hz); 13 C NMR (DMSO-d6): 17.2, 18.8, 25.0, 25.3, 28.8,30.2, 32.3, 34.6, 42.1, 44.0, 46.5, 53.3, 56.5, 60.7, 62.2, 65.8, 68.0, 74.5,76.0, 97.6, 124.9, 126.2, 129.2, 135.7, 136.5, 137.0, 219.0; LRMS for C 27 H 39 O5[M + H + H2O] + 461.2 m / z.

[0285] 3-(2-Bromoethyl)-15β-{2-[(oxacyclohexane-2-yl)oxy]ethoxy}estrost-1(10),2,4-tristan En-17-one (20)

[0286] At 0 °C, triphenylphosphine (202 mg, 0.77 mmol) and imidazole (212 mg, 3.11 mmol) were added to a DCM (20 mL) solution of compound 19 (170 mg, 0.41 mmol), followed by dropwise addition of a DCM (5 mL) solution of carbon tetrabromide (256 mg, 0.77 mmol) for 2 minutes. The solution was then stirred for 1 hour. The resulting solution was diluted with DCM (25 mL) and washed with saturated sodium bicarbonate solution. The organic layer was filtered through a cotton plug and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using EtOAc / hexane (1:9; containing 1% TEA) to give 69 mg (36%) of compound 20.

[0287] 1 H NMR (Acetone-d6): 1.17 and 1.18 (2s, 3H), 1.40-2.45 (m, 16H), 2.68-2.92 (m, 3H), 3.11 (t, 2H, J = 7.5 Hz), 3.46 (m, 1H), 3.56 (m, 2H), 3.66 (t,2H, J = 7.5 Hz), 3.82 (m, 3H), 4.32 (t, 1H, J = 5.4 Hz), 4.66 (m, 1H), 7.02(s, 1H), 7.06 (d, 1H, J = 7.9 Hz), 7.27 (d, 1H, J = 8.1 Hz); 13 C NMR (Acetone-d6): 17.0, 19.0, 25.4, 25.5, 25.9, 30.5, 32.8, 33.3, 34.9, 38.7, 42.7, 44.5,46.9, 54.2, 60.9, 66.3, 68.7, 68.9, 75.0, 98.1, 125.2, 126.0, 129.2, 136.4,136.5, 138.5, 217.9; LRMS for C 27 H 38 BrO4 [M + H + 2 H2O] + 536.1 and 538.1 m / z.

[0288] 3-(2-Bromoethyl)-15β-(2-hydroxyethoxy)estrost-1(10),2,4-trien-17-one(21)

[0289] p-TSA (2 mg, 0.01 mmol) was added to a 3 mL solution of compound 20 (65 mg, 0.13 mmol) in a DCM / MeOH (9:1) mixture at 0 °C. The solution was stirred at this temperature for 30 min. The resulting solution was diluted with DCM and then washed with a saturated sodium bicarbonate solution. The organic phase was filtered through a cotton plug and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using EtOAc / hexane (1:1 to 1:0) to give 35 mg (65%) of compound 21.

[0290] 1 H NMR (Acetone-d6): 1.16 (s, 3H), 1.40-2.46 (m, 10H), 2.67-2.95 (m,3H), 3.11 (t, 2H, J = 7.5 Hz), 3.47 (m, 1H), 3.51-3.70 (m, 6H), 4.31 (t, 1H,J = 5.4 Hz), 7.02 (s, 1H), 7.06 (d, 1H, J = 8.0 Hz), 7.26 (d, 1H, J = 7.9Hz); 13 C NMR (Acetone-d6): 17.1, 25.5, 26.0, 29.2, 32.8, 33.4, 34.7, 38.7,42.9, 44.4, 46.8, 54.3, 61.3, 71.3, 75.0, 125.2, 126.0, 129.2, 136.3, 136.6,138.6, 217.9; LRMS for C 22 H 30 BrO3 [M + H] + 421.1 and 423.1 m / z.

[0291] {[3-(2-bromoethyl)-17-oxoestrate-1(10),2,4-trien-15β-yl]oxy}acetic acid(22)

[0292] Jones' reagent (100 µL) was added dropwise to a 3 mL solution of compound 21 (33 mg, 0.08 mmol) in acetone at 0 °C, and the solution was stirred at this temperature for 2 hours. Then 2-propanol was added, and the mixture was stirred at 0 °C for another 10 minutes. The resulting solution was poured into water, extracted with EtOAc, washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give 35 mg of carboxylic acid 22 as a single product. This compound was used directly in the next step without further purification.

[0293] 1H NMR (CDCl3): 1.20 (s, 3H), 1.36-2.46 (m, 11H), 2.70-2.99 (m, 3H), 3.11 (t, 2H, J = 7.6 Hz), 3.55 (t, 2H, J = 7.7 Hz), 4.15 (q of AB system, 2H, J A = 7.0 Hz, J B = 16.7 Hz), 4.37 (t, 1H, J = 5.1 Hz), 6.96 (s, 1H), 7.00(d, 1H, J = 8.0 Hz), 7.25 (d, 1H, J = 8.8 Hz); 13 C NMR (CDCl3): 17.4, 25.4,26.1, 29.1, 32.7, 33.0, 34.6, 39.0, 42.9, 44.6, 47.3, 54.6, 66.4, 76.2,125.4, 126.0, 129.2, 136.4, 136.8, 138.4, 174.4, ~218.0; LRMS for C 22 H 28 BrO4 [M+ H] + 435.1 and 437.1 m / z.

[0294] 2-{[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-15β-yl]oxy}-N-(5-methyl-1, 3-Thiazolyl-2-yl)acetamide (PBRM-XIII)

[0295] To an anhydrous DMF (2 mL) solution of compound 21 (32 mg, 0.073 mmol), 1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholino-carbomony hexafluorophosphate (COMU) (35 mg, 0.08 mmol) was added, and the mixture was stirred for 5 minutes. Then, 2-amino-5-methylthiazole (17 mg, 0.15 mmol) and diisopropylethylamine (DIPEA) (26 µL, 0.15 mmol) were added. The resulting solution was stirred at room temperature for 2 hours. The solution was then poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure. The crude compound was purified by rapid chromatography using EtOAc / hexane (1:1) to give 25 mg (65%) of PBRM-XIII.

[0296] 1H NMR (Acetone-d6): 1.25 (s, 3H), 1.30-2.53 (m, 13H), 2.39 (d, 3H, J= 1.2 Hz), 2.81-3.06 (m, 3H), 3.11 (t, 2H, J = 7.5 Hz), 3.66 (t, 2H, J = 7.5Hz), 4.34 (q of AB system, 2H, J A = 22.5 Hz, J B = 7.3 Hz), 4.56 (t, 1H, J =5.3 Hz), 7.02 (s, 1H), 7.07 (d, 1H, J = 8.0 Hz), 7.09 (d, 1H, J = 1.2 Hz), 7.28 (d, 1H, J = 7.9 Hz), 10.0 (s, 1H); 13 C NMR (Acetone-d6): 10.5, 17.1,25.4, 26.1, 29.0, 32.9, 33.4, 34.8, 38.7, 42.4, 44.6, 46.8, 54.0, 68.2, 76.4,125.3, 126.0, 127.3, 129.2, 134.9, 136.4, 136.6, 138.4, 155.2, 167.2, 217.0;LRMS for C 26 H 32 BrN2O3S [M + H] + 531.1 and 533.1 m / z. HPLC purity = 99.0%.

[0297] 2-{[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-15β-yl]oxy}-N-(5-methyl- 1,3-Thiazolyl-2-yl)acetamide (PBRM-XIV)

[0298] Sodium borohydride (3.2 mg, 0.08 mmol) was added to a 2 mL solution of PBRM-XIII (15 mg, 0.028 mmol) in MeOH / DCM (9:1). The resulting solution was stirred at 0 °C for 1 hour, then poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over sodium sulfate, filtered, and evaporated under reduced pressure to give 13 mg (87%) of PBRM-XIV.

[0299] 1H NMR (Acetone-d6): 0.90 (m, 1H), 1.10 (s, 3H), 1.20-2.543 (m, 11H), 2.39 (d, 3H, J = 1.2 Hz), 2.80-3.04 (m, 3H), 3.10 (t, 2H, J = 7.5 Hz), 3.66(t, 2H, J = 7.5 Hz), 3.72 (t, 1H, J = 8.5 Hz), 4.15 (m, 1H), 4.18 (q of ABsystem, 2H, J A = 57.0 Hz, J B = 15.4 Hz), 7.00 (s, 1H), 7.05 (d, 1H, J = 8.0Hz), 7.08 (d, 1H, J = 1.2 Hz), 7.28 (d, 1H, J = 8.0 Hz), 9.72 (s, 1H); 13 C NMR(Acetone-d6): 10.5, 13.4, 25.9, 26.7, 29.5, 33.3, 35.0, 38.0, 38.7, 39.0,42.7, 44.6, 54.4, 67.4, 78.2, 80.4, 125.3, 125.9, 127.3, 129.2, 134.9, 136.3,136.7, 138.9, 155.0, 167.3; LRMS for C 26 H 34 BrN2O3S [M + H] + 533.1 and 535.1 m / z. HPLC purity = 99.8%.

[0300] Biological tests

[0301] I. Inhibitory efficacy

[0302] 17β-HSD1 inhibition assay (Method A):

[0303] T-47D breast cancer cells (purchased from the American Type Culture Collection (ATCC) (Manassas, Virginia, USA)) were grown in RPMI medium supplemented with 5% (v / v) dextran-coated activated charcoal-treated fetal bovine serum (FBS), L-glutamine (2 nM), penicillin (100 IU / mL), streptomycin (100 μg / mL), and insulin (50 ng / mL).

[0304] Cells were seeded in 24-well plates (8000-25000 cells / well). Stock solutions of each test compound were prepared in dimethyl sulfoxide (DMSO) and diluted to the appropriate concentration with culture medium before use. After incubation for 24 hours, the diluted solutions of each compound were added to the cells to obtain the solution for IC50 assay. 50 The appropriate final concentration for value determination is within the range of 0.5 nM to 1 μM. The final concentration of DMSO in the wells is adjusted to 0.1%.

[0305] These were then pre-incubated at 37°C for 1 hour. Additionally, [[...]] 3 The solutions of H-E1 (7 nM) and cold E1 (53 nM) yielded a total E1 concentration of 60 nM.

[0306] Cells were then incubated for 24 hours, and each inhibitor was evaluated in triplicate. After incubation, the culture medium was removed, and steroids were extracted with ether. The organic phase was evaporated to dryness under nitrogen. The residue was dissolved in DCM, spotted onto a silica gel thin-layer chromatography plate (EMDChemicals Inc., Manassas, Virginia, USA), and eluted with toluene / acetone (4:1) as the solvent system. The substrate [3H]-E1 / E1 and metabolites [3H]-E2 / E2 were identified and separated by comparison with reference steroids (E1 and E2). The labeled steroids were quantified using a scintillation counter. The percentage of conversion and percentage of inhibition were calculated as follows: conversion% = 100[3H]-E2 / ([3H]-E1 + [3H]-E2), inhibition% = 100(conversion% without inhibitor − conversion% with inhibitor) / conversion% without inhibitor. The concentration (IC50) at which 50% inhibition of E1 to E2 conversion was achieved was determined using GraphPad Prism 6 software.

[0307] Table 1. Concentrations in T-47D cells that inhibit 50% of the conversion of E1 to E2 by 17β-HSD1.

[0308]

[0309] Conclusion: The compound PBRM-II of the present invention, which introduces a specific amide moiety at C15, exhibits 26 times the activity of the prior art compound PBRM (in two experiments).

[0310] 17β-HSD1 inhibition assay (Method B):

[0311] T-47D cells were grown in a medium supplemented with insulin (50 ng / mL) and 5% FBS treated with dextran-coated activated charcoal (using this FBS instead of untreated 10% FBS to remove residual steroid hormones). Stock solutions of each test compound were prepared in DMSO and diluted to the appropriate concentration with medium before use. For assays, cells were seeded (3000 cells / well), incubated for 24 hours, and then diluted solutions of each inhibitor were added to the cells to achieve appropriate final concentrations (0.01, 0.1, 1, 5, 10, 50, 100, 500, and 1000 nM). The final concentration of DMSO in the wells was adjusted to 0.1%. The inhibitors and cells were pre-incubated at 37°C for 2 hours, and then solutions containing [14C]-E1 (10 nM) and cold E1 (50 nM) were added to obtain a final concentration of 60 nM. Cells were incubated for 24 hours, and each inhibitor was evaluated in triplicate. After incubation, the culture medium was removed, and the steroids (labeled and unlabeled E1 and E2) were extracted with diethyl ether. The organic phase was evaporated to dryness under nitrogen. The residue was dissolved in DCM, spotted onto silica gel TLC plates (EMD Chemicals Inc., Gibbstown, NJ, USA), and eluted with toluene / acetone (4:1) as the solvent system. The substrate [14C]-E1 / E1 and metabolite [14C]-E2 / E2 were identified and separated by comparison with reference steroids (E1 and E2). Radioactivity associated with each steroid was quantified using a Storm 860 Molecular Imager system (Molecular Dynamics, Sunnyvale, CA, USA). The percentage of conversion and the percentage of inhibition were calculated as follows: conversion% = 100 [14C]-E2 / ([14C]-E1 + [14C]-E2), inhibition% = 100 (conversion% without inhibitor − conversion% with inhibitor / conversion without inhibitor). The concentration (IC50) at which 50% of the E1 to E2 conversion was inhibited was determined using GraphPad Prism 6 software.

[0312] Table 2. Concentrations in T-47D cells that inhibit 50% of the conversion of E1 to E2 by 17β-HSD1.

[0313]

[0314]

[0315]

[0316] 1 In T47D cells, the conversion of estrone (60 nM) to estradiol by 17β-HSD1 (n=2) was investigated. 2 IC50 (PBRM) / IC 50 (Compound).

[0317] Conclusion: The compound PBRM-II of this invention, with a specific amide moiety introduced at C15, exhibits 23 times the activity of the prior art compound PBRM (in two experiments). Furthermore, PBRM-II (IC15) 50 =3.8 nM) is the most active 17β-HSD1 inhibitor in the tested PBRM analogue series, followed by PBRM-II-OH, PBRM-XII, PBRM-X and PBRM-XI (IC50, 3.8 nM). 50 The concentrations were 7.4, 9.6, 11.4, and 16.9 nM, respectively. All tested PBRM analogues were more active than PBRM. Compound CC-156 was found to have similar activity to PBRM.

[0318] The irreversibility of inhibitors

[0319] T47-D cells were cultured as described above. Cells were pre-incubated with the inhibitor at 37°C for 2 hours, the culture medium was slowly removed, and the cells were carefully washed three times with PBS (phosphate-buffered saline). Residual 17β-HSD1 activity was determined as described above by incubating cells with [14C]-E1 (10 nM) and cold E1 (50 nM) at 37°C for 24 hours. The steroids in the supernatant were then quantified using the above procedure. The percentage of transformation and the percentage of inhibition were calculated as described above.

[0320] Conclusion: The irreversibility of PBRM-2 has been confirmed, as the inhibitory activity was maintained after three washing steps. Figure 3 ).

[0321] In addition, IC values ​​were measured for both unwashed and washed samples. 50 Value. The concentration (IC50) required to inhibit 50% of the E1 to E2 conversion was determined using GraphPad Prism 6 software. 50 The results are shown in Table 3 below.

[0322] Table 3. Irreversibility (E1 to E2) of 17β-HSD1 inhibitors PBRM and PBRM-II

[0323]

[0324] 1 IC 50 (Washing available) 1 IC 50 (No washing); 2The irreversibility of PBRM was confirmed by X-ray diffraction analysis of the 17β-HSD1 / PBRM complex, indicating a strong covalent bond between the enzyme and the inhibitor (Li et al. J. Phys. Chem. Lett. 2018, 9, 5275). 3 The data showed no significant difference compared to no washing. 4 The reversibility of CC-156 was previously confirmed by X-ray analysis of the 17β-HSD1 / CC-156 complex, indicating that there is no strong covalent bond between the enzyme and the inhibitor (Mazumdar et al. Biochem. J. 2009, 424, 357).

[0325] As can be seen from Table 3 above, the inhibitory activity of PBRM-II was maintained after three washing steps, thus confirming its role as an irreversible inhibitor.

[0326] II. Antiproliferative activity of PBRM-II in T-47D cells

[0327] Select ER + The T-47D breast cancer cell line is named because it expresses ER, primarily ERα, and proliferates in the presence of estrogen compounds.

[0328] The cell line was maintained in a culture flask (175cm²) at 37°C and a humidified atmosphere of 5% CO2. 2 The growth area (BD Falcon) was measured. T-47D cells were grown in RPMI 1640 medium without phenol red, supplemented with 10% fetal bovine serum (FBS), penicillin (100 IU / mL), streptomycin (100 μg / mL), L-glutamine (2 mM) and 17β-estradiol (1 nM).

[0329] In this protocol, T-47D cells were suspended in RPMI supplemented with insulin (50 ng / mL) (instead of 17β-estradiol) and 5% activated charcoal-removed FBS to deprive the medium of estrogen. Cells were seeded at a density of 3000 cells / well in 96-well plates and allowed to adhere for 48 hours.

[0330] After this pre-incubation, the inhibitor and reference compound diluted in fresh culture medium were added to the wells and replaced every 2 days during the 7-day treatment period.

[0331] According to the manufacturer's instructions, use CellTitter 96. ®The Aqueous One Solution cell proliferation assay is an indirect colorimetric measurement of cell proliferation. In short, after treatment, 20 μL of MTS solution is added to each well (100 μL) of the plate and incubated at 37°C for 4 hours.

[0332] The absorbance at 490 nm was then measured using a Thermo Max microplate reader (Molecular Devices, Sunnyvale, California). The control (culture medium + DMSO) was set to 100% cell proliferation. Results were... Figure 1 Provided by China.

[0333] Conclusion: PBRM-II is non-estrogenous and more effective than PBRM in reducing the proliferation of estrogen-dependent T-47D cells.

[0334] III. Stability of PBRM-II in human liver microsomes

[0335] Studying compounds in human liver microsomes is a useful predictive tool for assessing their stability and potential metabolites.

[0336] Stability was determined at 37°C for 1 hour with or without 10 mM NADPH, in the presence of 40 μg of human liver S9 fraction from Corning (Melrose, MA, USA) and 10 μM substrate in a final volume of 100 μL of 50 mM Tris buffer (supplemented with 10 mM MgCl2).

[0337] The assay was terminated by adding 100 μL of methanol (MeOH), and the protein was precipitated by centrifugation at 13000g for 10 minutes.

[0338] The supernatants from the two independent experiments were combined and submitted for HPLC-MS analysis (Shimadzu LCMS-2020 APCI, Altima HP C18 (250 mm x 4.6 mm, 5 µm) column).

[0339] A 40-minute HPLC-MS run was performed at a flow rate of 1 mL / min. The elution gradient started at 70:30 (MeOH / H2O) and progressed to 100% MeOH over the first 15 minutes, followed by elution with 100% MeOH for 10 minutes, and then reequilibration with 70:30 (MeOH / H2O) for 15 minutes. APCI was used. +In ionization mode, a full scan of ionization was performed between 100 and 1000 m / z to detect compounds. The remaining compound (expressed as a percentage) was calculated by dividing the area under the curve of the substrate in the NADPH-containing assay by the area under the curve of the substrate in the NADPH-free assay, and then multiplying by 100. Numerical values ​​represent the average of two independent experiments. Figure 2 ).

[0340] Conclusion: PBRM-II is significantly more stable than PBRM and CC-156.

[0341] IV. Comparison of bioavailability between PBRM-II and PBRM

[0342] animal:

[0343] Female Balb / c mice, aged 6–7 weeks and weighing approximately 19 g, were purchased from Charles-River, Inc. (Saint-Constantine, Quebec, Canada). Animals were acclimatized to environmental conditions (temperature: 22±3°C; humidity: 50±20%; 12-hour light / 12-hour dark cycle, light starting at 07:15) for at least 5 days prior to the start of experiments. Three animals were housed per cage, with free access to water and a certified commercial rodent diet (Rodent diet #T.2018.15, Harlan Teklad, Madison, Wisconsin, USA), and water was provided on a free-flowing basis. Animal experiments were conducted in an animal facility accredited by the Canadian Council for Animal Care (CCAC) and the Laboratory Animal Care Assessment and Accreditation Association. Studies were conducted in accordance with the CCAC Guidelines for the Care and Use of Laboratory Animals. Institutional approval was obtained.

[0344] Measurement:

[0345] The study was conducted after oral administration of PBRM and PBRM-II at the following concentrations: 5, 15, 30, and 60 mg / kg body weight, dissolved in 0.1 mL of dimethyl sulfoxide and sunflower oil (8:92). The compounds were first dissolved in DMSO, and then sunflower oil was added to obtain a final DMSO concentration of 8%. Mice were fasted for 8 hours prior to administration of the compounds during this experiment. Blood samples for determining plasma compound concentrations were collected by cardiac puncture 2 hours after administration, with 3 samples collected from each dose. Blood samples were collected in Microvette potassium-EDTA (ethylenediaminetetraacetic acid) coated tubes (Sarstedt, Aktiegesellchaft & Co, Germany) and centrifuged at 3200 rpm for 10 minutes at 4 °C. Plasma was collected and stored at -80 °C until analysis by liquid chromatography / mass spectrometry / mass spectrometry (LC-MS / MS).

[0346] Measurement of PBRM and PBRM-II in plasma:

[0347] Inhibitor concentrations were determined by LC / MS / MS analysis using a procedure developed by the CHUQ (CHUL) Research Center (Bioanalytical Services). For plasma extraction, 100 μL of plasma sample was transferred to individual tubes and 600 μL of ammonium acetate (1 mM) was added. Then, 50 μL of methanol solution containing the deuterated steroid internal standard was added to each tube. The sample was transferred to a Strata-XSPE column (Phenomenex, Torrance, CA, USA), and each column was washed with water and methanol:water (10:90, v / v). The inhibitor was then eluted with 5 mL of methanol containing 1 mM ammonium acetate. The methanol was evaporated at 45 °C under an inert atmosphere, and the dried residue was redissolved in 100 μL of methanol:water (85:15, v / v). For inhibitor analysis, a 75 x 4.6 mm reversed-phase phenyl-hexyl column (Phenomenex, Torrance, USA) was used on the HPLC system at a flow rate of 0.8 mL / min. Inhibitors were detected using an API 4000 mass spectrometer (Applied Biosystems, Canada) equipped with TurboIonSpray. ESI positive ion mode was used.

[0348] Table 4. 17β-HSD1 inhibition (IC50) 50 Inhibitor plasma concentrations (IPCs) obtained in mice 2 hours after a single oral (oral) administration of four doses.

[0349]

[0350] 1 Targeting the conversion of estrone (60 nM) to estradiol by 17β-HSD1 in T47D cells.

[0351] As can be seen from the data in Table 4 above, the oral bioavailability (plasma concentration) of PBRM-II is very similar to that of PBRM.

[0352] V. Biodistribution of PBRM-II in six tissues in mice after oral administration (gavage)

[0353] In this experiment, mice were fasted for 10 hours before administration of the inhibitor. Female Balb / c mice were administered a single dose of PBRM-II (30 mg / kg) via gavage (PO) (3 animals / time point per dose), using sunflower oil:DMSO (dimethyl sulfoxide) (92:8) as the solvent. PBRM-II was first dissolved in DMSO, and then a suitable co-solvent (SO) was added to obtain a final DMSO concentration of 8% at 100 μL injection. Animals were euthanized by exsanguination under isoflurane at 2, 6, 12, and 24 hours after PBRM-II administration. Tissues (liver, kidney, uterus, ovary, and brain) and colon contents were then collected and stored at -80°C until the concentration of PBRM-II was determined by LC-MS / MS analysis using a procedure developed for steroid derivatives at the CHU Research Centre in Quebec. The results are shown in Table 5 below. Figure 4 .

[0354] Table 5. Concentration of PBRM-II in six tissues (ng / g tissue)

[0355]

[0356]

[0357] The biodistribution results of PBRM-II in six tissues showed that it was not significantly detected in brain tissue, indicating that it cannot cross the blood-brain barrier. PBRM-II was present in the five tissues analyzed (liver, kidney, uterus, ovary, and colon contents) but did not accumulate, and its concentration decreased over time. Furthermore, low levels of PBRM-II were detected at 24 h, indicating that the compound was completely cleared after 48 hours. More accumulation was observed in the ovarian and uterine tissues compared to some other tissues.

[0358] VI. Half-life (T) of PBRM-II and PBRM in human liver microsomes (HLM) 1 / 2 )

[0359] Incubation was performed in deep-well plates. Potassium phosphate buffer (100 mM, pH 7.4, +2 mM MgCl2, 600 µL) containing human liver microsomes (0.53 mg protein / mL) was pre-incubated for 10 min in a water bath set to 37°C (N=1) with either PBRM-II (4 µM) or a positive control (loperamide, 1 µM). T0 was obtained by transferring 47.5 µL of the pre-incubated microsomes containing the compound to a new 96-well plate placed on ice and quenching with 100 µL of (1:1) methanol / acetonitrile. Subsequently, 2.5 µL of 20 mM NADPH solution was added to the quenched T0 sample to maintain the same proportions as during incubation. The reaction was initiated by transferring 380 µL of the pre-incubated microsomes containing the compound to a new row of 96-well plates and adding 20 µL of NADPH (final concentration 1 mM). Using the remaining volume of pre-incubated microsomes containing the compound, an NADPH-free reaction was incubated to exclude non-NADPH metabolism or chemical instability in the incubation buffer. Simultaneously, a reaction without the compound was incubated as a blank control. At each time point (15, 60, 120, 180, and 240 minutes), the reaction was terminated by taking 50 µL of the incubation solution and quenching with 100 µL of (1:1) methanol / acetonitrile. The sample was centrifuged at 1500 rpm for 5 minutes, and the supernatant was transferred to a 96-well microplate for analysis by UPLC-QTof-MS. Remaining PBRM-II percentage vs. time is shown below. Figure 5A As stated above. Figure 5B A graph showing the percentage of remaining PBRM-II as Ln vs. time is displayed. Based on this graph, the half-life (T0) of 4 µM PBRM-II in human liver microsomes (HLM) was calculated. 1 / 2 The duration is 210 minutes.

[0360] The half-life of PBRM in human liver microsomes was determined using the same protocol as PBRM-II. Figure 5C and 5D The half-life of PBRM in human liver microsomes was calculated to be 103 minutes. Therefore, PBRM-II is more stable than PBRM in human liver microsomes.

[0361] Although the illustrative and presently preferred embodiments of the invention have been described in detail herein, it should be understood that the inventive concept may be embodied and applied in other ways, and the appended claims are intended to be construed as including these variations unless limited by the prior art.

Claims

1. A compound of formula (I) or its stereoisomer or salt: (I) in: A1 is selected from C(O) and CHR z1 ; A2 is selected from CH2 and O; R1 is selected from: hydrogen, (C1-C5) alkyl group optionally substituted with one or more Z-substituents, (C1-C5) alkoxy group optionally substituted with one or more Z-substituents, (C2-C5) alkenyl group optionally substituted with one or more Z-substituents, (C2-C5) alkynyl group optionally substituted with one or more Z-substituents; (C3-C8) cycloalkyl, aryl and heteroaryl groups; R2 is a heterocyclic aromatic ring having 5, 6, or 7 members selected from: CR z2 The following are N, S and O, provided that at least one member is N, S or O; R3 is a (C1-C5) alkyl group, which is reacted with one or more Br, Br 76 ;I、I 123 I 124 or I 131 replace; R z1 Selected from OH, NR x1 R x2 =N-OR x3 Halogens, (C1-C5)alkoxy groups, OC(O) (C1-C5)alkyl groups, and OSO2NR x4 NR x5 ; R z2 Selected from hydrogen, OH, or (C1-C) groups optionally substituted with one or more Z substituents. 10 ) alkyl, and (C1-C) substituted with one or more Z substituents optionally 10 )alkoxy; Z is selected from halogen, OH, (C1-C5)alkyl, (C1-C5)haloalkyl, (C1-C5)alkoxy, (C1-C5)haloalkoxy, and NR. x1 R x2 ; R x1 R x2 R x4 and R x5 The same or different, and selected from: hydrogen, (C1-C5)alkyl, (C1-C5)haloalkyl, (C1-C5)alkoxy and (C1-C5)haloalkoxy; R x3 Selected from H and (C1-C5) alkyl groups; m represents an integer value selected from 0 to 2; n represents an integer value selected from 0 to 2; p represents an integer value selected from 0 to 5; in: Aryl groups contain 5 or 6 CRs c The aromatic ring system of the members, where R c Selected from H, halogen, cyano, nitro, (C1-C5)alkyl, (C1-C5)haloalkyl, -O-(C1-C5)alkyl and -O-(C1-C5)haloalkyl; and Heteroaryl is an aromatic ring system comprising 5 or 6 members selected from: CR d , O, N, NH and S; where R d Selected from H, halogen, cyano, nitro, (C1-C5)alkyl, (C1-C5)haloalkyl, -O-(C1-C5)alkyl and -O-(C1-C5)haloalkyl.

2. The compound according to claim 1, wherein it is one of formula (Ia) or (Ib): (him) (One) Wherein R1 to R3, A1, A2, m, n and p are as defined in claim 1.

3. The compound according to claim 1 or 2, wherein it is one of formula (Ic) or (Id): (Ic) (Id) Wherein R1 to R3, A1, A2, m, n and p are as defined in claim 1.

4. The compound according to any one of claims 1 to 3, wherein A1 represents C(O) or CHOH.

5. The compound according to any one of claims 1 to 4, wherein m is not 0 and A2 = CH2.

6. The compound according to claim 5, wherein m is 1, A2=CH2 and n is 0.

7. The compound according to any one of claims 1 to 4, wherein m = 0 and A2 = -O-.

8. The compound according to claim 7, wherein m = 0, A2 = -O- and n is not 0.

9. The compound according to claim 8, wherein n=1.

10. The compound according to any one of claims 1 to 9, wherein R1 is hydrogen.

11. The compound according to any one of claims 1 to 10, wherein R3 is bromoethyl.

12. The compound according to any one of claims 1 to 11, wherein R3 is 2-bromoethyl.

13. The compound according to any one of claims 1 to 12, wherein R2 is a heterocyclic aromatic ring having 5 or 6 members.

14. The compound according to any one of claims 1 to 13, wherein R2 is a heterocyclic aromatic ring having one or two heteroatoms.

15. The compound according to claim 14, wherein one or both heteroatoms are N or S.

16. The compound according to any one of claims 1 to 15, wherein p is 0 when R2 represents a 5-membered heterocyclic aromatic ring.

17. The compound according to any one of claims 1 to 15, wherein p is 1 when R2 represents a 6-membered heterocyclic aromatic ring.

18. The compound according to any one of claims 1 to 17, wherein R2 is a thiazolyl ring, wherein Rz2 is as defined in claim 1.

19. The compound according to claim 18, wherein Rz2 is selected from H and (C1-C5) alkyl groups.

20. The compound according to any one of claims 1 to 15 and 17, wherein R2 represents a pyridyl ring, and wherein Rz2 is as defined in claim 1.

21. The compound according to claim 20, wherein Rz2 is selected from H and (C1-C5) alkyl groups.

22. The compound according to any one of claims 1 to 21, wherein the compound is selected from: 3-[3-(2-bromoethyl)-17-oxostera-1(10),2,4-trien-15β-yl]-N-(5-methyl-1,3-thiazolyl-2-yl)acrylamide; 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-15β-yl]-N-(5-methyl-1,3-thiazolyl-2-yl)acrylamide; 3-[3-(2-bromoethyl)-17-oxostera-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-thiazolyl-2-yl)acrylamide; 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-thiazo-2-yl)acetamide; 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-16β-yl]-N-(5-methyl-1,3-thiazolyl-2-yl)acetamide; 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl]acrylamide; 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl]acetamide; 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl]acrylamide; 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-16β-yl]-N-[(pyridin-3-yl)methyl]acetamide; 3-[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-15β-yl]-N-[(pyridin-3-yl)methyl]acrylamide; 3-[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-15β-yl]-N-[(pyridin-3-yl)methyl]acrylamide; 2-{[3-(2-bromoethyl)-17-oxostero-1(10),2,4-trien-15β-yl]oxy}-N-(5-methyl-1,3-thiazo-2-yl)acetamide; and 2-{[3-(2-bromoethyl)-17β-hydroxyestradiol-1(10),2,4-trien-15β-yl]oxy}-N-(5-methyl-1,3-thiazolyl-2-yl)acetamide; Or any salt or stereoisomer thereof.

23. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 22 and one or more pharmaceutically acceptable excipients or carriers.

24. A reagent kit comprising: (i) the compound as defined in any one of claims 1 to 22 or the pharmaceutical composition as described in claim 23, and (ii) Detectable markers.

25. A method for inhibiting the 17β-HSD1 enzyme in a subject, the method comprising the step of administering to the subject in need a therapeutically effective amount of a compound of formula (I) as defined in any one of claims 1 to 22 or a pharmaceutical composition as defined in claim 23.

26. A method for treating and / or preventing estrogen-dependent diseases or conditions, the method comprising the step of administering to a subject in need a therapeutically effective amount of a compound of formula (I) as defined in any one of claims 1 to 22 or a pharmaceutical composition as defined in claim 23.

27. The method of claim 25 or 26, wherein the subject suffers from malignant steroid dependence or a condition.

28. The method of claim 27, wherein the malignant steroid-dependent disease or condition is cancer.

29. The method of claim 25 or 26, wherein the subject suffers from a benign steroid dependence disease or condition.

30. The method of claim 29, wherein the benign steroid-dependent disease or condition includes endometriosis, uterine fibroids, uterine leiomyomas, adenomyosis, dysmenorrhea, menorrhagia, uterine bleeding, prostatodynia, benign prostatic hyperplasia, urinary dysfunction, polycystic ovary syndrome, lower urinary tract syndrome, multiple sclerosis, obesity, rheumatoid arthritis, colon cancer, tissue trauma, skin wrinkles, or cataracts.

31. The method according to any one of claims 25 to 30, wherein the compound of formula (I) is administered in combination with one or more other therapeutic agents suitable for treating steroid-dependent diseases.

32. Use of a compound of formula (I) as defined in any one of claims 1 to 22 or a pharmaceutical composition as defined in claim 23 in the inhibition of the 17β-HSD1 enzyme.

33. Use of a compound of formula (I) as defined in any one of claims 1 to 22 or a pharmaceutical composition as defined in claim 23 for the treatment and / or prevention of an estrogen-dependent disease or condition in a subject.

34. The use according to claim 32 or 33, wherein the subject suffers from malignant steroid dependence disease or condition.

35. The use according to claim 34, wherein the malignant steroid-dependent disease or condition is cancer.

36. The use according to claim 32 or 33, wherein the subject suffers from a benign steroid-dependent disease or condition.

37. The use according to claim 36, wherein the benign steroid-dependent disease or condition includes endometriosis, uterine fibroids, uterine leiomyomas, adenomyosis, dysmenorrhea, menorrhagia, uterine bleeding, prostatodynia, benign prostatic hyperplasia, urinary dysfunction, polycystic ovary syndrome, lower urinary tract syndrome, multiple sclerosis, obesity, rheumatoid arthritis, colon cancer, tissue trauma, skin wrinkles, or cataracts.

38. The use according to any one of claims 32 to 37, in combination with one or more other therapeutic agents suitable for treating steroid-dependent diseases.

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