EP4646421A1 - Fluoro-oxysterols positron emission tomography (pet) radiotracers - Google Patents

Abstract

Radiopharmaceuticals-based biomedical imaging plays a growing role in cancer management, including diagnosis, tumor staging and aggressiveness and treatment monitoring. Even though ¹⁸Fluoro-2-deoxy glucose and ¹⁸Fluoroestradiol are currently used for some types of breast cancer, a need remains to develop other candidate compounds which are useful as radiotracers in a different/ broader range of cancer types. Thus, it is described a family of ¹⁸F radiolabeled compounds of formula (I) (wherein one of R2, R4, R5, R6, R7 and R12 is ¹⁸F) for use as Positron Emission Tomography imaging agents for the detection of cancers and metastatic sites thereof, as well as for monitoring and predicting the efficacy of a cancer treatment, wherein the cancer is associated to cholesterol-5,6- epoxide hydrolase and/or lip-hydroxy steroid dehydrogenase type (2) overexpression. To overcome the issues linked to the lifetime of the radioisotope ¹⁸F and, to ensure an effective synthesis of the ¹⁸F radiolabeled compounds of formula (I), a group of novel intermediate compounds in the synthesis thereof is also described.

Description

FLUORO-OXYSTEROLS POSITRON EMISSION TOMOGRAPHY (PET)

RADIOTRACERS

The present invention refers to novel ¹⁸F-radiolabeled compounds which are precursors of the oncometabolite cholestan-6-oxo-3p,5a-diol (oncosterone, OCDO), as well as to a group of novel intermediate compounds in the synthesis of said ¹⁸F -radiolab eled compounds. Present invention also refers to a process to obtain said ¹⁸F-radiolabeled compounds. Said ¹⁸F -radiolab eled compounds are useful in diagnostic methods and, in particular as imaging compounds for use in diagnostic methods using Positron Emission Tomography (PET).

BACKGROUND OF THE INVENTION

Breast cancer (BC) remains the leading cause of cancer related mortality in women with approximately 0.6 million of deaths in 2020. Targeted therapies, such as Tamoxifen (Tam) for treating tumors expressing the estrogen receptor (ER), or agents that target the overexpressed growth factor receptor HER2 (human epidermal growth factor receptor) have strongly contributed to improve the outcomes of women with primary BC. However, intrinsic and acquired resistance constitutes a major limitation to the beneficial effect of therapies currently used and, in addition, no effective targeted therapies are currently available for triple negative breast cancer (TNBC), a cancer subtype which does not express ER, progesterone receptor (PR) and HER2. Consequently, the discovery of innovative therapeutic strategies, as well as biomarkers, for the detection of BC and prediction of treatment response are major health concerns.

It has been reported that a particular cholesterol pathway is deregulated in BC. This deregulated cholesterol pathway relays on the transformation of 5a,6a-epoxycholestanols (5,6a-EC) and 5p,6P-epoxycholestanols (5,6P-EC) that are two oxygenation products of cholesterol. It was reported that on normal breast 5,6a-EC can be metabolized into a steroidal alkaloid named dendrogenin A (DDA) which displays tumor suppressor properties. However, in BC, 5,6-EC are mainly metabolized into cholestane-3p,5a,6P-triol (CT) by the cholesterol-5,6-epoxide hydrolase (ChEH), an enzymatic complex formed by two cholesterogenic enzymes, 7-dehydrocholesterol reductase (DHCR7) and 3P-hydroxysteroid- A8,A7-isomerase (D8D7I or EBP). Subsequently CT is further metabolized into 6-oxo- chestan-3p,5a-diol (cholestan-6-oxo-3p,5a-diol, OCDO, Oncosterone) by the l ip- hydroxysteroid-dehydrogenase-type II (HSD2). OCDO was shown to be an oncometabolite (i.e. metabolite linked to cancer onset and development) in BC and to display tumor promoter properties, stimulating the growth and the invasiveness of mouse and human ER(+) and triple negative BC cells through the modulation of glucocorticoid receptor (GR) and liver-X-Receptor (LXR).

Radiopharmaceuticals-based biomedical imaging is playing a growing role in health prevention and disease treatment. Indeed, radiopharmaceuticals-based biomedical imaging is being used in cancer management including diagnosis, tumor staging and aggressiveness and treatment monitoring, but also in other diseases. It has been described, for instance, the use of radiolabeled cholesterol analogues for imaging of adrenal adenomas and carcinomas of the adrenal gland also imaging of plaques on walls of arteries, in subjects suffering, or suspected of suffering from conditions featuring excessive uptake and storage of cholesterol, such as Cushing’s syndrome, primary aldosteronism, hyperandrogenism, adenoma, gonadal disease, pheochromocytoma, an atherosclerotic disease, a disorder of cholesterol metabolism and distribution, or ectopic cholesterol production. Other examples include Fluoride- 18 based positron emission tomography radiotracers such as [¹⁸F]-fluoro-2-deoxyglucose (¹⁸FDG; a marker of glycolysis) and 16 alpha-[¹⁸F]-fluoroestradiol (¹⁸FES; an estrogen receptor (ER) ligand), which are currently used for patients suffering from BC. ¹⁸FDG is notably used for metastatic examination whereas ¹⁸FES allows the detection of functional ER in vivo and is used as a predictor of response to endocrine therapy in patients with advanced or metastatic ER-positive breast cancer.

In that regard, there is a clear need for candidate compounds which may be used in the diagnosis and detection of cancer types for which the development of biomarkers for the detection thereof are not currently available.

SUMMARY OF THE INVENTION

One aspect of the invention therefore relates to a ¹⁸F-radiolabeled compound of formula (I): wherein:

R³ is H or OH; and R², R⁴, R⁵, R⁶, R⁷ and R¹² are independently selected from ¹⁸F or H;

R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide; wherein, when R² is ¹⁸F, then R³ is OH and all R⁴, R⁵, R⁶, R⁷ and R¹² are H; when R⁴ is ¹⁸F, then R⁸ and R⁹ are each OH and all R², R³, R⁵, R⁶, R⁷ and R¹² are H; when R⁵ is ¹⁸F, then R³ is OH and all R², R⁴, R⁶, R⁷ and R¹² are H; when R⁶ is ¹⁸F, then R³ is OH and all R², R⁴, R⁵, R⁷ and R¹² are H; when R⁷ is ¹⁸F, then R³ is OH and all R², R⁴, R⁵, R⁶ and R¹² are H; and when R¹² is ¹⁸F, then R³ is OH and all R², R⁴, R⁵, R⁶ and R⁷ are H; and wherein at least one of R², R⁴, R⁵, R⁶, R⁷ and R¹² is a ¹⁸F.

Another aspect of the present invention refers to a composition comprising a ¹⁸F- radiolabeled compound of formula (I), as described in the present invention, and one or more pharmaceutically acceptable excipients.

The structure of the ¹⁸F-radiolabeled compounds of formula (I), as described in the present invention, makes them useful as PET imaging agents for the detection of cancers and tumors, as well as the detection of possible metastatic sites, and additionally for monitoring the treatment of a cancer, wherein said cancers and tumors are associated to the overexpression of the enzymes ChEH and/or HSD2, overexpression resulting into oncosterone biogenesis, which is a metabolite linked to cancer onset and development.

Another additional aspect of the present invention refers to an intermediate compound in the synthesis of a ¹⁸F-radiolabeled compound of formula (I), according to the present invention, wherein said intermediate compound has formula (II): wherein:

R¹, R⁵, R⁶, R⁷ and R¹² are independently selected from a group R¹⁰ or H;

R³ is independently selected from a group R¹⁰ or OH;

R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide; and

R¹⁰ is selected from the group consisting of a mesylate group, a tosyl group and a triflate group; wherein when R¹ is a group R¹⁰, then R³ is OH and all R⁵, R⁶, R⁷ and R¹² are H; when R³ is a group R¹⁰, then R⁸ and R⁹ are each OH, all R¹, R⁵, R⁶, R⁷ and R¹² are H, being R¹⁰ a mesylate group or a triflate group; when R⁵ is a group R¹⁰, then R³ is OH and all R¹, R⁶, R⁷ and R¹² are H; when R⁶ is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁷ and R¹² are H; when R⁷ is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁶ and R¹² are H;and when R¹² is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁶ and R⁷ are H; and wherein at least one of R¹, R³, R⁵, R⁶, R⁷ and R¹² is a group R¹⁰.

Yet another aspect of the invention refers to a ¹⁸F-radiolabeled compound of formula (I) or of a composition comprising the same according to the present invention, for use in a method for detecting the presence of a cancer in a patient, or in a method for detecting the presence of metastatic sites in a patient suffering from a primary tumor, wherein the cancer and tumor are associated to overexpression of cholesterol-5,6-epoxide hydrolase (ChEH) and/orl ip- hydroxysteroid dehydrogenase type 2 (HSD2); and wherein said method comprises the steps of: administering the ¹⁸F -radiolab eled compound of formula (I) or a composition comprising the same to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal.

Another further aspect of the invention refers to a ¹⁸F-radiolabeled compound of formula (I) or a composition comprising the same according to the present invention, for use in a method for monitoring the efficacy of a treatment administered to a patient suffering from a cancer associated to overexpression of cholesterol-5,6-epoxide hydrolase (ChEH) and/or l ip- hydroxysteroid dehydrogenase type 2 (HSD2); wherein said method comprises the steps of: administering the ¹⁸F -radiolab eled compound of formula (I) or a composition comprising the same to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal; and repeating said steps at different moments of the treatment.

Another further aspect of the invention refers to a ¹⁸F-radiolabeled compound of formula (I) or a composition comprising the same according to the present invention, for use in a method for predicting the efficacy of a treatment targeting oncosterone biogenesis or with a treatment targeting an oncosterone downstream effector, wherein said method comprises the steps of: administering the ¹⁸F -radiolab eled compound of formula (I) or a composition comprising the same according to the present invention, to a patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting a positron emission tomography (PET) signal; administering to the patient said treatment targeting oncosterone biogenesis or said treatment targeting an oncosterone downstream effector when the PET signal collected detects a cancer in said patient; wherein the treatment targeting oncosterone biosynthesis or biogenesis is an anti-estrogen binding site (AEBS) ligand, an EBP inhibitor, a DHCR7 inhibitor, or a ChEH inhibitor; and wherein the treatment targeting an oncosterone downstream effector is a glucocorticoid receptor (NR3C1) modulator, or a Liver-X-Receptor (NR1H2, NR1H3) modulator.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have now succeeded in developing a family of new ¹⁸F-radiolabeled compounds which are useful as PET imaging agents and can be used for the detection of cancers and tumors, as well as the detection of possible metastatic sites, for monitoring the treatment of a cancer, or even for predicting the efficacy of a cancer treatment, wherein said cancers and tumors are associated to ChEH and/or HSD2 overexpression.

As used herein, the term “PET” refers to Positron Emission Tomography, which is an imaging technique.

¹⁸F -radiolab eled compounds

In that regard, one aspect of the invention therefore relates to a ¹⁸F -radiolab eled compound of formula (I): wherein:

R³ is H or OH; and R², R⁴, R⁵, R⁶, R⁷ and R¹² are independently selected from ¹⁸F or H;

R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide; wherein, when R² is ¹⁸F, then R³ is OH and all R⁴, R⁵, R⁶, R⁷ and R¹² are H; when R⁴ is ¹⁸F, then R⁸ and R⁹ are each OH and all R², R³, R⁵, R⁶, R⁷ and R¹² are H; when R⁵ is ¹⁸F, then R³ is OH and all R², R⁴, R⁶, R⁷ and R¹² are H; when R⁶ is ¹⁸F, then R³ is OH and all R², R⁴, R⁵, R⁷ and R¹² are H; and when R⁷ is ¹⁸F, then R³ is OH and all R², R⁴, R⁵, R⁶ and R¹² are H; and when R¹² is ¹⁸F, then R³ is OH and all R², R⁴, R⁵, R⁶ and R⁷ are H; and wherein at least one of R², R⁴, R⁵, R⁶, R⁷ and R¹² is a ¹⁸F.

Said compounds of formula (I) are imaging agents, for PET imaging techniques, which are able to target a cancer associated to ChEH and/or HSD2 overexpression.

In some aspects of the invention, the compounds of formula I are those wherein:

R² is ¹⁸F, R³ is OH, all R⁴, R⁵, R⁶, R⁷ and R¹² are H, and R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide; or wherein

R⁴ is ¹⁸F, R⁸ and R⁹ are each OH and all R², R³, R⁵, R⁶, R⁷ and R¹² are H;

In other aspects of the invention the compounds of formula I are those wherein R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide, and wherein:

R⁵ is ¹⁸F, R³ is OH and all R², R⁴, R⁶, R⁷ and R¹² are H; or

R⁶ is ¹⁸F, R³ is OH and all R², R⁴, R⁵, R⁷ and R¹² are H; or

R⁷ is ¹⁸F, R³ is OH and all R², R⁴, R⁵, R⁶ and R¹² are H; or

R¹² is ¹⁸F, R³ is OH and all R², R⁴, R⁵, R⁶ and R⁷ are H. More preferred compounds of formula I are those wherein R² is ¹⁸F, R³ is OH, all of R⁴, R⁵, R⁶, R⁷ and R¹² are H, and R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide.

An even more preferred compound of formula I is that wherein R² is ¹⁸F, R³ is OH, all of R⁴, R⁵, R⁶, R⁷ and R¹² are H and R⁸ and R⁹ are each OH.

Particularly preferred compounds for use according to the invention are those listed in Table 1 hereafter: Compositions

Another aspect of the invention refers to a composition comprising a ¹⁸F-radiolabeled compound of formula (I), as described in the present invention, and one or more pharmaceutically acceptable excipients.

The term “excipient” as used herein means a substance formulated alongside the active agent or active ingredient in a pharmaceutical composition or medicament. Acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, 21^st Edition 2011. The choice of excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice. The excipient must be acceptable in the sense of being not deleterious to the recipient thereof. The at least one pharmaceutically acceptable excipient may be for example, a binder, a stabilizer, an antioxidant, a diluent, a carrier, a lubricant, a disintegrator, a wetting agent, a dispersing agent, a suspending agent, and the like.

The term “pharmaceutical vehicle” as used herein means a carrier or inert medium used as solvent or diluent in which the pharmaceutically active agent is formulated and/or administered.

In some aspects of the invention, said pharmaceutically acceptable excipient may selected among one or more pharmaceutically acceptable solvents, vehicles or stabilizers. In a particular aspect of the invention said stabilizer is an antioxidant that allows improving the stability of the ¹⁸F-radiolabeled compounds of formula (I), as described in the present invention, against radiolysis. Examples of stabilizers or antioxidants useful in the context of the present invention are ascorbate, gentisic acid, vitamin E, N-acetyl cysteine, butylated hydroxyl toluene and N-tert-Butyl-a-phenylnitrones.

Positron Emission Tomography (PET)

The level of oncosterone (cholestan-6-oxo-3p,5a-diol, OCDO) as well as the expression levels of the enzyme HSD2 and of EBP, the catalytic subunit of ChEH, was found very low in normal adjacent tissues (NAT) to tumors, and HSD2 was found absent from normal cells constitutives of the breast, showing that HSD2 appeared during BC carcinogenesis. In addition, the analysis of transcriptomic databases showed that the overexpression of the enzymes HSD2 and ChEH, responsible for the synthesis of OCDO, are associated with a bad prognosis in several cancer types (ref. 7), in addition to BC. Moreover, the expression of OCDO forming enzymes HSD2 and ChEH is negatively correlated with patient survival.

In that regard, the structure of the ¹⁸F -radiolab eled compounds described herein makes them useful specifically and can be used, as well as the compositions comprising the same described above herein, as PET imaging agents for the detection of cancers and tumors, as well as the detection of possible metastatic sites, and additionally for monitoring the treatment of a cancer, wherein said cancers and tumors are associated to ChEH and/or HSD2 overexpression.

The term “cancers and tumors associated to ChEH and/or HSD2 overexpression” as included in the present disclosure refers to cancers and tumors which are associated to overexpression of one or more of HSD2 and ChEH enzymes and, said overexpression resulting into oncosterone biogenesis.

In some embodiments, the cancer or tumor associated to cholesterol-5,6-epoxide hydrolase (ChEH) and/or 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression may be selected from the group consisting of cancer of the skin, tissues, organs, bone, cartilage, blood and vessels. In some embodiments, the cancer is a bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, colon cancer, esophagus cancer, gastrointestinal cancer, gum cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharynx cancer, neck cancer, ovary cancer, prostate cancer, skin cancer, stomach cancer, testis cancer, tongue cancer, thyroid cancer and uterus cancer. In some embodiments, the cancer is breast cancer, leukemia, thyroid cancer or a melanoma. In some embodiments, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; non-encapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extramammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

In some embodiments, the cancer or tumor associated to cholesterol-5,6-epoxide hydrolase (ChEH) and/or 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression may be selected from the group consisting of urothelial bladder cancer, breast cancer, invasive breast cancer, adrenal gland cancer, leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, esophageal carcinoma, chromophobe renal cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous cystadenoma, pancreatic adenocarcinoma, rectal adenocarcinoma, uterine corpus endometrial carcinoma, stomach adenocarcinoma, esophageal carcinoma, colon adenocarcinoma, prostate adenocarcinoma, testicular germ cells tumors, thyroid cancer, thyroid carcinoma, uterine carcinosarcoma, melanoma and osteosarcoma.

In a preferred aspect of the invention the cancer or tumor associated to cholesterol-5,6- epoxide hydrolase (ChEH) and/or 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression may be selected from the group consisting of urothelial bladder cancer, invasive breast cancer, adrenal gland cancer, acute myeloid leukemia, chromophobe renal cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous cystadenoma, pancreatic adenocarcinoma, rectal adenocarcinoma, uterine corpus endometrial carcinoma, acute lymphoblastic leukemia and osteosarcoma, stomach adenocarcinoma, esophageal carcinoma, colon adenocarcinoma, prostate adenocarcinoma, testicular germ cells tumors, thyroid carcinoma and uterine carcinosarcoma.

In an aspect of the invention the cancer or tumor associated to cholesterol-5,6-epoxide hydrolase (ChEH) and 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression may be selected from the group consisting of urothelial bladder cancer, invasive breast cancer, adrenal gland cancer, acute myeloid leukemia, chromophobe renal cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous cystadenoma, pancreatic adenocarcinoma, rectal adenocarcinoma, uterine corpus endometrial carcinoma, acute lymphoblastic leukemia and osteosarcoma, and stomach adenocarcinoma.

In another aspect of the invention the cancer or tumor associated to l ip-hydroxysteroid dehydrogenase type 2 (HSD2) overexpression is esophageal carcinoma.

In yet another aspect of the invention the cancer or tumor associated to cholesterol-5,6- epoxide hydrolase (ChEH) may be selected from the group consisting of colon adenocarcinoma, prostate adenocarcinoma, testicular germ cells tumors, thyroid carcinoma and uterine carcinosarcoma.

In a preferred embodiment the cancer or tumor associated to cholesterol-5,6-epoxide hydrolase (ChEH) and/or 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression is breast cancer or invasive breast cancer.

The compounds of formula I are ¹⁸F -radiolab eled precursors of the oncometabolite cholestan-6-oxo-3p,5a-diol (oncosterone). Since the overexpression of enzymes HSD2 and/or ChEH results in the production of the oncometabolite oncosterone, the uptake of the ¹⁸F -radiolab eled compounds of formula I may be detected in those tumor tissue where oncosterone is produced.

This makes the ¹⁸F -radiolab eled compounds of formula I particularly useful in methods for diagnosing the status and dissemination of a cancer or tumor, detecting metastatic sites and assess the efficacy of a treatment being administered to a patient of said cancer or tumor, by PET imaging.

According to the present invention the term “presence of a cancer” refers to the detection of cancer cells in the different tissues of the patient. On the other hand, the term “presence of a metastatic site” refers to the spread of cancer cells from a primary tumor (i.e. the first tumor site) where they were first formed to another part of the body which is called, thus, a metastatic site. The new metastatic site is the same type of cancer as the primary tumor.

The term “cancer or tumor associated to 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression” refers to a tumor or a cancer tissue featuring an increased expression of HSD2 in tumor compared to normal adjacent normal tissues or to normal samples from non- cancerous patients (ref. 7).

The term “cancer or tumor associated to cholesterol-5,6-epoxide hydrolase (ChEH) overexpression refers to a tumor or a cancer tissue featuring an increased expression of ChEH subunits (EBP and/or DHCR7) when compared to normal adjacent normal tissues or to normal samples from non-cancerous patients (ref. 7).

Accordingly, another aspect of present invention refers to a ¹⁸F-radiolabeled compound of formula (I) or a composition comprising the same according to the present invention, for use in a method for detecting the presence of a cancer or tumor in a patient, or detecting the presence of metastatic sites in a patient suffering from a primary tumor, wherein the cancer and tumor are associated to cholesterol-5,6-epoxide hydrolase (ChEH) and/or l ip- hydroxysteroid dehydrogenase type 2 (HSD2) overexpression; and wherein said method comprises the steps of: administering the ¹⁸F -radiolab eled compound of formula (I) or a composition comprising the same, to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal.

In other terms the present invention refers to a method for detecting the presence of a cancer or tumor in a patient, or detecting the presence of metastatic sites in a patient suffering from a primary tumor, wherein the cancer and tumor are associated to cholesterol-5,6-epoxide hydrolase (ChEH) and/or 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression; and wherein said method comprises the steps of: administering a ¹⁸F-radiolabeled compound of formula (I) or a composition comprising the same according to the present invention, to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal.

According to a further feature of the present invention, there is provided the use of a compound of formula (I) or a composition comprising the same according to the present invention for detecting the presence of a cancer or tumor in a patient, or detecting the presence of metastatic sites in a patient suffering from a primary tumor, wherein the cancer and tumor are associated to cholesterol-5,6-epoxide hydrolase (ChEH) and/or l ip- hydroxysteroid dehydrogenase type 2 (HSD2) overexpression; and wherein said method comprises the steps of: administering a ¹⁸F-radiolabeled compound of formula (I) or a composition comprising the same according to the present invention to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal.

Another aspect of present invention refers to a ¹⁸F-radiolabeled compound of formula (I) or a composition comprising the same according to the present invention for use in a method for monitoring the efficacy of a treatment administered to a patient suffering from a cancer associated to cholesterol-5,6-epoxide hydrolase (ChEH) and/or l ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression; wherein said method comprises the steps of: administering the ¹⁸F -radiolab eled compound of formula (I) or a composition comprising the same to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal; and repeating said steps at different moments of the treatment.

In other words, the present invention refers to a method for monitoring the efficacy of a treatment administered to a patient suffering from a cancer associated to cholesterol-5,6- epoxide hydrolase (ChEH) and/or 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression; wherein said method comprises the steps of: administering a ¹⁸F-radiolabeled compound of formula (I) or a composition comprising the same according to the present invention to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal; and repeating said steps at different moments of the treatment. Present invention also provides the use of a compound of formula (I) or a composition comprising the same according to the present invention for monitoring the efficacy of a treatment administered to a patient suffering from a cancer associated to cholesterol-5,6- epoxide hydrolase (ChEH) and/or 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression; wherein said method comprises the steps of: administering a ¹⁸F-radiolabeled compound of formula (I) or a composition comprising the same according to the present invention to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal; and repeating said steps at different moments of the treatment.

In particular, the treatment may be a chemotherapeutic treatment, a hormone therapy, an immunotherapy, a stem cell transplant, radiation therapy, photodynamic therapy, or a combination thereof, among others.

Preferably said method for monitoring the efficacy of a treatment administered to a patient suffering from a cancer comprises repeating the steps of the method prior and after the treatment has been administered. Also preferably, said method comprises repeating the steps of the method at different time intervals prior, during and after the treatment has been administered. Said time intervals may be a fixed time interval or a variable time interval of, for example, after 1, 2, 3, 5 or more days, 1, 2, 3 or more weeks, 1, 2, 3 or more months.

Another further aspect of the invention refers to a ¹⁸F-radiolabeled compound of formula (I) or a composition comprising the same according to the present invention, for use in a method for predicting the efficacy of a treatment targeting oncosterone biogenesis or of a treatment targeting an oncosterone downstream effector, wherein said method comprises the steps of: administering the ¹⁸F -radiolab eled compound of formula (I) or a composition comprising the same according to the present invention, to a patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting a positron emission tomography (PET) signal; administering to the patient said treatment targeting oncosterone biogenesis or said treatment targeting an oncosterone downstream effector when the PET signal collected detects a cancer in said patient; wherein the treatment targeting oncosterone biogenesis is an anti-estrogen binding site (AEBS) ligand, an EBP inhibitor, a DHCR7 inhibitor, or a ChEH inhibitor; and wherein the treatment targeting an oncosterone downstream effector is a glucocorticoid receptor (NR3C1) modulator, or a Liver-X-Receptor (NR1H2, NR1H3) modulator.

In other words, the present invention refers to a method for predicting the efficacy of a treatment targeting oncosterone biogenesis or of a treatment targeting an oncosterone downstream effector, wherein said method comprises the steps of: administering the ¹⁸F -radiolab eled compound of formula (I) or a composition comprising the same according to the present invention, to a patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting a positron emission tomography (PET) signal; administering to the patient said treatment targeting oncosterone biogenesis or said treatment targeting an oncosterone downstream effector when the PET signal collected detects a cancer in said patient; wherein the treatment targeting oncosterone biosynthesis or biogenesis is an anti-estrogen binding site (AEBS) ligand, an EBP inhibitor, a DHCR7 inhibitor, or a ChEH inhibitor; and wherein the treatment targeting an oncosterone downstream effector is a glucocorticoid receptor (NR3C1) modulator, or a Liver-X-Receptor (NR1H2, NR1H3) modulator.

Present invention also provides the use of a ¹⁸F -radiolab eled compound of formula (I) or a composition comprising the same according to the present invention for predicting the efficacy of a treatment targeting oncosterone biogenesis or with a treatment targeting an oncosterone downstream effector, wherein said method comprises the steps of: administering the ¹⁸F -radiolab eled compound of formula (I) or a composition comprising the same according to the present invention, to a patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting a positron emission tomography (PET) signal; administering to the patient said treatment targeting oncosterone biogenesis or said treatment targeting an oncosterone downstream effector when the PET signal collected detects a cancer in said patient; wherein the treatment targeting oncosterone biosynthesis or biogenesis is an anti-estrogen binding site (AEBS) ligand, an EBP inhibitor, a DHCR7 inhibitor, or a ChEH inhibitor; and wherein the treatment targeting an oncosterone downstream effector is a glucocorticoid receptor (NR3C1) modulator, or a Liver-X-Receptor (NR1H2, NR1H3) modulator.

The term “treatment targeting oncosterone biosynthesis” or “treatment targeting oncosterone biogenesis” refers, according to the present invention, to a treatment which, when administered to a patient suffering from a cancer, lowers the amount of oncosterone generated by the patient.

In some aspects of the present invention, the said treatment targeting oncosterone biosynthesis or biogenesis is an anti-estrogen binding site (AEBS) ligand, an EBP inhibitor, a DHCR7 inhibitor, or a ChEH inhibitor, in particular, selected from the group consisting of tamoxifen, tesmilifene, raloxifen, terbinafine, haloperidol, trifluoroperazine, clomiphene, toremifene DSP-0390, TASIN (Truncated APC-Selective Inhibitors) compounds, dendrogenin A (DDA), PBPE (l-[2-[4-(phenylmethyl)phenoxy]ethyl]-pyrrolidine), perospirone, nefazodone, aripiprazole, trazodone buspirone, fluoxetine, risperidone, AY9944, BM15766 and cariprazine (see ref. 16 and 17).

On the other hand, the term “treatment targeting an oncosterone downstream effector” refers, according to the present invention, to compounds which directly bind downstream effectors of OCDO (GR or LXR ligands) and therefore inhibit the tumor promoter properties of OCDO that involve onconsterone downstream effectors; or to compounds that may promote the degradation of nuclear receptors involved in oncosterone protumor activities, such as, for example Glucocorticoid receptor Proteolysis targeting chimeras (PROTACs).

In some aspects of the present invention, the said treatment targeting an oncosterone downstream effector is a glucocorticoid receptor (NR3C1) modulator, or a Liver-X-Receptor (NR1H2, NR1H3) modulator. In some aspects of the invention, said glucocorticoid receptor (NR3C 1) modulator is selected from the group consisting of CORT108297, Fosdagrocorat, AZD5423, AZD7594, AZD9567, LEO 134310, Mapracorat and Mifepristone.

In other aspects of the invention, said Liver-X-Receptor (NR1H2, NR1H3) modulator is selected from the group consisting of GW3965, TO091317, LXR-623, flavonoids, betasitosterol, fucosterol, campesterol, GSK3987, riccardin, saringosterol, RGX-104 and GSK2033.

Preferably, in some aspects, the treatment targeting oncosterone biogenesis is an antiestrogen binding site (AEBS) ligand, an EBP inhibitor, a DHCR7 inhibitor, or a ChEH inhibitor, selected from the group consisting of tamoxifen, tesmilifene, raloxifene, terbinafine, haloperidol and trifluoroperazine, clomiphene, toremifene, DSP-0390 and TASIN (Truncated APC-Selective Inhibitors) compounds, dendrogenin A (DDA), PBPE (1- [2-[4-(phenylmethyl)phenoxy]ethyl]-pyrrolidine), perospirone, nefazodone, aripiprazole, trazodone buspirone, fluoxetine, risperidone, AY9944, BM 15766 and cariprazine; and the treatments targeting an oncosterone downstream effector is a glucocorticoid receptor (NR3C1) modulator selected from the group consisting of CORT108297, Fosdagrocorat, AZD5423, AZD7594, AZD9567, LEO 134310, Mapracorat and Mifepristone; or a Liver-X- Receptor (NR1H2, NR1H3) modulator selected from the group consisting of GW3965, TO091317, LXR-623, flavonoids, beta-sitosterol, fucosterol, campesterol, GSK3987, riccardin, saringosterol, RGX-104, dendrogenins and GSK2033.

Preferably the method comprises subjecting said patient to a positron emission tomography (PET) scanner combined with a computer tomography (CT) scanner.

The term “patient” refers to a warm-blooded animal, more preferably a human, who/which is awaiting or receiving medical care or is or will be the object of a medical procedure.

The term “human” refers to subjects of both genders and at any stage of development (i.e. neonate, infant, juvenile, adolescent, adult). In one embodiment, the human is an adolescent or adult, preferably an adult.

The term “administration”, or a variant thereof (e.g. “administering”), means providing the active agent or active ingredient (compound of formula I or the cancer treatment), alone or as part of a pharmaceutically acceptable composition, to the patient. By “pharmaceutically acceptable” is meant that the ingredients of a pharmaceutical composition are compatible with each other and not deleterious to the patient thereof.

The methods for detecting the presence of a cancer in a patient or detecting the presence of metastatic sites in a patient suffering from a primary tumor, and the methods for monitoring the efficacy of a treatment administered to a patient suffering from a cancer, comprise administering a ¹⁸F -radiolab eled compound of formula (I) according to the present invention to said patient, alone or as part of a pharmaceutically acceptable composition.

In a particular embodiment, the compound of formula (I) as previously defined is administered to said patient, alone or as part of a pharmaceutically acceptable composition, by intra-venous, subcutaneous or oral route. Preferably the compound of formula (I) as previously defined is administered to said patient, alone or as part of a pharmaceutically acceptable composition, by intra-venous route.

Such suitable administration forms as well as methods and carriers, diluents and excipients for use in the preparation thereof, will be clear to the skilled person; reference is made to the latest edition of Remington’s Pharmaceutical Sciences.

Intermediate compounds of formula

On the other hand, since the compounds of formula I of present invention contain the fluorine radioisotope ¹⁸F which decays with a half-life of less than 2 hours (ti/2= HO min), it is important to be able to develop intermediate compounds which are stable, and which allow to obtain the ¹⁸F-radioprobes of formula I relatively fast and with high yields.

In that regard, another aspect of the present invention refers to an intermediate compound in the synthesis of a ¹⁸F -radiolab eled compound of formula (I) according to claim 1, wherein said intermediate compound has formula (II):

wherein:

R¹, R⁵, R⁶, R⁷ and R¹² are independently selected from a group R¹⁰ or H; R³ is independently selected from a group R¹⁰ or OH;

R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide; and

R¹⁰ is selected from the group consisting of a mesylate group, a tosyl group and a tritiate group; wherein when R¹ is a group R¹⁰, then R³ is OH and all R⁵, R⁶, R⁷ and R¹² are H; when R³ is a group R¹⁰, then R⁸ and R⁹ are each OH, and all R¹, R⁵, R⁶, R⁷ and R¹² are

H, being R¹⁰ is a mesylate group or a tritiate group; when R⁵ is a group R¹⁰, then R³ is OH and all R¹, R⁶, R⁷ and R¹² are H; when R⁶ is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁷ and R¹² are H; and when R⁷ is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁶ and R¹² are H; when R¹² is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁶ and R⁷ are H; and wherein at least one of R¹, R³, R⁵, R⁶, R⁷ and R¹² is a group R¹⁰.

Preferred compounds of formula II are those wherein R¹ is a group R¹⁰, R³ is OH, all R⁵, R⁶, R⁷ and R¹² are H, R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide; and R¹⁰ is selected from the group consisting of a mesylate group, a tosyl group and a triflate group; or

R³ is a group R¹⁰, R⁸ and R⁹ are each OH, all R¹, R⁵, R⁶, R⁷ and R¹² are H, R⁸ and R⁹ are each OH and R¹⁰ is a mesylate group or a triflate group.

More preferred compounds of formula II are those wherein R¹ is a group R¹⁰, R³ is OH, all R⁵, R⁶, R⁷ and R¹² are H; R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide, and R¹⁰ is selected from the group consisting of a mesylate group, a tosyl group and a triflate group.

Even more preferred compounds are those wherein R¹ is a group R¹⁰, R³ is OH, all R⁵, R⁶, R⁷ and R¹² are H; R⁸ and R⁹ are each OH; and R¹⁰ is selected from the group consisting of a mesylate group, a tosyl group and a triflate group.

Also preferred compounds of formula II are those wherein

R⁵ is a group R¹⁰, R³ is OH and all R¹, R⁶, R⁷ and R¹² are H; or

R⁶ is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁷ and R¹² are H; or

R⁷ is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁶ and R¹² are H; or

R¹² is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁶ and R⁷ are H; and wherein R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide; and R¹⁰ is selected from the group consisting of a mesylate group, a tosyl group and a triflate group.

In a particularly preferred embodiment R¹⁰ is a mesylate group.

Particularly preferred intermediate compounds of formula II according to the invention are those listed in Table 2 hereafter:

Methods to obtain the intermediate compounds of formula (II) and the 18-Fluorinated compounds of formula (I).

Said intermediate compounds of formula II may be obtained according to a method disclosed in the present invention, wherein said method comprises: a. providing a compound of formula (Ila) or a compound of formula (lib) : wherein R¹ , R⁵ , R⁶ , R⁷ and R¹² are independently selected from a group -OH or H;

R³ is independently selected from a group -OR¹¹ or -OH;

R¹¹ is an alcohol protecting group; and wherein when R³ is a group -OH, then all of R¹ , R⁵ , R⁶ and R⁷ are H and R¹² is H or OH; when R³ is a group -OR¹¹, then only one of R¹ , R⁵ , R⁶ , R⁷ and R¹² is -OH; b. activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group, a tosyl group and a triflate group when R³ is a group - OR¹¹ or when both R³ and R¹² are OH; or activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group and a triflate group when R³ is a group -OH; c. optionally conducting an epoxidation of a double bond; d. hydrolyzing the group -OR¹¹, when present, to obtain a group -OH; e. optionally hydrolyzing the epoxide when present; f. oxidizing the double bond, when present, to obtain a vicinal diol.

The steps of the method to obtain said intermediate of formula (II) may be carried out in different order and said method may, or may not, comprise all the steps, for example, depending on whether the steps are optional or not, and whether the group for which the step is carried out is present or not.

In other words, the method to obtain said intermediate of formula (II) above disclosed comprises: a. providing a compound of formula (Ila) or a compound of formula (lib) : wherein

R¹ , R⁵ , R⁶ , R⁷ and R¹² are independently selected from a group -OH or H;

R³ is independently selected from a group -OR¹¹ or -OH;

R¹¹ is an alcohol protecting group; and wherein when R³ is a group -OH, then all of R¹ , R⁵ , R⁶ and R⁷ are H and R¹² is H or OH; when R³ is a group -OR¹¹, then only one of R¹ , R⁵ , R⁶ , R⁷ and R¹² is -OH; b. activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group, a tosyl group and a triflate group when R³ is a group - OR¹¹ or when both R³ and R¹² are OH; or activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group and a triflate group when R³ is a group -OH; wherein when step (a) is providing a compound of formula (Ila) and a double bond is present, the method further comprises either:

(i) conducting the steps of; c. conducting an epoxidation of the double bond; d. hydrolyzing the group -OR¹¹, when present, to obtain a group -OH; and e. optionally hydrolyzing the epoxide; or (ii) conducting the steps of; d. hydrolyzing the group -OR¹¹, when present, to obtain a group -OH; and f. oxidizing the double bond to obtain a vicinal diol. and wherein when step (a) is providing a compound of formula (lib) and a double bond is not present conducting the steps of: d. hydrolyzing the group -OR¹¹, when present, to obtain a group -OH; e. optionally hydrolyzing the epoxide.

In some aspects of the invention, the method to obtain said intermediate of formula (II) comprises: a. providing a compound of formula (lib), wherein R³ is a group OH, and all of R¹ , R⁵ , R⁶ , R⁷ and R¹² are H; b. activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group and a triflate group; e. hydrolyzing the epoxide.

In other aspects of the invention, the method to obtain said intermediate of formula (II) comprises: a. providing a compound of formula (Ila), wherein R³ is a group -OR¹¹, R¹ , R⁶ , R⁷ and R¹² are H, and R⁵ is -OH; b. activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group, a tosyl group and a triflate group; c. conducting an epoxidation of a double bond; d. hydrolyzing the group -OR¹¹ to obtain a group -OH; e. optionally hydrolyzing the epoxide.

In some aspects of the invention step (c) is carried out prior to step (b). For example, in one aspect of the invention, the method to obtain said intermediate of formula (II) comprises: a. providing a compound of formula (Ila), wherein R³ is a group -OR¹¹, R¹ is OH, and R⁵ , R⁶ , R⁷ and R¹² are H; c. conducting an epoxidation of a double bond; b. activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group, a tosyl group and a triflate group; d. hydrolyzing the group -OR¹¹ to obtain a group -OH; e. optionally hydrolyzing the epoxide.

In other additional aspects of the invention step (d) is carried out prior to step (c), and the method to obtain said intermediate of formula (II) comprises: a. providing a compound of formula (Ila), wherein R³ is a group -OR¹¹, R¹ , R⁵ and R¹² are all H, and being one of R⁶ and R⁷ -OH and the other H; b. activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group, a tosyl group and a triflate group; d. hydrolyzing the group -OR¹¹ to obtain a group -OH; c. conducting an epoxidation of a double bond; e. optionally hydrolyzing the epoxide.

In other additional aspects of the invention, steps (c) and (e) are not present, and the method to obtain said intermediate of formula (II) comprises: a. providing a compound of formula (Ila), wherein R³ is a group -OR¹¹, R¹ , R⁵ and R¹² are all H, and being one of R⁶ and R⁷ -OH and the other H; b. activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group, a tosyl group and a triflate group; d. hydrolyzing the group -OR¹¹ to obtain a group -OH; f. oxidizing the double bond to obtain a vicinal diol.

In other aspects of the invention, steps (c) and (e) are not present, and the method to obtain said intermediate of formula (II) comprises: a. providing a compound of formula (Ila), wherein R³ is a group -OR¹¹, R¹ , R⁶ , R⁷ and R¹² are H, and R⁵ is -OH; b. activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group, a tosyl group and a triflate group; d. hydrolyzing the group -OR¹¹ to obtain a group -OH; f. oxidizing the double bond to obtain a vicinal diol.

In other aspects of the invention, the method to obtain said intermediate of formula (II) comprises: a. providing a compound of formula (Ila), wherein R³ is a group -OH, R¹ , R⁵ , R⁶ and R⁷ are H, and R¹² is -OH; b. activating the primary hydroxyl moiety with an alcohol activating group selected from the group consisting of a mesylate group, a tosyl group and a triflate group; and f. oxidizing the double bond to obtain a vicinal diol.

In yet other aspects of the invention, the method to obtain said intermediate of formula (II) comprises: a. providing a compound of formula (Ila), wherein R³ is a group -OH, R¹ , R⁵ , R⁶ and R⁷ are H, and R¹² is -OH; b. activating the primary hydroxyl moiety with an alcohol activating group selected from the group consisting of a mesylate group, a tosyl group and a triflate group; and c. conducting an epoxidation of the double bond; e. optionally hydrolyzing the epoxide.

The group R¹¹ may be any suitable alcohol protecting group as described in the literature, for instance by Greene and Wutts (Greene's Protective Groups in Organic Synthesis, 2006, Wiley&Sons, Inc.). In some aspects of the invention R¹¹ is an acyl group, a benzyl group or a silyl group. In some aspects of the invention R¹¹ is selected from the group consisting of acetyl, benzoyl, pivaloyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triisopropylsilyl, triethylsylil and benzyl. In a particular aspect of the invention R¹¹ is an acetyl or a triisopropyl silyl (TIPS) group. The activation of the hydroxyl group with an alcohol activating group may be carried out by any known method, according to the known literature, using a mesylate (methanesulfonate group), a tosylate (toluenesulfonyl group) or a tritiate (trifluoromethanesulfonate group) derivative such as a halide, preferably a chloride, or an anhydride, in the presence of a base, such as pyridine.

The hydrolysis of the epoxide may also be conducted by any methods according to the known literature. For example, said hydrolysis may be carried out in the presence of perchloric acid.

In some aspects of the present invention, R³ is a group -OR¹¹, R¹ is OH, and R⁵ , R⁶ , R⁷ and R¹² are H, said method to obtain an intermediate compound of formula II according to the present invention further comprises prior to step (a): conducting an allylic oxidation of a compound of formula (lie) to obtain an allylic alcohol: wherein R¹¹ is an alcohol protecting group as defined above herein; and conducting an epoxidation of the double bond to obtain a compound of formula (lib).

The allylic oxidation may be carried out by any suitable method according to the literature. In one example, the allylic oxidation comprises obtaining a keto derivative in allylic position and carrying out a Luche reduction of the keto derivative with sodium borohydride and CeCl₃.

The epoxidation of the double bond may also be carried out by any suitable method according to the literature. General methods for epoxidation include for example treatment with oxone® (potassium peroxymonosulfate) (see ref. 7), or with magnesium bis(monoperoxyphthalate) hexahydrate (see ref 8). In one example, said epoxidation is carried out in the presence of a peroxide, preferably a peroxide alcohol or a peroxycarboxylic acid. In some aspects of the invention said epoxidation is carried out in the presence of tBuOOH catalyzed by VO(acac)2 or with m- chloroperbenzoic acid. Other suitable methods to conduct the epoxidation are those known in the art which are useful to perform stereoselective epoxidation providing preferentially 5,6P-EC, such as: using a ruthenium(II) bioxazoline complex under aerobic conditions (see ref. 9), using bis(dipivaloylmethanato)manganese(II) in the presence of molecular oxygen and an aldehyde (Mn(dpm)2/isobutyraldehyde/O2 (see ref 10), using magnesium monoperoxyphthalate in the presence of manganese tetra-o-dichlorophenylporphyrin complexes (see ref 77), using chromyl diacetate (ref 12), or using a catalytic system involving metalloporphyrins (see ref 13).

Yet another aspect of the present invention refers to a method for obtaining a ¹⁸F- radiolabeled compound of formula (I) according to the present invention, wherein said method comprises: providing an intermediate compound of formula II according to the present invention or an intermediate compound obtainable according to the methods disclosed in the present invention; reacting said intermediate compound of formula II with a ¹⁸F-fluorinating agent in a polar solvent.

In one preferred embodiment the ¹⁸F-fluorinating agent is a ¹⁸F-cryptand complex and the method comprises refluxing said ¹⁸F-cryptand complex in a polar solvent. In other embodiments of the invention said ¹⁸F -fluorinating agent is K¹⁸F, or tetra-n-butylammonium 18-fluoride (TBA¹⁸F) or Cs¹⁸F. Preferably said polar solvent is acetonitrile, 2-methyl-butan- 2-ol, DMF (dimethylformamide) or tBuOH (tert-butyl alcohol). More preferably said polar solvent is acetonitrile. Most preferably the¹⁸F-cryptand complex is Kryptofix 222® (also known as K222®).

The present invention will be better understood with reference to the following examples and figures. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention. BRIEF DESCRIPTION OF THE FIGURES

Figure 1: MCF-7 cells lysate (150 pg) were incubated with 10 pM of [¹⁴C]-5,6P-EC at 37°C for 10 minutes with or without the indicated concentrations of 7a-Fluoro-5,6P-EC (compound 7, which corresponds to compound la of formula I) ranging from 10 to 200 pM. ChEH activity was assayed by measuring the conversion of [¹⁴C]-5,6P-EC to [¹⁴C]-CT by thin-layer chromatography (TLC) and quantified as described in Materials and Methods. (A) On the left panel, a representative autoradiogram of a TLC run from three independent experiments; (B) on the right panel, the dose-response curves of the inhibition of ChEH activity measured by TLC with 7a-Fluoro-5,6P-EC (compound 7, compound la of formula I) at the indicated concentrations. The curves were used to calculate the IC50. Ki was calculated using Cheng-Prusoff equation.

Figure 2: HEK293T-HSD2 cells lysate (20 pg) were incubated with 1 pM of [¹⁴C]-CT at 37°C for 10 minutes with or without 50 pM of CT, 7a-Fluoro-CT (compound 9 - compound 5a of formula I) or 3a-Fluoro-CT (compound 18 - compound 6a of formula I). The OCDO synthase (OCDOS) activity of HSD2 was assayed by measuring the conversion of [¹⁴C]-CT to [¹⁴C]-OCDO by thin-layer chromatography (TLC) and quantified as described in Materials and Methods. (A) a representative autoradiogram of a TLC run from three independent experiments; (B) the inhibition of OCDOS activity was measured by TLC with CT, 7a-Fluoro-CT (compound 9 - compound 5a of formula I) or 3a-Fluoro-CT (compound 18 - compound 6a of formula I) at 50 pM.

Figure 3: HEK293T-HSD2 cells lysate (20 pg) were incubated with 1 pM of [¹⁴C]-CT at 37°C for 10 minutes with or without increasing dose of 7a-Fluoro-CT (compound 9 - compound 5a of formula I) from 1 to 50 pM. The OCDOS activity was assayed by measuring the conversion of [¹⁴C]-CT to [¹⁴C]-OCDO by thin-layer chromatography (TLC) and quantified as described in Materials and Methods. (A) On the left panel, a representative autoradiogram of a TLC run from three independent experiments; (B) on the right panel, the inhibition of l ip -HSD2 activity measured by TLC with 7a-Fluoro-CT (compound 9 - compound 5a of formula I).

Figure 4: MCF-7 cells were incubated with 1 pM of [¹⁴C]-CT for 5 hours with or without the indicated concentrations of 7a-Fluoro-CT (compound 9 - compound 5a of formula I). HSD2 activity (OCDO synthase) was assayed by measuring the conversion of [¹⁴C]-CT to [¹⁴C]-OCDO by thin-layer chromatography (TLC) and quantified as described in Materials and Methods. (A) On the left panel, a representative autoradiogram of a TLC run from three independent experiments; (B) on the right panel, the inhibition of HSD2 activity measured by TLC with 7a-Fluoro-CT (compound 9 - compound 5a of formula I) at the indicated concentrations.

Figure 5: (A) 19F-NMR analysis of compound 7 - compound la of formula I - (a) and of lipid extracts of MDA-MB-231 or 4T1 exposed to solvent vehicle (b and d) or 10 pM of compound 7 - compound la of formula I - for 24 hours (c and e). (B) 19F-NMR analysis of compound 9 - compound 5a of formula I - (a) and of lipid extracts of MDA-MB-231 and 4T1 exposed to solvent vehicle (b and d) or 10 pM of compound 9 - compound 5a of formula I - for 24 hours (c and e).

Figure 6: (A and B) GC-MS analysis of compound 7 - compound la of formula I - (6A) GC chromatogram; (6B) MS spectrum of the peak at 12.23 min. (C and D) GC-MS analysis of compound 9 - compound 5a of formula I. (6C) GC chromatogram; (6D) MS spectrum of the peak at 13.33 min.

Figure 7: Quantification of compound 7 - compound la of formula I - and compound 9 - compound 5a of formula I - uptake in cancer cells by GC/MS. (A) compound 7 - compound la of formula I - was incubated at lOpM for 24 hr with MCF7 and MDA-MB-231 (MB231) cells. Cells were washed and compound 7 - compound la of formula I - was extracted and quantified by GC/MS as described on the “Materials and Methods” section. (B) Cells were treated with 10 pM of compound 9 - compound 5a of formula I. The presence of compound 9 - compound 5a of formula I - was analyzed by GC/MS. The results are reported as pg/million cells for the quantification of 7 - compound la of formula I - (A) and as ng/million cells for the quantification of compound 9 - compound 5a of formula I - (B) in cancer cells and as % of uptake per million cells (C).

Figure 8: Quantification of compound 9 - compound 5a of formula I - by GC/MS in MDA- MB-231 tumors implanted in mice and treated by subcutaneous injection (peri -turn oral treatment) of compound 9 - compound 5a of formula I - (7 pg) for 24 hours. The results are reported as ng/g tumor (A) or as % of injected dose (ID)/g tumor (B).

Figure 9: Quantification of compound 9 - compound 5a of formula I - by GC/MS in MDA- MB-231 tumors implanted into mice treated by intravenous injection in the tail of mice of compound 9 - compound 5a of formula I - (1.75 pg) for 60 or 90 minutes. The results are reported as ng/g tumor (A) or as % of injected dose (ID)/g tumor (B).

EXAMPLES

Materials and methods

Chemistry

Reagents and materials were purchased from Sigma-Aldrich Co and were used without further purification. The reactions were monitored by TLC on Merck silica gel 60 F254 (0.040-0.063 mm) layers. The spots were detected by spraying with sulfuric acid/methanol (1 : 1) and heating. Column chromatography was performed using silica gel 60 (0.063-0.2 mm). Purification was performed by flash chromatography on CombiFlash NextGen 300 (Serlabo) or by HPLC on a silica column (Prontosil, Bischoff). Melting points were determined with a Kotler apparatus and are uncorrected. ^JH NMR and ¹³C NMR spectra were recorded on a Bruker spectrometer (AVANCE II 300 MHz and AVANCE 400 MHz). Solutions were prepared in deuterochloroform (CDC13) or deuterated tetrahydrofuran (THF- ds). Deuterated solvents were used as internal reference (7.26 and 77.0 for CDCh). Mass spectra were recorded in positive mode on DSQ II (Thermo Fisher Scientific) using chemical ionization (NH3). High resolution mass spectrometry (HRMS) was performed in positive mode on GCT premier (Waters) using chemical ionization (CH4). The observed mass yields referred to purified products and are not optimized. All moisture-sensitive reactions were carried out under an argon atmosphere using oven-dried glassware and anhydrous solvents. All the organic layers were dried using anhydrous sodium sulfate.

Synthesis of 7p-hydroxy-cholesteryl-3p-acetate (3)

To a solution of 7-keto-cholesteryl-3P-acetate (2) (884 mg; 2 mmol) in THF (12 mL) and MeOH (8 mL) containing CeC13.7H2O (745 mg; 2 mmol) was added NaBHj (190 mg; 5 mmol). The mixture was stirred at rt for 15 min and concentrated under vacuum. The residue was dissolved in diethyl ether, washed sequentially with HC1 (5% aq soln), NaHCCE (5% aq soln) and water, dried over anhydrous MgSCU, filtered, and evaporated. The residue was purified by flash column chromatography with EtOAc-hexane (30-70) as eluent to yield 3 as a white solid (650 mg; 96%) mp 106.7-108.4, lit 110-111 °C. IR (neat) v: 3472, 2836, 2861, 1708, 1463, 11260, 1036, 1023, 757 cm’¹. ^XH NMR (400 MHz, CDC1₃) 6 5.33 (t, J= 2.0 Hz, 1H, 6-CH), 4.64 (tdd, J= 10.7, 6.2, 4.3 Hz, 1H, 3-CH ), 3.87 (dt, J = 7.8, 2.2 Hz, 1H, 7-CH ), 2.05 (s, 3H, -CH3COO),1.08 (s, 3H, 19-CH₃), 0.94 (d, J= 6.5 Hz, 3H, 21-CH₃ ), 0.88 (dd, J= 6.6, 6H, 26-CH₃ and 27-CH₃), 0.71 (s, 3H, 18-CH₃).¹³C NMR (101 MHz, CDCI3) 8 170.47, 142.38, 126.30, 73.46, 73.23, 55.91, 55.47, 48.20, 42.93, 40.81, 39.53, 39.51, 37.61, 36.67, 36.52, 36.21, 35.73, 28.53, 28.01, 27.72, 26.37, 23.84, 22.80, 22.55, 21.37, 21.04, 19.08, 18.78, 11.82.

Synthesis of 7p-hydroxy-5,6p-epoxycholestan-3p-acetate (4)

To a solution of 3 (669 mg, 1.5 mmol) and Vo(acac)2 (16 mg, 0.06 mmol) in anhydrous CH2Q2 (10 ml) was added tBuOOH (2.25 mmol, 0.4 ml, 5.5 M in decan) at 0 °C. After the reaction was stirred at rt for 4 h, solvent was removed under reduced pressure. The residue was purified by flash column chromatography with EtOAc-hexane (30-70) as eluent to yield 5 (598 mg, 86%) as a white solid. Mp: 128.7-130.6. °C. 'HNMR (400 MHz, CDCI3) 64.79 (ddt, J= 11.8, 9.8, 4.7 Hz, 1H), 3.54 (d, J= 9 Hz, 1H, 7-H), 3.17 (d, J= 1.6 Hz, 1H, 6-H), 2.05 (s, 3H, -CH3COO),1.03 (s, 3H), 0.91(d, J = 6.6, 3H, CH3), 0.88 (dd, J = 6.6, 6H, 26- CH₃ and 27-CH₃), 066(s, 3H). ¹³C NMR (101 MHz, CDCI3) 6 170.47, 74.60, 70.93, 67.32, 66.77, 55.45, 55.27, 49.42, 43.01, 39.53, 39.49, 38.28, 37.57, 36.35, 36.16, 35.67, 34.39, 28.55, 28.00, 27.23, 27.12, 23.83, 22.79, 22.54, 21.89, 21.26, 18.79, 16.78, 11.77.

Synthesis of 7p-methanesulfonate-5,6p-epoxycholestan-3p-acetate (5)

To a solution of 4 (1.5 mmol) in dry pyridine (5 ml) at 0°C was added MsCl (4.5 mmol) drop wise under argon. After the reaction was stirred at rt for 2 h, some ice was added and the reaction solvent under reduced pressure. The residue was dissolved with diethyl ether (50 ml) and washed sequentially with HC1 (5% aq soln), NaHCCh (5% aq soln) and water, dried over anhydrous MgSC , filtered, and evaporated. The residue was purified by flash column chromatography with EtOAc-hexane (10-90) as eluent to yield 5 (787 mg, 97%) as a white solid. Mp: 82.3 °C. IR (neat): v: 2948, 2866, 1731, 1466, 1332, 1241, 1169, 1033, 902, 822 cm’¹. ^XH NMR (400 MHz, CDCI3) 84.84 - 4.71 (m, 2H, 3-CH and 7-CH), 3.5 (s, 1H, 6-CH) 3.09 (s, 3H), 2.04 (s, 3H, -CH3COO), 1.06 (s, 3H, 18-CH3), 0.91 (d, J= 6.6, 3H, CH3), 0.88 (dd, J= 6.6, 6H, 26-CH3 and 27-CH₃), 066 (s, 3H). ¹³C NMR (101 MHz, CDCI3) 6 170.44, 83.74, 70.75, 67.19, 63.81, 55.14, 54.49, 49.36, 43.31, 39.97, 39.61, 39.24, 37.38, 36.33, 36.21, 35.61, 35.54, 34.54, 28.53, 28.13, 27.08, 26.16, 23.85, 22.92, 22.69, 22.07, 21.34, 18.95, 17.01, 11.84.

Synthesis of 7p-methanesulfonate-5,6p-epoxycholestan-3p-ol (compound 6 - compound lb of formula II)

To a solution of 5 (538 mg, 1 mmol) in THF-MeOH (1 : 1, 10 ml) was added K2CO3 (276 mg, 2 mmol). The reaction mixture was stirred at rt for 24 h and concentrated under reduced pressure. The residue was dissolved with diethyl ether (50 ml) and washed water (20 ml), dried over anhydrous MgSC , filtered, and evaporated. The residue was purified by flash column chromatography with EtOAc-hexane (30-70) as eluent to yield 5 (486 mg, 98%) as a white solid. Mp: 125.6 °C. IR (neat): v: 3675, 3384, 2951, 1458, 1325, 1166, 1077, 928, 899 cm’¹. 'H NMR (400 MHz, CDCI3) 8 4.77 (dd, J= 9.1, 1.5 Hz, 1H, 7-CH), 3.73 (m,lH, 3-CH), 3.49 (d, J= 1.4 Hz, 1H, 6-CH), 3.10 (s, 3H), 1.05 (s, 3H, 21-CH3), 0.91(d, J= 6.6, 3H, 19-CH3), 0.88 (dd, J= 6.6, 6H, 26-CH₃ and 27-CH₃), 0.66 (s, 3H, 18-CH3). ¹³C NMR (101 MHz, CDCI3) 6 83.90, 68.90, 67.58, 64.03, 55.14, 54.47, 49.70, 43.31, 41.44, 39.92, 39.59, 39.28, 36.90, 36.20, 35.59, 35.56, 34.39, 30.83, 28.52, 28.12, 26.15, 23.84, 22.91, 22.68, 22.15, 18.94, 17.03, 11.82.

Synthesis of 7a-fluoro-5,6p-epoxycholestan-3p-ol: compound 7 - compound la of formula I)

To a solution of compound 6 - compound lb of formula II - (0.25 mmol) in anhydrous acetonitrile (6 mL) was added kryptofix 222 (2 mmol) and potassium fluoride (2 mmol). The mixture was stirred under reflux (oil bath; 110°C) for 30 minutes. Water (5 mL) was added and the solvent was removed under vacuo. The residue was resuspended in chloroform (50 mL). Organic layer was washed two times with brine, dried with anhydrous sodium sulfate, filtered and evaporated. The white crude product was purified by HPLC using a chloroform gradient in hexane (5% CHCI3 for 10 minutes, then to 100% CHCI3 in 40 minutes; flow rate= 2 mL/min) with a retention time of 48 min. White solid (60% yield). Rf (Et2O) = 0.6.¹⁹F- NMR 6= -209.17 (dd, ²J_H-F= 31 Hz, ²J_H-F 50.3 Hz) ppm. 'H-NMR (300 MHz, CDC13): 6= 4.84 (1H, td, .7=3,9 Hz, J 49.9 Hz, .7=2,6 Hz, 7P-H), 3.71-3.81 (1H, m, 3a-H), 3.18 (1H, d, J= 3.1 Hz, 6a-H), 1.00 (3H, 19-CH₃), 0.89 (3H, d, J=6.6 Hz, 21-CH₃), 0.86 (6H, d, J=6.6 Hz, 26-CH3 and 27-CH₃), 0.64 (3H, I8-CH3) ppm. ¹³C-NMR (300 MHz, CDC13): 6= 89.3 (d, ¹JC-F= 167.8 Hz, C-7), 69.2 (C-3), 64.5 (C-5), 61.7 (d, ²J_C-F= 40.1 Hz, C-6), 57.0, 49.0 (d, ³JC~F= 2.9 Hz, C-14), 42.5, 42.3, 41.5, 39.5, 39.3, 36.6, 36.1, 35.8, 34.4 (d, ³J_C-F= 17.0 Hz, C-8), 34.3, 30.8, 28.1, 28.0, 23.8, 23.5, 22.8, 22.6, 21.8, 18.7, 17.0, 11.4 ppm. HRMS (DCI-CH4): m/z calcd for C27H45O2F [M⁺] 420.3404, found 420.3394.

Synthesis of 7p-methanesulfonate-cholestane-3p,5a,6p-triol (compound 8 - compound 5b of formula II)

To a solution of compound 6 - compound lb of formula II (0.25 mmol) in 5.5 mL of tetrahydrofurane/H2O/acetone (v/v/v; 4: 1 :0.5) was added perchloric acid (0.5 mL). The mixture was stirred at room temperature for 30 minutes and then diluted with chloroform (50 mL). Organic layer was washed with brine, aqueous sodium hydrogenocarbonate (5%) and brine, dried with anhydrous sodium sulfate, filtered and evaporated. The white crude product was purified by flash chromatography (CombiFlash NextGen) on a silica column (4G) using a tetrahydrofuran gradient in chloroform (0% THF for 5 minutes, then to 100% THF in 20 minutes and 100% THF for 5 minutes; flow rate= 13 mL/min) with a retention time of 22 min. White solid (80% yield). Rf (hexane/THF)= 0.44. 'H-NMR (300 MHz, CDC13): 5= 5.01 (1H, dd, J=3.9 Hz, J=10.6 Hz, 7a-H), 3.99-4.09 (3H, m, 3a-H), 3.94 (1H, d, .7=3,9 Hz, 6a-H), 1.16 (3H, 19-CH₃), 0.91 (3H, d, J=6.5 Hz, 21-CH₃), 0.86 (6H, d, J=6.6 Hz, 26-CH3 and 27-CH₃), 0.69 (3H, I8-CH3) ppm. ¹³C-NMR (300 MHz, CDC13): 5= 85.2 (C-7), 76.4 (C-5), 76.0 (C-6), 66.6 (C-3), 55.0, 54.0, 44.4, 43.6, 40.1, 39.5, 39.3, 39.1, 37.4, 35.9, 35.8, 35.4, 32.1, 30.0, 28.3, 27.8, 26.1, 23.5, 22.4, 22.2, 21.1, 18.5, 16.9, 11.8 ppm. ¹⁹F-NMR {^JH} 5= -169.8 ppm. HRMS (DCI-CH4): m/z calcd for C28H49O6S [M-H]⁺ 513.3250, found 513.3244.

Synthesis of 7a-fluoro-cholestane-3p,5a,6p-triol (compound 9 - compound 5a of formula I)

To a solution of 7 (0.25 mmol) in 5.5 mL of tetrahydrofurane/H2O/acetone (v/v/v; 4: 1 :0.5) was added perchloric acid (0.5 mL). The mixture was stirred at room temperature for 30 minutes and then diluted with chloroform (50 mL). Organic layer was washed with brine, aqueous sodium hydrogenocarbonate (5%) and brine, dried with anhydrous sodium sulfate, filtered and evaporated. The white crude product was purified by flash chromatography (CombiFlash NextGen) on a silica column (4G) using an ethyl acetate gradient in chloroform (0% EtOAC for 2 minutes, then to 100% EtOAc in 10 minutes and 100% EtOAC for 2 minutes; flow rate= 13 mL/min) with a retention time of 12 min. White solid (75% yield). Rf (Et2O)= 0.23. ¹⁹F-NMR {‘H} 5= -192.3. 'H-NMR (300 MHz, CDC13): 5= 4.51 (1H, td, 7=3.9 Hz, ²7//./.=46.8 HZ, J= 2.7 Hz, 7a-H), 4.00-4.11 (1H, m, 3a-H), 3.59 (1H, d, ³JH-F 10.2 Hz, 7=4.3 Hz, 6a-H), 1.14 (3H, 19-CH₃), 0.97 (3H, d, J=6.5 Hz, 21-CH₃), 0.9 (6H, d, J=6.5 Hz, 26-CH₃ and 27-CH₃), 0.74 (3H, 18-CH₃) ppm. MS (DCI-NH₃) m/z: [M-NH4⁺- H₂O]=438.4; [M-NH4⁺]=456.4.

Synthesis of 3p-methanesulfonate-5,6p-epoxycholestane (16)

To a solution of 5,6P-epoxy-cholestane-3P-ol 15 (0.25 mmol) in pyridine (0.5 mL) at 0°C was added methanesulfonate chloride (0.75 mmol). The mixture was stirred at room temperature for 1 hour. Ice was added for the hydrolysis of methanesulfonate chloride excess and the solvent was removed under vacuo. The residue was resuspended in ethyl acetate. Organic layer was washed with aqueous hydrochloride acid IN, with saturated aqueous sodium hydrogenocarbonate dried with anhydrous sodium sulfate, filtered and evaporated. The white crude product was purified by flash chromatography (CombiFlash NextGen) on a silica column (12G) using a chloroform gradient in hexane (0% CHC1₃ for 1 minute, then to 100% CHC1₃ in 13 minutes; flow rate= 30 mL/min) with a retention time of 13 min. White solid (70% yield), mp - °C. Rf (CHC1₃)= 0.4. ‘H-NMR (300 MHz, CDC13): 5= 4.64-4.74 (1H, m, 3a-H), 3.08 (1H, d, 7=2.2 Hz, 6a-H), 3.00 (3H, s, SO-CH₃), 1.00 (3H, 19-CH₃), 0.89 (3H, d, J=6.6 Hz, 21-CH₃), 0.86 (6H, d, J=6.6 Hz, 26-CH₃ and 27-CH₃), 0.63 (3H, 18-CH₃) ppm. ¹³C-NMR (300 MHZ, CDC13): 5= 78.8 (C-3), 63.5 (C-6), 62.2 (C-5), 56.2, 56.1, 50.9, 42.3, 39.7, 39.5, 39.0, 38.9, 36.3, 36.1, 35.7, 34.8, 32.4, 29.6, 28.3, 28.1, 28.0, 24.2, 23.8, 22.8, 22.6, 21.9, 18.7, 16.9, 11.7 ppm. HRMS (DCI-CH₄): m/z calcd for C₂8H₄8O₄S [M⁺] 480.3273, found 480.3268.

Synthesis of 3p-methanesulfonate-cholestane-5a,6p-diol (compound 17 - compound 6b of formula II)

To a solution of 16 (0.25 mmol) in 5.5 mL of tetrahydrofuran e/H2O/acetone (v/v/v; 4: 1 :0.5) was added perchloric acid (0.5 mL). The mixture was stirred at room temperature for 30 minutes and then diluted with chloroform (50 mL). Organic layer was washed with brine, aqueous sodium hydrogenocarbonate (5%) and brine, dried with anhydrous sodium sulfate, filtered and evaporated. The white crude product was purified over silica gel using CHC1₃- hexane (8-2). White solid (70% yield). ‘H-NMR (CDC1₃): 5= 5.04-5.15 (1H, m, 3a-H), 3.54 (1H, t, 7=2.8 Hz, 6a-H), 3.0 (3H, s, SO) 2.39 (1H, dd, 7=11.3 Hz, 7=12.8 Hz), 1.18 (3H, 19- CH₃), 0.90 (3H, d, J=6.6 Hz, 21-CH₃), 0.86 (6H, d, J=6.6 Hz, 26-CH₃ and 27-CH₃), 0.67 (3H, 18-CH₃) ppm. ¹³C-NMR (CDC13): 5= 80.3 (C-3), 76.2 (C-5), 75.8 (C-6), 56.3, 55.8, 45.6, 42.7, 39.8, 39.5, 38.6, 38.3, 38.1, 36.1, 35.8, 34.6, 32.2, 30.1, 28.2, 28.0, 27.9, 24.1, 23.9, 22.8, 22.6, 21.1, 18.7, 16.7, 12.1 ppm. HRMS (DCI-CH₄): m/z calcd for C₂8H₅o0₅S [M⁺] 498.3379, found 498.3369.

Synthesis of 3a-fluoro-cholestane-5a,6p-diol (compound 18 - compound 6a of formula I)

To a solution of compound 17 - compound 6b of formula I - (0.25 mmol) in anhydrous acetonitrile (6 mL) was added kryptofix 222 (2 mmol) and potassium fluoride (2 mmol). The mixture was stirred under reflux (oil bath; 110°C) for 30 minutes. Water (5 mL) was added and the solvent was removed under vacuo. The residue was resuspended in chloroform (50 mL). Organic layer was washed two times with brine, dried with anhydrous sodium sulfate, filtered and evaporated. The white crude product was purified by flash chromatography (CombiFlash NextGen) on a silica column (4G) using a chloroform gradient in hexane (0% CHC1₃ for 1 minute, then to 100% CHC1₃ in 6.5 minutes and 100% CHC1₃ for 5 min; flow rate= 13 mL/min) with a retention time of 9 min. White solid (55% yield). ¹⁹F-NMR J ¹ H } 5= -169.8. ^JH-NMR (CDC13): 5= 5.09 (1H, dquint, /.r 48.3 HZ, ³J/M/=2.6 HZ, 3 -H), 3.59 (1H, t, J=2.3 Hz, 6a-H), 1.10 (3H, 19-CH₃), 0.91 (3H, d, J=6.6 Hz, 21-CH₃), 0.86 (6H, d, J=6.6 Hz, 26-CH₃ and 27-CH₃), 0.68 (3H, 18-CH₃) ppm. ¹³C-NMR (CDC13): 5 ppm. HRMS (DCI-CH4): m/z calcd for C27H47O2F [M⁺] 422.356, found 422.3541.

General procedure for the mesylation of Hydroxy-steroids 19, 28, 36 or 43.

To a solution of 19 (commercially available), 28, 36 or 43 (commercially available) (1.5 mmol) in dry pyridine (5 ml) at 0°C was added MsCl (4.5 mmol for compound 19, 28 and 36 or 1.5 mmol for compound 43) drop wise under argon. After the reaction was stirred at rt for 2 h, some ice was added and the reaction solvent under reduced pressure. The residue was dissolved with diethyl ether (50 ml) and washed sequentially with HC1 (5% aq soln), NaHCO₃ (5% aq soln) and water, dried over anhydrous MgSCU, filtered, and evaporated. The residue was purified by flash column chromatography. General procedure for the fluorination of Methanesulfonate-steroids 22, 25, 31, 33, 39, 41, 45 or 47.

To a solution of methanesulfonate-steroid (22, 25, 31, 33, 39, 41, 45 or 47) (0.25 mmol) in anhydrous acetonitrile (6 mL) was added kryptofix 222 (2 mmol) and potassium fluoride (2 mmol). The mixture was stirred under reflux (oil bath; 110°C) for 30 minutes. Water (5 mL) was added and the solvent was removed under vacuo. The residue was resuspended in chloroform (50 mL). Organic layer was washed two times with brine, dried with anhydrous sodium sulfate, filtered and evaporated. The white crude product was purified by flash chromatography.

General procedure for the hydrolysis of Epoxy-steroids 23, 32, 40, 45 or 46.

To a solution of Epoxy-steroids (23, 32, 40, 45 or 46) (0.25 mmol) in 5.5 mL of THF/EEO/acetone (v/v/v; 4: 1 :0.5) was added perchloric acid (0.5 mL). The mixture was stirred at room temperature for 30 minutes and then diluted with chloroform (50 mL). Organic layer was washed with brine, aqueous sodium hydrogenocarbonate (5%) and brine, dried with anhydrous sodium sulfate, filtered and evaporated. The white crude product was purified over silica gel using EtOAc-hexane as eluant.

General procedure for the hydrolysis of steroid-3p-acetate 21.

To a solution of steroids-3P-acetate (21) (0.25 mmol) in THF-MeOH (1 : 1,10 mL) was added potassium carbonate (0.5 mmol). The reaction mixture was stirred at rt for 24 h and concentrated under reduced pressure. The residue was dissolved with diethyl ether (50 ml) and washed water (20 ml), dried over anhydrous MgSCU, filtered, and evaporated. The residue was purified by flash column chromatography.

General procedure for the epoxidation of steroids 20, 30, 38 or 44. m-Chloroperbenzoic acid (mCPBA) (1.6 mmol) in methylene chloride (4 mL) was added dropwise to a solution of steroids 20, 30, 38 or 44 (1 mmol) in methylene chloride (10 mL) over a period of 1 min at room temperature. The reaction mixture was stirred over 4 hours and then washed with aqueous sodium sulfite (10%) and sodium hydrogenocarbonate (5%) and dried over anhydrous MgSCU, filtered, and evaporated. The residue was purified by flash column chromatography and 5,6P-epoxysteroid was separated from its diasteroisomer 5,6a- epoxysteroid by RP-HPLC. General procedure for the oxidation of steroids 24, 30, 38 or 44 with HIO4.

Pure THF (15 mL) in aqueous iodide metaperiodate (5.5 mL, 10 mmol) was added with stirring to a solution of 24, 30, 38 or 44 (1 mmol). The mixture was stirred at room temperature and turned yellow, orange and brown in a day. The completion of the reaction was shown by TLC. The mixture was washed with excess 10% aqueous sodium thiosulfate and a white precipitate appeared in a colorless solution. The resulting mixture was extracted with ethyl acetate (40 mL). The extract was washed with water, 5% aqueous sodium bicarbonate and brine. The organic layer was then dried over MgSO4, and the solvent removed under reduced pressure. The crude product was purified by flash column chromatography.

General procedure for the deprotection of steroids silyl ethers 29 or 37.

The deprotection of steroids silyl ethers 29 or 37 was performed by using 20 mol % of Zinc (II) trifluoromethanesulfonate (Zn(OTf)2) at room temperature in methanol as previously described (ref. 3).

Biology methods

Cell culture

MCF7, 4T1, MDA-MB-231 and HEK293T were from the American Tissue Culture Collection and cultured until passage 30. MCF7 cells were grown in RPMI 1640 supplemented with 5% fetal bovine serum (FBS). 4T1 cells were grown in RPMI 1640 supplemented with 10% FBS, HEK293T and MDA-MB-231 cells were grown in DMEM 10% FBS. All media were supplemented with penicillin and streptomycin (50 U/mL). Cells were cultured in a humidified atmosphere with 5% CO2 at 37°C. Cell lines were tested once a month for mycoplasma contamination using My coalert Detection (Lonza).

Animals.

Mice were handled and cared for according to the ethical guidelines of our institution and following the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996) and the European Directive EEC/86/609, under the supervision of authorized investigators. All mice were maintained in specific pathogen-free conditions and were only included in protocols following 2 weeks of quarantine. Nude mice (NU/NU) mice were from Charles River Laboratories, Saint-Germain-sur-L’Arbresle, France. Metabolism of sterols in MCF7 Cells.

MCF7 cells were plated into six-well plates (1.5x 105 cells per well) in the appropriate complete medium. Two days after seeding, this medium was replaced with complete medium and cells were treated with either 0.6 pM [¹⁴C]-5,6P-EC or 1 pM [¹⁴C]-CT for 5 h in the presence or absence of increasing concentrations of the tested compounds. After incubation, cells were washed with PBS, trypsinized and counted. Neutral lipids were extracted with a chloroform-methanol mixture as described previously and then separated by TLC using ethyl acetate as the eluent. The radioactive sterols were revealed by autoradiography. For quantification, silica zones at the expected Rf values corresponding to authentic [¹⁴C]-labeled standards were scraped and radioactivity was measured using a P- counter, as previously described (see ref. 14).

Cell Transfection.

For cell transfection, 5 x io⁶ HEK293T cells were transfected with 5 pg of a plasmid encoding HSD2 (RG207796; OriGene) or the empty plasmid using the NEON Transfection System according to the manufacturer’s recommendations.

Preparation of cell lysate for the measurement of OCDO synthase activity

Cell lysates were prepared from HEK293T cells transiently expressing human HSD2. Briefly, 2.10⁶ cells were resuspended in 150 pL of activity buffer (Tris-HCl 25 mM PH 7.4, glycerol 20%, sucrose 25 mM, NaCl 200 mM, MgCh 1 mM, CaCh 1 mM) with 1% protease inhibitor mixture (Sigma-Aldrich). Cells were lysed by four cycles of freeze/thaw (nitrogen- rt) and samples were centrifuged at 10000 rpm for 10 minutes at 4°C. Supernatant were collected, aliquoted and stored at -80°C before use. The protein concentration was measured using the Bradford method (see ref. 15).

Preparation of cell lysate for the measurement of ChEH activity

5.10⁶ MCF7 cells were resuspended in 1 mL of buffer (Tris-HCl 50 mM PH 7.4, KC1 150 mM) with 1% protease inhibitor mixture (Sigma- Aldrich). Cells were lysed by four freeze/thaw cycles (liquid nitrogen-rt) and samples were centrifuged at 4000 rpm for 10 minutes at 4°C. The supernatants were collected, aliquoted and stored at -80°C before use. The protein concentration was measured using the Bradford method. Measure of the ChEH enzymatic activity

ChEH activity was carried out exactly as previously described (ref 6).

Measure of the OCDO synthase activity assay

Enzymatic activity was measured in the HSD2 activity buffer (final volume 0.2 mL) containing the substrate ([¹⁴C]-CT (1 pM), cell lysate proteins (20 pg), NAD⁺ (0.5 mM) and DMSO 1% in the absence or presence of the tested compounds. After 10 minutes of incubation at 37°C, the reaction was stopped by immersing the sample in ice-water and adding 1.5 ml chloroform/methanol (2: 1) and 300 pl of aqueous KC1 (8.8%). The organic layer was washed with water (1 mL) and reduced to dryness under a flux of nitrogen. Lipids were then separated by TLC using ethyl acetate as eluent. The radiolabeled sterols were revealed by autoradiography. For quantification, silica zones at the expected Rf values corresponding to authentic [¹⁴C]-labeled standards were scraped and radioactivity was measured using a P-counter, as previously described (see ref. 14).

Preparation of cell extracts for GC/MS analysis

MDA-MB-23 1 and MCF7 cells (1 x 10⁶) were seeded in T75 flask. Twenty -four hours later, cells were treated with either 10 pM compound 9 - compound 5a of formula I, or 10 pM compound 7 - compound la of formula I. After a 48 h incubation, the cells were washed with PBS, trypsinized and counted. Cell suspension was centrifuged at 1200 rpm for 5 minutes at 4°C and lipid extraction was performed on cell pellet. 1 ml of methanol containing the internal standard [d6]-CT and [d6]-OCDO (50 ng each) and 2 ml of chloroform were added. The suspensions were vortexed 30 s, 1ml of 8.8% aqueous KC1 was added. After centrifugation at 2500 rpm for 5 minutes at 4°C, organic layers were collected and the solvent was evaporated under a stream of nitrogen. Lipid extracts were analyzed by ¹⁹F- NMR or further purified for GC-MS analysis. The residue was dissolved in 1 ml toluene and oxysterols were separated from cholesterol by solid phase extraction. Silica cartridges (100 mg), previously equilibrated with n-hexane, were loaded with toluene-dissolved samples. Cholesterol and non-cholesterol neutral sterols were eluted with 1% propan-2-ol in hexane before eluting OS with 30% propan-2-ol in n-hexane. Solvent was evaporated under nitrogen and samples stored at -80°C before GC/MS analysis. Preparation of tumor extracts for GC/MS analysis

Exponentially growing MDA-MB-231 were harvested, washed twice in PBS then resuspended in PBS. MDA-MB-231 cells (5 x io⁶ cells) were injected subcutaneously (s.c.) into the flank of nude mice. When tumors were palpable, animals (n = 10-20 mice per group, as indicated) were randomized to receive either subcutaneous injection of compound 7 - compound la of formula I (7 pg), compound 9 - compound 5a of formula I (7 pg) or solvent vehicle (l%o ethanol in PBS) for 24 hours or intravenous injection of compound 9 - compound 5a of formula I (1.75 pg) or solvent vehicle for 1 hour. At the end of experiments, mice were sacrificed, and tumors were excised and weighed. Tumors were homogenized at 4 °C using a Precellys 24 homogenizer system (Bertin technologies, Montigny-le- Bretonneux, France): briefly, tumors were disposed in cold tubes containing metal beads and lysis buffer (50 mM Tris-HCl, 150 mM KC1 pH 7.4) 5 vol per g of tissue at 4 °C. The suspension was agitated 3x 20 s at 6500 r.p.m. Samples were then centrifuged (10 min at 4000 g, 4 °C), the supernatant was collected in new tubes and the protein concentration was measured by Bradford method. Lipid extraction was performed using the Bligh and Dyer liquid/liquid (B&D) extraction method. Samples were mixed with a volume of methanol containing the internal standard [d6]-CT and [d6]-5,6P-EC (50 ng each) and two volumes of chloroform. The suspensions were vortexed 30 s and then centrifuged at 10,000 rpm for 10 minutes at 4°C. Organic layers were collected and the solvent was evaporated under a stream of nitrogen, then the residue was dissolved in 1 ml toluene and oxysterols were separated from cholesterol by solid phase extraction. Silica cartridges (100 mg), previously equilibrated with n-hexane, were loaded with toluene-dissolved samples. Cholesterol and non-cholesterol neutral sterols were eluted with 1% propan-2-ol in hexane before eluting OS with 30% propan-2-ol in n-hexane. Solvent was evaporated under nitrogen and samples stored at -80°C before GC/MS analysis.

Detection and quantification of compound 7 - compound la of formula I - and compound 9 - compound 5a of formula I - by GC/MS

7a-Fluoro-5,6P-epoxycholestan-3P-ol (compound 7 - compound la of formula I) and 7a- Fluoro-cholestane-3p,5a,6P-triol (compound 9 - compound 5a of formula I) were derivatized using pyridine-hexamethyldisilazane-trimethylchlorosilane (3:2: 1) and analyzed by GC/MS. Quantification of these compounds was carried out using stable isotope dilution mass spectrometry. For GC/MS analysis, samples were redissolved in 100 pl hexane and 1 pl was used for analysis in a trace gas chromatographer coupled to a mass spectrometer (ISQ Thermo Fisher Scientific). Samples were separated on an RTX-5MS fused silica column (15 m x 250 pm x 0.25 pm). The oven temperature program was as follows: 180°C for 1 min, 20°C/min to 300°C where the temperature was kept for 12 min. Helium was used as the carrier gas, with a flow rate of 1 ml/min. The molecules were ionized by electron impact at 70 eV. 5,6-EC were monitored with ions at mass/charge ratio (m/z) 321 and 564 (7aF-CT), 402 and 491 (7aF-5,6p-EC), 409 and 462 (d6-CT), 391 and 481 (d7-5,6p-EC). Quantitative GC/MS determinations were calculated from triplicate injections and from the linear response range of standard curves established for oxysterol/IS pairs. D6-CT and d7- 5,6P-EC were used as internal standard for the quantification of 7aF-5,6P-EC and 7aF-CT respectively.

Results

1. Chemistry

Considering the half-life of fluorine- 18 (ti/2= 110 min), the introduction of this radionucleide must be performed at the final step of the chemical synthesis. Consequently, our approach involves the production of structural analogues of 5,6P-epoxycholestan-3P-ol (EC-P) and cholestane-3p,5a,6p-triol (CT) bearing an electrophilic methanesulfonate group. Indeed, mesylate precursors are suitable for the rapid introduction of 18-Fluorine at the final step. The chemical reactivity of allylic protons and hydroxyl function of cholesterol prompted us to add methanesulfonate group at position 3, 4 and 7 of the sterol backbone.

1.1 Chemical modification at position 7 of the steroid backbone

The synthesis of 7P-methanesulfonate-5,6P-epoxycholestan-3P-ol (compound 6 - compound lb of formula II) was achieved in five steps from commercially available cholesteryl-3P-acetate (1) (scheme 1):

Scheme 1

1 2 3 Scheme 1 (cont.)

Scheme 1: i: (ref. 2) ii: 2.5 equivNaBIH , 1 equiv CeCh 7H2O, THF-MeOH, 30 min (95%), iii: VO(acac)2 (4% mol), 2 equiv of tBuOOH (5 M in decane), CH2Q2, rt, 5h, (85%); iv: 3 equiv MsCl, pyridine, O°C-rt, 2h (97%); v: 2 equiv K2CO3, THF-MeOH, rt, 24h (98%); vi: 8 equiv KF, 8 equiv K222, ACN, reflux 30 min (60%); vii: vi: HC1O4, THF/H2O/acetone, rt, 30 min.

Thus, allylic oxidation of 1 was prepared according the literature (ref. 2) to 7-keto- cholesteryl-3P-acetate 2. Luche reduction of 7-keto-cholesteryl-3P-acetate 2 with sodium borohydride and CeCh gave 7P-Hydroxy-cholesteryl-P-acetate 3. Allylic alcohol epoxidation with tBuOOH catalysed by VO(acac)2, furnished the epoxide 4 as sole product. Mesylation of alcohol and deprotection of the remaining acetate afforded the desired 7P- mesylate precursor 6 - compound lb of formula II. We then evaluated the fluorination of compound 6 - compound lb of formula II by comparing fluorinating agents (CsF, kryptofix 222/KF and TBAF) that are frequently used in radiochemistry. In our hands, refluxing K222/KF and TBAF with compound 6 - compound lb of formula II - trigger its complete consumption toward 7a-fluoro-5,6P- epoxycholestan-3P-ol (compound-7 - compound la of formula I) (85%) and an unidentified byproduct (15%) whereas CsF was less effective leading to the presence of the precursor 6 (30%), 7 (55%) and the byproduct (15%). Kinetics strongly differs between fluorinating agents with K222/KF being the most effective (reaction time= 15 minutes) followed by TBAF (reaction times=l hour) and then CsF (reaction time= 12 hours). A large excess of fluorinating agent (8 equiv) also improves the reaction compared to a slight excess (2 equiv). For this reaction, acetonitrile was more suitable compared to tBuOH considering that solvolysis of compound 6 - compound lb of formula II - was observed in tBuOH.

For the production of fluorinated analogue of CT, we first performed the hydrolysis of compound 6 - compound lb of formula II - with perchloric acid leading to 7P- methanesulfonate-cholestane-3p,5a,6P-triol 8 - compound 5b of formula II. However, compound 8 - compound 5b of formula II - failed to undergo fluorination under exposure with fluorinating agents. The reaction leads to an unidentified compound that do not correspond to the elimination product in ¹HNMR.

Finally, 7a-fluoro-cholestane-3p,5a,6P-triol (compound 9 - compound 5a of formula I) was produced through acidic hydrolysis of compound 7 - compound la of formula I - with perchloric acid. It is noteworthy that compound 9 - compound 5a of formula I - can be produced in situ by adding directly perchloric acid in the reaction medium just after the formation of compound 7 from compound 6. This observation suggests that 7a-¹⁸Fluoro-CT could be produced in situ from 7a-¹⁸Fluoro-5,6P-EC by acidic hydrolysis.

1.2. Chemical modification at position 3 of the steroid backbone

Reaction of commercially available 5,6P-EC 15 with methanesulfonate chloride gave 3P- methanesulfonate-5,6P-EC 16. Acidic hydrolysis of 16 with perchloric acid lead to 3P- methanesulfonate-CT 17 - compound 6b of formula II. Fluorination of 17 with kryptofix 222/KF (path iii starting from compound 17 in scheme 2) afforded 3a-Fluoro-CT 18- compound 6a of formula I (Scheme 2) whereas the fluorination of 16 in similar conditions (path iii starting from compound 16 in scheme 2) was unsuccessful and the corresponding 3a-¹⁸Fluoro-5,6P-EC could not be obtained (barred in scheme 2).

Scheme 2

17 18 Scheme 2: i : 3 equiv MsCl, pyridine, 0°C-rt, 24h (90%) ; ii : HCIO4, THF/FEO/acetone, rt,

30 min (75%) ; iii : 8 equiv KF, 8 equiv K222, ACN, reflux 30 min (60%).

1.3. Chemical modification at position 19 of the steroid backbone

The synthesis of the intermediate compounds of formula (II), 2b (compound 22) and 7b (compound 25) and of the 18-fluorinated compounds of formula (I), 2a (compound 23) and 7a (compound 26), was carried out according to the following synthetic path (Scheme 3). The details for each of the steps of the synthesis of each of the compounds indicated in the scheme may be found in the section “materials and methods” above herein.

Scheme 3: i: 3 equiv MsCl, pyridine, O°C-rt, 2h ; ii: 1.6 equiv mCPBA, DCM, rt, 4h, iii: 2 equiv K2CO3, THF-MeOH, rt, 24h; iv: 8 equiv KF, 8 equiv K222, ACN, reflux 30 min ; v:

10 equiv HICU aq, ThF, rt, 24h. vi: HCIO4, THF/FhO/acetone, rt, 30 min.

1.4. Chemical modification at position 20 of the steroid backbone

The synthesis of the intermediate compounds of formula (II), 3b (compound 31) and 8b (compound 33) and of the 18-fluorinated compounds of formula (I), 3a (compound 32) and 8a (compound 34), was carried out according to the following synthetic path (Scheme 4). The details for each of the steps of the synthesis of each of the compounds indicated in the scheme may be found in the section “materials and methods” above herein.

Scheme 4

Scheme 4: i: ref. 4 ii: 3 equiv MsCl, pyridine, 0°C-rt, 2h ; iii: Zn(Otf)220 mol %, MeOH, rt; iv: 1.6 equiv mCPBA, DCM, rt, 4h ; v: 8 equiv KF, 8 equiv K222, ACN, reflux 30 min ; vi: 10 equiv HICU aq, THf, rt, 24h; vii: HCIO4, THF/FFO/acetone, rt, 30 min. (ref 4) 1.5. Chemical modification at position 24 of the steroid backbone

The synthesis of the intermediate compounds of formula (II), 4b (compound 39) and 9b (compound 41) and of the 18-fluorinated compounds of formula (I), 4a (compound 40) and 9a (compound 42), was carried out according to the following synthetic path (Scheme 5). The details for each of the steps of the synthesis of each of the compounds indicated in the scheme may be found in the section “materials and methods” above herein.

Scheme 5 Scheme 5: i: ref. 5; ii: 3 equiv MsCl, pyridine, O°C-rt, 2h ; iii: Zn(0tf)220 mol %, MeOH, rt; iv: 1.6 equiv mCPBA, DCM, rt, 4h ; v: 8 equiv KF, 8 equiv K222, ACN, reflux 30 min ; vi: 10 equiv HIO4 aq, THf, rt, 24h; vii: HCIO4, THF/ FO/acetone, rt, 30 min (ref 5). 1.6. Chemical modification at position 27 (or 25(R)-26) of the steroid backbone

The synthesis of the intermediate compounds of formula (II), 10b (compound 45) and 11b (compound 47) and of the 18-fluorinated compounds of formula (I), 10a (compound 46) and Ila (compound 48), was carried out according to the following synthetic path (Scheme 6). The details for each of the steps of the synthesis of each of the compounds indicated in the scheme may be found in the section “materials and methods” above herein.

Scheme 6

47

48 Scheme 6 (cont.)

47

Scheme 6: i: 1 equiv MsCl, pyridine, 0°C-rt, 2h ; ii: 1.6 equiv mCPBA, DCM, rt, 4h, iii: 8 equiv KF, 8 equiv K222, ACN, reflux 30 min ; iv: 10 equiv HIO4 aq, THF, rt, 24h. v: HCIO4, THF/H2O/acetone, rt, 30 min.

2. Biological tests

We have successfully produced fluorinated analogues of 5,6P-EC (compound 7 - compound la of formula I) and of CT (compound 9 - compound 5a of formula I - and compound 18 - compound 6a of formula I). In their 18-F form, these compounds should constitute valuable candidates for the imaging of breast cancer through the targeting of ChEH and HSD2 that are overexpressed in breast cancer and negatively associated with patient survival³. Consequently, we evaluated whether fluorinated analogues of 5,6P-EC (compound 7 - compound la of formula I) and of CT (compound 9 - compound 5a of formula I - and compound 18 - compound 6a of formula I) maintain their ability to interact with ChEH and HSD2 respectively, which constitute important parameters driving the attraction of compounds by tumor cells.

2.1 Measurement of enzymatic activities

2.1.1 Inhibition of the ChEH activity

We tested the impact of 7 - compound la of formula I - on the hydrolysis of [¹⁴C]-5,6P-EC in breast cancer cells (MCF7) lysate. For this purpose, [¹⁴C]-5,6P-EC (10 pM) were incubated with MCF7 cells lysate (150 pg) for 10 minutes alone or with increasing concentrations (10-200 pM) of 7 - compound la of formula I. After lipids extraction and separation by TLC, we observed that 7 - compound la of formula I - inhibits ChEH activity in a concentration-dependent manner (Figure 1) (ICso= 71.2 pM; Ki=21.8 pM). Consequently, 7 - compound la of formula I - is an inhibitor of ChEH and since it is an isoster of 5,6P-EC we can conclude that it interacts with ChEH which constitutes a parameter of attractivity of 7 - compound la of formula I - by BC cells, which deserves further evaluation.

2.1.2 Inhibition ofHSD2 activity on cell lysates and whole cell.

We evaluated the impact of compound 9 - compound 5a of formula I, compound 18 - compound 6a of formula I - and CT at 50 pM on the conversion of [¹⁴C]-CT into [¹⁴C]- OCDO in HEK293T-HSD2 lysate (Figure 2). Both CT and compound 9 - compound 5a of formula I - inhibits the production of [¹⁴C]-OCDO from [¹⁴C]-CT by more than 75% whereas compound 18 - compound 6a of formula I - has a modest activity with approximately 20% of inhibition at 50 pM compared to control (Figure 2).

We then performed a dose-response experiment with compound 9 - compound 5a of formula I - on HSD2 activity. For this purpose, [¹⁴C]-CT was incubated with HEK293T-HSD2 lysate for 10 minutes alone or with increasing concentrations (1-50 pM) of compound 9 - compound 5a of formula I. After lipids extraction and separation by TLC, we observed that compound 9 - compound 5a of formula I - inhibits the OCDO synthase activity in a dose dependent manner (Figure 3) (ICso= 17 pM).

We next performed a whole cell OCDO synthase inhibition assay on MCF7 cells. We found that compound 9 - compound 5a of formula I - blocks in a concentration-dependent manner OCDO formation (Figure 4) with an apparent IC50 around 20 pM, which is close to the IC50 measured on cell lysates.

Compound 9 - compound 5a of formula I - is an inhibitor of HSD2 activity and since it is an isoster of CT we can conclude that it interacts with HSD2, which and this constitutes a parameter of attractivity of compound 9 - compound 5a of formula I - by BC cells and deserves further evaluation of 9.

2.2 Internalization of 7aF-5,6fl-EC (7 - compound la of formula I) and 7aF-CT (compound 9 - compound 5a of formula I) on breast cancer cells in vitro and in vivo

The 18-Fluoro- epoxy cholestanols (EC) derivatives of formula (I), such la, and the 18- Fluoro-cholestan-triol (CT) derivatives of formula (I), such as 5a, of the invention have different overexpressed targets in breast cancer. Indeed, as seen previously, 18-Fluoro-EC compounds target ChEH (a complex formed by DHCR7 and EBP) whereas 18-Fluoro-CT compounds target HSD2. In that regard, the detection of breast cancer using the 18-Fluoro- EC of formula (I), la, by targeting of ChEH on one hand, and the detection of breast cancer using the 18-Fluoro-CT of formula (I) 5a, through the targeting of HSD2 on the other hand, was tested.

2.2.1 Internalization of 7aF-5,6fl-EC (7 - compound la of formula I) and 7aF-CT (compound 9 - compound 5a of formula I) on breast cancer cells in vitro

2.2.1.1 Evaluation of uptake by 19F-NMR

Succeeding in tumor imaging involves that the radiopharmaceutical was internalized by the tumor. Consequently, we first investigated if the uptake of compounds 7 - compound la of formula I - and compound 9 - compound 5a of formula I - by BC cells was possible using a 19F-NMR detection method of cell extracts. For this purpose, human MDA-MB-231 and mouse 4T1 TNBC cells were treated for 24 hours with 10 pM of 7 or 9. The analysis of the lipidic extract of cells by 19F-NMR allows the detection of specific signal for 7 (5= -209 ppm) and for 9 (5= -190 ppm) (Figure 5A and 5B).

This shows that 7 - compound la of formula I - and compound 9 - compound 5a of formula I - are internalized by both human and murine breast cancer cells, which deserves further evaluation with these compounds.

2.2.1.2 Evaluation of the cellular uptake of compounds 7 - compound la of formula I - and compound 9 - compound 5a of formula I - by GC-MS

To complete the preceding qualitative analysis by a quantification of compounds 7 - compound la of formula I - and compound 9 - compound 5a of formula I - in cells, we set up a GC-MS method. Our method offers a good separation between 7 (RT= 12.23 min) (Figure 6A-B) and 9 (RT= 13.63 min) (Figure 6C-D).

Calibration curves were performed using deuterated 5,6P-EC and deuterated CT for the quantification of 7 - compound la of formula I - and compound 9 - compound 5a of formula I - respectively. These curves were determined using increasing concentrations of analyte from 0.1 to lO ng/pL. Deuterated d7-5,6P-EC and d6-CT (1 ng/pl) were used as internal standard for the quantification of compound 7 - compound la of formula I - and compound 9 - compound 5a of formula I - respectively. The calibration curves were linear over a range 0.1-10 ng/pl for the analytes: Y= -0.0440957+0.842418*X with R^A2 = 0.9970 and W: 1/X for compound 7 (compound la of formula I); and

Y= -0.0932045+1.24028*X with R^A2 = 0.9971 and W: 1/X for compound 9 (compound 5a of formula I).

MCF7 and MDA-MB-231 cells were treated for 24 hours with 10 pM of 9. GC-MS data showed that both compounds 7 - compound la of formula I - and compound 9 - compound 5a of formula I - are internalized by breast cancers cells (Figure 7A-C).

The internalization of compound 9 - compound 5a of formula I - corresponds to 20% of the amount incubated per millions of cells (Figure 7C).

2.2.2 Internalization of compound 9 - compound 5 a of formula I - by breast tumors in vivo

2.2.2.1 Treatment of animals by subcutaneous peritumoral injection of compound 9 - compound 5a of formula I

We tested if 9 - compound 5a of formula I - can be detected on MDA-MB-231 tumors implanted on nude mice. As shown in Figure 8, treatment of tumors with compound 9 - compound 5a of formula I - lead to intratumor accumulation of this compound (200.3+31.2 ng/g tumor) with an uptake of 2.615+0.46 percent of the injected dose per gram of tumor (Figure 8B).

2.2.2.2 Treatment of animals by intravenous administration of compound 9 - compound 5 a of formula I

Radiotracers for cancer imaging are mainly injected intravenously for a short time of exposure. Nude mice xenografted with human breast cancer were exposed for 60 or 90 minutes to solvent vehicle or 7aF-CT injected intravenously. As shown in Figure 9, 7aF- CT, compound 9 - compound 5a of formula I - was detected in tumors as soon as 60 minutes after administration (11.76+1.47 ng/g tumor) and its intratumor level does not increase for longer time of exposure (90 min; 9.605+3.8 ng/g tumor) (Figure 9).

60 minutes after administration, the uptake of compound 9 - compound 5a of formula I - by the tumor was 0.67+0.17 percent of the injected dose per gram of tumor (Figure 9B). Consequently, our data demonstrate that these fluorinated oxysterols are internalized by breast cancer both in vitro and in vivo highlighting that these compounds in their ¹⁸F forms are promising as radiopharmaceuticals for the detection of cancers overexpressing HSD2.

Compound 9 - compound 5a of formula I - gives a good tumor %ID (% Injected dose) value that is in the range of what is expected for a potent imaging probe.

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Claims

1. A ¹⁸F -radiolab eled compound of formula (I): wherein:

R³ is H or OH; and R², R⁴, R⁵, R⁶, R⁷ and R¹² are independently selected from ¹⁸F or H;

R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide; and when R² is ¹⁸F, then R³ is OH and all R⁴, R⁵, R⁶, R⁷ and R¹² are H; when R⁴ is ¹⁸F, then R⁸ and R⁹ are each OH and all R², R³, R⁵, R⁶, R⁷ and R¹² are H; when R⁵ is ¹⁸F, then R³ is OH and all R², R⁴, R⁶, R⁷ and R¹² are H; when R⁶ is ¹⁸F, then R³ is OH and all R², R⁴, R⁵, R⁷ and R¹² are H; when R⁷ is ¹⁸F, then R³ is OH and all R², R⁴, R⁵, R⁶ and R¹² are H; and when R¹² is ¹⁸F, then R³ is OH and all R², R⁴, R⁵, R⁶ and R⁷ are H; and wherein at least one of R², R⁴, R⁵, R⁶, R⁷ and R¹² is a ¹⁸F.

2. The ¹⁸F-radiolabeled compound of formula (I) according to claim 1 selected from the group consisting of la, 2a, 3a, 4a, 5a, 6a, 7a, 8a, 9a, 10a and I la:

3. A composition comprising a ¹⁸F -radiolab eled compound of formula (I), according to any of claims 1 or 2, and at least one or more pharmaceutically acceptable excipient.

4. An intermediate compound in the synthesis of a ¹⁸F -radiolab eled compound of formula

(I) according to any of claims 1 or 2, wherein said intermediate compound has formula

(II): wherein: R¹, R⁵, R⁶, R⁷ and R¹² are independently selected from a group R¹⁰ or H;

R³ is independently selected from a group R¹⁰ or OH;

R⁸ and R⁹ are each OH or are linked to an oxygen atom forming together an epoxide; and

R¹⁰ is selected from the group consisting of a mesylate group, a tosyl group and a tritiate group; and wherein when R¹ is a group R¹⁰, then R³ is OH and all R⁵, R⁶, R⁷ and R¹² are H; when R³ is a group R¹⁰, then R⁸ and R⁹ are each OH, and all R¹, R⁵, R⁶, R⁷ and R¹² are H, being R¹⁰ a mesylate group or a tritiate group; when R⁵ is a group R¹⁰, then R³ is OH and all R¹, R⁶ and R⁷ and R¹² are H; when R⁶ is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁷ and R¹² are H; when R⁷ is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁶ and R¹² are H; and when R¹² is a group R¹⁰, then R³ is OH and all R¹, R⁵, R⁶ and R⁷ are H; and wherein at least one of R¹, R³, R⁵, R⁶, R⁷ and R¹² is a group R¹⁰.

5. The intermediate compound according to claim 4, wherein R¹⁰ is a mesylate group.

6. The intermediate compound according to any of claims 4 or 5 selected from the group consisting of lb, 2b, 3b, 4b, 5b, 6b, 7b, 8b, 9b, 10b and 11b:

7. A ¹⁸F -radiolab eled compound of formula (I) according to any of claims 1 or 2, or a composition according to claim 3, for use in a method for detecting the presence of a cancer in a patient, wherein the cancer is associated to cholesterol-5,6-epoxide hydrolase (ChEH) and/or 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression; and wherein said method comprises the steps of: administering the ¹⁸F-radiolabeled compound of formula (I), or a composition according to claim 3, to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal.

8. A ¹⁸F -radiolab eled compound of formula (I) according to any of claims 1 or 2, or a composition according to claim 3, for use in a method for detecting the presence of metastatic sites in a patient suffering from a primary tumor, wherein said primary tumor is associated to cholesterol-5,6-epoxide hydrolase (ChEH) and/or l ip- hydroxysteroid dehydrogenase type 2 (HSD2) overexpression; and wherein said method comprises the steps of: administering the ¹⁸F-radiolabeled compound of formula (I), or a composition according to claim 3, to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal.

9. A ¹⁸F -radiolab eled compound of formula (I) according to any of claims 1 or 2, or a composition according to claim 3, for use in a method for monitoring the efficacy of a treatment administered to a patient suffering from a cancer associated to cholesterol- 5,6-epoxide hydrolase (ChEH) and/or 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression; wherein said method comprises the steps of: administering the ¹⁸F-radiolabeled compound of formula (I), or a composition according to claim 3, to said patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting the positron emission tomography (PET) signal; and repeating said steps at different moments of the treatment.

10. A ¹⁸F -radiolab eled compound of formula (I) according to any of claims 1 or 2, or a composition according to claim 3, for use in a method for predicting the efficacy of a treatment targeting oncosterone biogenesis or with a treatment targeting an oncosterone downstream effector, wherein said method comprises the steps of: administering the ¹⁸F-radiolabeled compound of formula (I) according to any of claims 1 or 2, or a composition according to claim 3, to a patient; subjecting said patient to a positron emission tomography (PET) scanner; collecting a positron emission tomography (PET) signal; administering to the patient said treatment targeting oncosterone biogenesis or said treatment targeting an oncosterone downstream effector when the PET signal collected detects a cancer in said patient; wherein the treatment targeting oncosterone biogenesis is an anti-estrogen binding site (AEBS) ligand, an EBP inhibitor, a DHCR7 inhibitor, or a ChEH inhibitor; and wherein the treatment targeting an oncosterone downstream effector is a glucocorticoid receptor (NR3C1) modulator, or a Liver-X-Receptor (NR1H2, NR1H3) modulator.

11. The ¹⁸F-radiolabeled compound of formula (I) for use according to any of claims 7 to 10, wherein the cancer or primary tumor associated to cholesterol-5,6-epoxide hydrolase (ChEH) and/or 1 ip-hydroxy steroid dehydrogenase type 2 (HSD2) overexpression is selected from the group consisting of urothelial bladder cancer, invasive breast cancer, adrenal gland cancer, acute myeloid leukemia, chromophobe renal cell carcinoma, liver hepatocellular carcinoma, lung adenocarcinoma, squamous cell lung carcinoma, ovarian serous cystadenoma, pancreatic adenocarcinoma, rectal adenocarcinoma, uterine corpus endometrial carcinoma, acute lymphoblastic leukemia and osteosarcoma, stomach adenocarcinoma, esophageal carcinoma, colon adenocarcinoma, prostate adenocarcinoma, testicular germ cells tumors, thyroid carcinoma and uterine carcinosarcoma.

12. A method for obtaining an intermediate compound according to any of claims 4 to 6, wherein said method comprises: a. providing a compound of formula (Ila) or a compound of formula (lib) : wherein

R¹ , R⁵ , R⁶ , R⁷ and R¹² are independently selected from a group -OH or H;

R³ is independently selected from a group -OR¹¹ or -OH;

R¹¹ is an alcohol protecting group; and wherein when R³ is a group -OH, then all of R¹ , R⁵ , R⁶ and R⁷ are H and R¹² is H or OH; when R³ is a group -OR¹¹, then only one of R¹ , R⁵ , R⁶ , R⁷ and R¹² is -OH; b. activating a hydroxyl moiety with an alcohol activating group selected from the group consisting of a mesylate group, a tosyl group and a triflate group when R³ is a group - OR¹¹ or when both R³ and R¹² are OH; or activating a hydroxy moiety with an alcohol activating group selected from the group consisting of a mesylate group and a triflate group when R³ is a group -OH; c. optionally conducting an epoxidation of a double bond; d. hydrolyzing the group -OR¹¹, when present, to obtain a group -OH; e. optionally hydrolyzing the epoxide when present; f. oxidizing the double bond, when present, to obtain a vicinal diol.

13. The method for obtaining an intermediate compound according to claim 12, wherein the alcohol protecting group is an acyl group or a silyl group.

14. The method for obtaining an intermediate compound according to any of claims 12 or 13, wherein, when R³ is a group -OR¹¹, R¹ is OH, and all R⁵ , R⁶ , R⁷ and R¹² are H, said method further comprises prior to step (a): conducting an allylic oxidation of a compound of formula (lie): to obtain an allylic alcohol; and conducting an epoxidation of the double bond to obtain a compound of formula (lib).

15. A method for obtaining a ¹⁸F -radiolab eled compound of formula (I) according to any of claims 1 or 2, wherein said method comprises: providing an intermediate compound of any of claims 4 to 6 or an intermediate compound obtainable according to the method of any of claims 12 to 14; reacting said intermediate compound with a ¹⁸F -fluorinated agent in a polar solvent.