Cyclophilin modulators

Abstract

The present invention provides compounds for use in selectively modulating cyclophilin mediated processes in general. In particular, the invention provides compounds for use in treating diseases such as hyper-proliferative disorders, immunity and inflammation disorders and metabolic dysfunction associated steatohepatitis. Additionally or alternatively, the present invention particularly provides compounds for use in engaging an organism's immune system to target a specific cell, specific cells, a cell-type, an organelle or an organ in that organism.

Description

[0001] CYCLOPHILIN MODULATORS

[0002] Field of the Invention

[0003] Compounds are provided that modulate Cyclophilin activity, including compounds that selectively bind to CypA, CypB and CypD over other Cyclophilin isoforms. In particular, the compounds bind to CypA, CypB and CypD, but show selective binding to CypB or CypD over CypA, preferably selective binding to CypB over CypA.

[0004] The invention also provides methods of using cyclophilin inhibitory compounds to modulate cyclophilin mediated processes in vitro and in vivo, including methods of treating diseases, such as hyper-proliferative disorders (e.g. cancer), immunity and inflammation disorders and metabolic dysfunction associated steatohepatitis (MASH). Additionally or alternatively, the compounds may be used to engage an organism’s immune system to target a specific cell, specific cells, a cell-type, an organelle or an organ in that organism. For instance, where those specific cells are a cancer.

[0005] Background of the Invention

[0006] Breast cancer is the most common cancer worldwide; predicted to account for ~31 % of new cancer cases in 2022 and be the second most common cause of cancer death among women.

[0007] Triple negative breast cancer (TNBC) is characterised by the absence of expression of the oestrogen and progesterone receptors, alongside absent or low expression of the HER2 receptor. It accounts for about 12-20% of all breast cancer cases.

[0008] Most breast cancer therapies target one or more of the oestrogen, progesterone or HER2 receptors. As these receptors are scarce or absent in TNBC, typical first-line treatments rely on non-specific interventions such as surgical resection, chemotherapy and radiotherapy.

[0009] These non-specific treatments have resulted in response rates as high as 70% for some subtypes of TNBC, but have high toxicity and relapse rates, with early recurrence and lower 5-year overall survival compared to patients with other breast cancers. For instance, up to 75% of TNBC patients die within 3 months after recurrence.

[0010] Compared to other breast cancer subtypes, TNBC is heterogeneous, aggressive and highly metastatic. As such, the prognosis can be poorer.

[0011] Thus, there remains a need for therapeutics (preferably targeted therapeutics) that can treat hyper-proliferative diseases such as cancers, particularly TNBC.

[0012] Cyclophilins (Cyps) are a family of proteins where family members can both act as molecular chaperones (vesicular transport adaptor activity), and act as peptidyl prolyl cis-trans isomerases, known to catalyse the isomerization of peptidyl-prolyl amide bonds in unfolded and native proteins (PPIase activity).

[0013] Cyclophilins are known to be implicated in many diseases and injuries such as viral and parasitic infections, cardiovascular diseases, ischemia / reperfusion injury, diabetes, chronic and acute inflammatory disorders, cancers, neurodegenerative disorders, traumatic brain injury and other diseases associated with mitochondrial disorders.

[0014] For example, CypA is believed to play a critical role in the replication of the hepatitis C virus. Changes in expression of various Cyp isoforms have been associated with various cancers. CypA is overexpressed in human pancreatic cancer cells, non-small cell lung cancer and endometrial carcinoma. Both CypA and CypB are associated with breast cancer.

[0015] CypA inhibitors have demonstrated clinical efficacy for treating hepatitis C infection and the mechanism of action suggests that similar efficacy would be observed for other viruses such as Dengue, West Nile or yellow fever viruses. Research has implicated extracellular CypA and CypB in inflammation- mediated diseases such as asthma, severe sepsis, rheumatoid arthritis, COPD, age related macular degeneration among others.

[0016] Initial studies suggest that CypB and / or CypD (particularly CypB) inhibition may also protect the liver from fibrosis, including fibrosis after metabolic dysfunction associated steatohepatitis (MASH), a common indication for the need for liver transplantation after chronic hepatitis C.

[0017] CypD is an essential regulator of the opening of the mitochondrial permeability transition pore (MPTP) located in the mitochondrial matrix.

[0018] The activation of the MPTP has been implicated in broad acute and traumatic events such as: spinal cord injury, traumatic brain injury, ischaemia / reperfusion injury, stroke, myocardial infarction, acute pancreatitis.

[0019] The activation of the MPTP has also been implicated in chronic diseases such as congestive heart failure, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, macular degeneration, epilepsy, diabetic retinopathy, liver disease and other diseases due to mitochondrial dysfunction.

[0020] NKTR is a cyclophilin-related protein receptor found in various cell types, including cells of the immune system, such as natural killer cells and a subset of T cells. In natural killer and T cells, NKTR mediates killing of target cells.

[0021] Due to the high similarity among the different cyclophilin isoforms, the design of different ligands that selectively bind to a specific cyclophilin subtype represents a major challenge in medicinal chemistry. At present, the cyclophilin inhibitors that have entered clinical studies are pan-selective inhibitors. This may limit engagement of CypB or CypD, due to the large relative abundance of CypA. Thus, a need exists for cyclophilin inhibitors which selectively bind to CypB and / or CypD over CypA. As shown in Figure 1 , the Cyps active site consists in two main sub-pockets, "Pro” and "Abu”. Both pockets have been targeted by various Cyps binders. Beyond the two main pockets the cyclophilin’s active site contains a more remote "3 o’clock” pocket, which is more variable between different Cyp isoforms.

[0022] Cyclosporine A (CsA) is a non-specific inhibitor of the Cyps family. It is widely used for inhibition of CypA, however, CsA also inhibits CypB, CypD and other Cyps.

[0023] CsA also has immunosuppressant effects and is used as a medication to treat rheumatoid arthritis, psoriasis, Crohn’s disease, nephrotic syndrome and to prevent organ transplants rejection.

[0024] So, while CsA could be used to inhibit CypA and CypB to treat cancer, CsA is unsuitable for this use due to its immunosuppressive properties. These properties are not a result of CsA binding to Cyps but from the interaction between the Cyp-CsA complex with calcineurin.

[0025] Additionally, CsA only crosses the blood-brain barrier to a very limited extent and has well documented side-effects such as kidney and liver dysfunction, pancreatitis, diarrhoea, convulsions, and ulcers which prevent its use in a number of Cyp-implicated conditions.

[0026] PCT / EP2019 / 073106, De Simone et al., Chemical Science, 2019, 10, 542 and Ahmed-Belkacem et al., Nature Communications, 7, 12777 (2016) also disclose various cyclophilin inhibitors - these documents are incorporated herein by reference.

[0027] Cyclophilin inhibitors have demonstrated therapeutic activities in many disease models, but no drug candidates have yet advanced completely through development to market. Thus, there remains a need for compounds providing inhibition of Cyclophilins with enhanced potency and / or isoform selectivity and / or improved toxicity over existing compounds.

[0028] The present invention provides cyclophilin modulators; small molecules that bind simultaneously to the Cyps active site (Abu and Pro pockets), and the 3 o’clock pocket. The binding mode of these novel compounds achieves enhanced potency, isoform selectivity and / or reduced genotoxicity.

[0029] Summary of the Invention

[0030] The present invention provides novel compounds with improved cyclophilin selectivity and binding properties. The compounds selectively bind to CypA, CypB and CypD, over other Cyclophilin proteins. In particular, the compounds bind to CypA, CypB and CypD, but show selective binding to CypB or CypD over CypA, preferably selective binding to CypB over binding to CypA.

[0031] Without wishing to be bound by theory, it is believed that the compounds are also able to modulate the cyclophilin family member NKTR in a manner which enhances its expression and / or function.

[0032] In mammalian cells (particularly human cells), CypA is localised extracellularly, and is present at significantly higher concentrations than CypB or CypD, which are respectively localised to the endoplasmic reticulum and the mitochondria.

[0033] Without wishing to be bound by theory, it is considered that compounds that selectively bind to CypB and / or CypD over CypA, preferably selectively binding CypB over CypA are advantageous over compounds that are not selective between CypA, CypB and CypD (such as CsA). Otherwise, the high concentration and extracellular accessibility of CypA is considered to effectively sequester the compound from the extracellular milieu before it can bind to CypB and / or CypD. Thus, this binding profile ensures that CypB and / or CypD are modulated by the compound. As noted above, the ability to modulate CypB and / or CypD makes such compounds suitable for treating certain disease states.

[0034] The invention also provides pharmaceutical compositions containing the novel compounds, as well as a method for the treatment and / or prevention cyclophilin-mediated diseases and disorders using the novel compounds - particularly treating and / or preventing CypB and / or CypD mediated diseases and disorders using the compounds of the present invention.

[0035] Additionally or alternatively, the inventors were surprised to discover that these compounds display a biological activity that is previously unknown for cyclophilin inhibitors. The compounds of the present invention can potentiate cytolysis of cells (particularly cancer cells, especially TNBC cells) by lymphocytes. This has been demonstrated in vitro, where the compounds have been used to pre-treat and concurrently treat natural killer (NK) cells and CD8+ T cells, increasing their ability to kill cancer cells, thus suppressing growth rate in cancer cell lines. This activity provides a use of the compounds in boosting immune cell cytolytic ability and cell cytolysis, especially in anticancer treatment regimens, particularly against TNBC.

[0036] As such, the compounds of the present invention may be used as immunoadjuvants in the treatment or prevention of a disease, in particular oncoadjuvants.

[0037] This effect is present when used as a standalone treatment, but additionally or alternatively and without wishing to be bound by theory, it is believed that they will act synergistically when combined with either traditional cancer therapies or traditional immunotherapies. Suitable combination therapies include those that enhance tumour-killing lymphocytes (e.g. ex-vivo activated NK cells, T cells and B cells or genetically modified lymphocytes including CAR NK and CAR T-cell infusion, as oncoadjuvants increase CAR-T cell persistence), or immune checkpoint inhibitors, or agents which counteract resistance to immune checkpoint inhibition or agents which activate or enhance the anti-cancer ability of lymphocytes or other immune cells with anti-cancer function, or agents which reverse T-cell exhaustion or agents which reprogram immunosuppressive tumour-associated macrophages to non-suppressive phenotypes or agents which reverse immunosuppressive signalling within the tumour microenvironment.

[0038] This immunoadjuvant / oncoadjuvant effect in the treatment or prevention of a disease (either alone or in combination with either traditional cancer therapies or immunotherapies) is particularly suitable for treating cancers with high level of tumour infiltrating lymphocytes (TILs), such as TNBC.

[0039] Without wishing to be bound by theory, it is believed that this immunoadjuvant activity is due to the compounds modulating the cyclophilin family member NKTR in a manner which enhances its expression and / or function.

[0040] Some aspects of the present invention are to provide compounds that can selectively modulate the biological activity of human cyclophilins (particularly CypB and / or CypD) and / or act as immunoadjuvants, in particular oncoadjuvants. As such, these compounds promote medical treatment of diseases mediated by cyclophilins dysfunction (particularly CypB and / or CypD).

[0041] Additionally or alternatively, some embodiments of the present invention advantageously include novel moieties suitable for providing the desired immunoadjuvant activity and / or cyclophilin selective activity, while avoiding the use of an unsubstituted aniline moiety.

[0042] This is advantageous, given that unsubstituted anilines are readily metabolised into genotoxic imminoquinones. However, it is the unsubstituted aniline moiety (or its equivalent) that engages the Abu pocket (see Figure 1 ). The Abu pocket shows more variability than the Pro pocket within the cyclophilin family, thus compounds with moieties that can engage this pocket provide some of the selectivity between cyclophilin subtypes. Thus, while replacing the unsubstituted aniline moiety to reduce / remove genotoxicity is beneficial, doing so may result in either a non-selective Cyp modulator, or may remove Cyp activity entirely.

[0043] However, in some embodiments of the present invention, compounds are provided that do not have an unsubstituted aniline moiety at position A that show a lack of genotoxicity while retaining the desired Cyp selectivity profile.

[0044] Without wishing to be bound by theory, it is believed that the compounds of the present invention do not possess immunosuppressive activity via inhibition of calcineurin phosphatase activity (unlike CsA), given that urea- arylpyrrolidine structures have been found to lack calcineurin inhibition activity (Ahmed-Belkacem et al., Nature Communications, 7, 12777 (2016) - herein incorporated by reference).

[0045] In a first aspect, the present invention provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof: wherein

[0046] R1is selected from a group consisting of halo, -OCH3, -SCH3, -CF3, -CF2H, cyclopropyl, -Et and isopropyl,

[0047] A is selected from a group consisting of: a) , wherein;

[0048] R2is selected from a group consisting of -NH2, -OH, -SH, -CF2H and halo, and

[0049] Y1is selected from a group consisting of methine (=CH-) and nitrogen (=N-); where Y1is methine, its hydrogen is optionally substituted with fluoro or chloro, and b) A 6-membered unsaturated ring having 0, 1 or 2 nitrogen atoms, where the ring is substituted at the para position with an amine, optionally where the ring is independently substituted with 1 or 2 fluoro or chloro groups.

[0050] B is: wherein

[0051] Q is selected from a group consisting of carbon, oxygen, sulfur and nitrogen,

[0052] Z1is selected from a group consisting of carbon and nitrogen,

[0053] Z2is selected from a group consisting of carbon and nitrogen,

[0054] R3selected from a group consisting of -CH3, -CF3, -CF2H and a Co-3-alkyl substituted with a moiety selected from a group consisting of -COOH, -SOOH, -SOOOH, -SOONHCH3, -NHSOOCH3, -CONHSOOCH3, -NHCONHSOOCH3, -SOOCi-3-alkyl, -CONHOH, a 3, 4, 5 or 6-membered saturated ring and a 5 or 6-membered unsaturated ring having 0, 1 , 2, 3 or 4 heteroatoms selected from nitrogen, oxygen or sulfur and optionally substituted with carbonyl, sulfonyl or hydroxy.

[0055] In some embodiments, R1is selected from a group consisting of -SCH3, -Br, - Cl, -CF3,-CF2H and cyclopropyl, preferably -SCH3, -Br and -Cl, more preferably -SCH3 and -Br, more preferably R1is -SCH3. Without wishing to be bound by theory, it is considered that these moieties provide compounds with an optimised binding coefficient to the relevant cyclophilins.

[0056] In some embodiments, A is either: a) , wherein;

[0057] R2is selected from a group consisting of -NH2, -OH, -SH, -CF2H and halo, preferably a group consisting of -NH2 and -OH, further preferably where R2is -OH, and

[0058] Y1is selected from a group consisting of methine (=CH-) and nitrogen (=N-); where Y1is methine, its hydrogen is optionally substituted with fluoro or chloro, preferably Y1is methine, preferably, where A is: b) wherein

[0059] Y2is selected from a group consisting of methine (=CH-) and nitrogen (=N-); where Y2is methine, the hydrogen is optionally substituted with chloro or fluoro, further preferably chloro, and

[0060] Y3is selected from a group consisting of methine (=CH-) and nitrogen (=N-); where Y3is methine, the hydrogen is optionally substituted with chloro or fluoro, further preferably chloro.

[0061] Preferably, Y3is selected from a group consisting of methine (=CH-) and nitrogen (=N-); where Y3is methine, the hydrogen is substituted with chloro or fluoro, further preferably chloro.

[0062] Alternatively, wherein Y2and Y3are selected from a group consisting the following: i. Y2is nitrogen and Y3is nitrogen, ii. Y2is nitrogen and Y3is methine substituted with chloro (=C(CI)- ), iii. Y2is methine substituted with chloro (=C(CI)-) and Y3is unsubstituted methine (=CH-), and iv. Y2is methine substituted with fluoro (=C(F)-) and Y3is methine substituted with fluoro (=C(F)-).

[0063] In some embodiments, A is

[0064] Y2is selected from a group consisting of methine (=CH-) and nitrogen (=N-); where Y2is methine, the hydrogen is optionally substituted with chloro or fluoro, further preferably chloro, and

[0065] Y3is selected from a group consisting of methine (=CH-) and nitrogen (=N-); where Y2is methine, the hydrogen is optionally substituted with chloro or fluoro, further preferably chloro.

[0066] Preferably, Y3is selected from a group consisting of methine (=CH-) and nitrogen (=N-); where Y2is methine, the hydrogen is substituted with chloro or fluoro, further preferably chloro. Alternatively, wherein Y2and Y3are selected from a group consisting the following: a) Y2is nitrogen and Y3is nitrogen, b) Y2is nitrogen and Y3is methine substituted with chloro, c) Y2is methine substituted with chloro and Y3is unsubstituted methine and d) Y2is methine substituted with fluoro and Y3is methine substituted with fluoro.

[0067] In an embodiment, A is selected from a group consisting of:

[0068] n

[0069] R6is selected from a group consisting of -H, halo and -CF3, preferably -H, fluoro and chloro, further preferably -H and chloro, and wherein;

[0070] R7is selected from a group consisting of -H, halo and -CF3, preferably -H and halo, further preferably -H, fluoro and chloro, and

[0071] R8is selected from a group consisting of -H, halo and -CF3, preferably halo and -CF3, further preferably halo, further preferably fluoro and chloro.

[0072] Alternatively, wherein R7and R8are either: i. both fluoro, or ii. R7is -H and R8is chloro

[0073] Suitably, A may be selected from a group consisting of: wherein

[0074] R6is selected from a group consisting of -H, halo and -CF3, preferably -H, fluoro and chloro, further preferably -H and chloro, and , wherein; R7is selected from a group consisting of -H, halo and -CF3, preferably

[0075] -H and halo, further preferably -H, fluoro and chloro, and

[0076] R8is selected from a group consisting of -H, halo and -CF3, preferably halo and -CF3, further preferably halo, further preferably fluoro and chloro.

[0077] Alternatively, wherein R7and R8are either: iii. both fluoro, or iv. R7is -H and R8is chloro

[0078] In some embodiments, A is selected from a group consisting of:

[0079] wherein A is selected from a group consisting of: In some embodiments, A is selected from a group consisting of: preferably, wherein A is selected from a group consisting of:

[0080]

[0081] In some embodiments where A is the 6-membered unsaturated ring, A is not:

[0082] Without wishing to be bound by theory, it is believed that where A is the 6- membered unsaturated ring and is not: the compounds provide reduced genotoxicity, while maintaining activity and selectivity. This may be due to destabilising the nitrenium ion.

[0083] In some embodiments, B is wherein

[0084] Q is selected from a group consisting of carbon, sulfur and nitrogen, preferably sulfur and nitrogen,

[0085] Z1is selected from a group consisting of carbon and nitrogen,

[0086] Z2is selected from a group consisting of carbon and nitrogen, R3selected from a group consisting of -CH3, -CF3, -CF2H and a Co-3-alkyl substituted with a moiety selected from a group consisting of -COOH, a 3, 4, 5 or 6-membered saturated ring and a 5 or 6-membered unsaturated ring having 0, 1 , 2, 3 or 4 heteroatoms selected from nitrogen, oxygen or sulfur and optionally substituted with carbonyl, sulfonyl or hydroxy.

[0087] Preferably, R3is selected from a group consisting of -CH3, a 3, 5 or 6- membered saturated ring, and a 6-membered unsaturated ring.

[0088] Without wishing to be bound by theory, it is believed that an R3with an acid bioisostere (e.g. a Co-3-alkyl substituted with a moiety selected from a group consisting of -COOH, -SOOH, -SOOOH, -SOONHCH3, -NHSOOCH3, - CONHSOOCH3, -NHCONHSOOCH3, -SOOCi-3-alkyl, -CONHOH, tetrazole, 5-oxo-1 ,2,4-oxadiazole, 5-oxo-1 ,2,4-thiadiazole, 2-thioxo-1 ,3,4-oxadiazole, 1 ,2,4-triazole, Imidazole, preferably -CH2-COOH or -CH2-tetrazole) assists withs selective binding to NKTR over other cyclophilin proteins.

[0089] In some embodiments, B is selected from a group consisting of: is selected from a group consisting of:

[0090] selected from a group consisting of:

[0091] Without wishing to be bound by theory, it is believed that the claimed moieties at B (particularly the above embodiments listed as “preferably” and “further preferably”) provide an optimised balance between binding constant to the relevant cyclophilins and partitioning coefficient. This permits optimisation between cell permeability and solubility.

[0092] In exemplary embodiments, the compound is selected from a group consisting of:

[0093] 13 In an alternative embodiment, the compound is selected from a group consisting of Compounds 1 , 2, 4, 7, 8, 9, 10 and 11 , due to decreased genotoxicity, while maintaining activity and selectivity.

[0094] In a further alternative embodiment, the compound is selected from a group consisting of Compounds 1 , 2, 3, 7, 8, 9, 10 and 11 , due to increased cyclophilin selectivity. Preferably, the compound is selected from a group consisting of Compounds 1 , 2, 7, 8, 9, 10 and 11 , due to decreased genotoxicity, while maintaining activity and selectivity.

[0095] In a further alternative embodiment, the compound is selected from a group consisting of Compounds 1 , 2, 7 and 8, due to increased cyclophilin selectivity and activity; and positive DMPK activity.

[0096] In a further alternative embodiment, the compound is selected from Compounds 1 and 7, due to comparatively high cyclophilin selectivity and activity; and positive DMPK activity.

[0097] In a further alternative embodiment, the compound is selected from Compounds 1 , 3, 5, 6, 7, 8 and 11 , due to strong binding constants to NKTR. Preferably, the compound may be selected from Compounds 1 , 3, 5, 7 and 8. Further preferably, the compound is selected from Compounds 1 and 3, due to their ability to enhance natural killer cell killing. In some embodiments of any aspect of the invention, the present invention excludes any one or more of the following compounds:

[0098] 14 15 16

[0099] In a second aspect, the present invention provides a compound of formula (II) or a pharmaceutically acceptable salt thereof: wherein

[0100] R1’ is selected from a group consisting of bromo and -SCH3,

[0101] A’ is selected from a group consisting of:

[0102] B’ is selected from a group consisting of: In some embodiments, A’ is selected from a group consisting of

[0103]

[0104] In some embodiments, A’ is selected from a group consisting of: preferably, wherein A’ is selected from a group consisting of:

[0105]

[0106] In some embodiments where A’ is the 6-membered ring, A’ is not:

[0107] In some embodiments, B’ is selected from a group consisting of:

[0108] In a third aspect, the present invention provides a compound of Formula (III) or a pharmaceutically acceptable salt thereof: wherein

[0109] R1” is selected from a group consisting of bromo and -SCH3, A" is selected from a group consisting of:

[0110] In one embodiment of the invention, a compound of the first, second or third aspect, or a pharmaceutically acceptable salt thereof can modulate CypA, CypB and CypD activity in biochemical and cell-based assays and exhibit therapeutic activity in medical conditions in which CypA, CypB and / or CypD modulation is suitable to treat the medical conditions. Preferably, where a compound of the first, second or third aspect, or a pharmaceutically acceptable salt thereof can selectively modulate CypB and / or CypD activity over CypA (preferably selectively modulating CypB over CypA) activity in biochemical and cell-based assays and exhibit therapeutic activity in medical conditions in which CypB and / or CypD modulation is suitable to treat the medical conditions.

[0111] In one embodiment of the invention a compound of the first, second or third aspect, or a pharmaceutically acceptable salt thereof can enhance the ability of cells of the immune system to recognise and kill a specific cell, a cell type, an organelle or an organ. Without wishing to be bound by theory, it is believed that this is accomplished at least in part by modulating the cyclophilin family member NKTR in a manner which enhances its expression and / or function.

[0112] In a fourth aspect, the present invention provides a pharmaceutically acceptable composition comprising a compound of the first, second or third aspect, or a pharmaceutically acceptable salt thereof. The composition may comprise the compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. Suitably, the composition may be for use in the treatment of a CypA- mediated, CypB-mediated and / or CypD-mediated condition. Preferably, the condition is a CypB-mediated and / or CypD-mediated condition.

[0113] The amount of the compound in the composition is such that it is effective to measurably modulate CypA, CypB and / or CypD (preferably an amount that is selective for effective to measurably modulate CypB and / or CypD over CypA, further preferably measurably modulating CypB over CypA) in a biological sample or in a patient. Additionally or alternatively, the amount of the compound in the composition is such that it is effective to act as an immunoadjuvant / oncoadjuvant in a biological sample or in a patient.

[0114] In some embodiments, the composition further comprises a cancer therapy or an immunotherapy. In other embodiments, the composition is configured to be provided to a patient in need thereof concurrently or consecutively with a cancer therapy or an immunotherapy.

[0115] Suitable cancer therapies or immunotherapies include those that enhance tumour-killing lymphocytes (e.g. ex-vivo activated NK cells, T cells and B cells or genetically modified lymphocytes including CAR NK and CAR T-cell infusion, as oncoadjuvants increase CAR-T cell persistence), or immune checkpoint inhibitors, or agents which counteract resistance to immune checkpoint inhibition or agents which activate or enhance the anti-cancer ability of lymphocytes or other immune cells with anti-cancer function, or agents which reverse T-cell exhaustion or agents which reprogram immunosuppressive tumour-associated macrophages to non-suppressive phenotypes or agents which reverse immunosuppressive signalling within the tumour microenvironment.

[0116] In a fifth aspect, the present invention provides a compound of the first, second or third aspect, or a pharmaceutically acceptable salt thereof for use in medicine, preferably in the treatment of a CypA-mediated, CypB-mediated and / or CypD-mediated condition or for modulation of CypA, CypB, CypD CypA-like, CypB-like and / or CypD-like protein induced signalling in cells wherein such signalling modulation is beneficial for the treatment of a condition. More preferably, in the selective treatment of a CypB-mediated and / or CypD-mediated condition over a CypA-mediated condition or for selective modulation of CypB, CypD CypB-like and / or CypD-like protein induced signalling in cells over CypA and / or CypA-like protein induced signalling in cells, wherein such signalling modulation is beneficial for the treatment of a condition, wherein CypA signalling modulation is neutral or negative for the treatment of the condition. Additionally or alternatively, the compound or pharmaceutically acceptable salt thereof for use is for use as an immunoadjuvant or oncoadjuvant.

[0117] Suitably, such a use in medicine may be in combination with a cancer therapy or an immunotherapy. Suitable cancer therapies or immunotherapies include those that enhance tumour-killing lymphocytes (e.g. ex-vivo activated NK cells, T cells and B cells or genetically modified lymphocytes including CAR NK and CAR T-cell infusion, as oncoadjuvants increase CAR-T cell persistence), or immune checkpoint inhibitors, or agents which counteract resistance to immune checkpoint inhibition or agents which activate or enhance the anti-cancer ability of lymphocytes or other immune cells with anti-cancer function, or agents which reverse T-cell exhaustion or agents which reprogram immunosuppressive tumour-associated macrophages to non-suppressive phenotypes or agents which reverse immunosuppressive signalling within the tumour microenvironment.

[0118] In a fifth aspect, the present invention provides a method for modulating CypA, CypB and / or CypD (preferably selective for CypB and / or CypD over CypA, further preferably selective for CypB over CypA) in a patient or in a biological sample, comprising the step of administering to said patient or contacting said biological sample with a compound of the first, second or third aspect, or a pharmaceutically acceptable salt thereof. The compounds are characterized by such a high and selective affinity to CypA, CypB and / or CypD, preferably selectively CypB and / or CypD over CypA (further preferably selectively for CypB over CypA), which ensures a reliable binding and preferably modulation of CypB and / or CypD. In certain embodiments, the substances are mono-specific in order to guarantee an exclusive and directed recognition with the single CypB target.

[0119] In certain embodiments, the method for modulating CypA, CypB and / or CypD is performed in-vitro.

[0120] In certain embodiments, the invention provides a method for preventing, treating or ameliorating in a subject a disease, disorder, or condition that is causally related to the aberrant activity of CypA, CypB and / or CypD, preferably selectively CypB and / or CypD over CypA activity (further preferably selectively CypB over CypA activity), which comprises administering to a subject a therapeutically effective amount of a compound of any formulae herein, or a pharmaceutically acceptable salt thereof.

[0121] In certain embodiments, the present invention provides a method for modulating CypA, CypB and / or CypD, preferably selectively CypB and / or CypD activity over CypA activity (further preferably selectively CypB activity over CypA activity) in a patient comprising administering to the patient a compound of any formulae herein, or a pharmaceutically acceptable salt thereof, or composition of the present invention. In another embodiment, the present invention provides a method for modulating CypA, CypB and / or CypD, preferably selectively CypB and / or CypD activity over CypA activity, further preferably selectively CypB activity over CypA activity in a biological sample comprising administering a compound of any formulae herein, or a pharmaceutically acceptable salt thereof, or a composition of the present invention.

[0122] The condition may be selected from a group consisting of hyper-proliferative disorders (e.g., cancer), fibrosis (e.g. fibrosis of the kidney, liver, lung or pancreas), cardiac failure, viral infections, chronic and acute inflammatory disorders, spinal cord injury, traumatic brain injury, ischaemia / reperfusion injury, stroke, myocardial infarction, acute pancreatitis, chronic mitochondrial disorders (e.g. congestive heart failure, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, macular degeneration, epilepsy, diabetic retinopathy, liver disease and other diseases due to mitochondrial dysfunction), neurodegenerative disorders, diabetes and metabolic dysfunction associated steatohepatitis (MASH).

[0123] In particular, the condition may be cancer, Alzheimer's disease, Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), Dementia, Multiple Sclerosis, Huntington's disease. Preferably, the cancer is a cancer with a high level of tumour infiltrating lymphocytes (TILs). More preferably, the cancer is TNBC.

[0124] The condition may be, pneumonia, bacteremia, trauma, tuberculosis, parasitic disease, neuro-inflammation, schizophrenia, depression, neurodegenerative disease, and pain.

[0125] Where the condition is a viral infection, it may be selected from Human Immunodeficiency Virus (HIV), Hepatitis A-D, Human Papilloma virus (HPV), Dengue, West Nile, yellow fever and Herpes, including Herpes Simplex I and II; as well as the Epstein Barr Virus.

[0126] Where the condition is an inflammatory disorder, it may be selected from asthma, severe sepsis, rheumatoid arthritis, COPD and age-related macular degeneration.

[0127] The condition may be a condition treatable using immunotherapy.

[0128] In certain embodiments, the invention provides for the treatment of diseases, disorders, and conditions characterized by excessive or abnormal cell proliferation. Such diseases include a proliferative or hyper-proliferative disease. Examples of proliferative and hyper-proliferative diseases include cancer and myeloproliferative disorders. In another aspect, the invention provides for a kit consisting of separate packs of an effective amount of a compound according to the invention and / or pharmaceutically acceptable salts thereof, or a composition of the invention and optionally, an effective amount of a further active ingredient such as a cancer therapy or immunotherapy.

[0129] The kit may comprise suitable containers, such as boxes, individual bottles, bags or ampoules. The kit may, for example, comprise separate ampoules, each containing an effective amount of a compound according to the invention and / or pharmaceutically acceptable salts or a composition of the invention, and an effective amount of a further active ingredient in dissolved or lyophilized form.

[0130] Compounds and / or pharmaceutically acceptable salts thereof, or compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. Preferably, they are administered orally, intraperitoneally or intravenously. Most preferably, pharmaceutically acceptable compositions of this invention are formulated for oral administration. Exemplary oral dosage forms are capsules, tablets, aqueous suspensions or solutions. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation.

[0131] The compounds and compositions, according to the method of the present invention, are administered using any amount and any route of administration effective for treating or lessening the severity of a disorder provided above. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition, the particular agent, its mode of administration, and the like.

[0132] Compounds and / or pharmaceutically acceptable salts thereof, or compositions of the present invention may be administered to humans and other animals. Brief Description of the Figures

[0133] Figure 1 : Type-ll binding mode of a compound of the present invention in the PPIase domain of CypA.

[0134] Figure 2: Correlation between the SPR CypD Kd values of Table 2 and the HepG2 ECso values of Table 3, confirming the mode of action relies on cyclophilin targeting.

[0135] Figure 3: Effect of Compounds on Proliferation of Cell Lines Representing 3 TNBC Subtypes, showing fold increase in the number of cells after 120 h treatment with compound, compared to the initial number of cells analysed by 2 -way ANOVA and Dunnett post hoc, n = 4.

[0136] Figure 3A: Effect of compounds on proliferation of the MDA-MB-231 cell line (mesenchymal stem-like subtype).

[0137] Figure 3B: Effect of compounds on proliferation of the HCC1806 cell line (basal-like 2 subtype) and

[0138] Figure 3C: Effect of compounds on proliferation of the BT549 cell line (mesenchymal-like subtype).

[0139] Figure 4: Effect of Compounds on Lymphocyte Cytolysis of Target TNBC Cell Line.

[0140] Figure 4A: Apoptosis induced in MDA-MB-231 cells with compound alone. AUC analysed by 1-way ANOVA and Dunnett post hoc with comparison to vehicle, n=3.

[0141] Figure 4B: Apoptosis induced in MDA-MB-231 cells with compound and NK92 cells. AUC analysed by 1-way ANOVA and Dunnett post hoc with comparison to vehicle, n=3. Figure 4C: Proliferation of MDA-MB-231 cells with compound and NK92 cells. Fold increase in the number of cells after 72 h treatment with compound compared to the initial number of cells analysed by 2-way ANOVA with Dunnett post hoc, n = 3.

[0142] Figure 4D: Apoptosis induced in MDA-MB-231 cells with compound and primary NK cells. AUC analysed by 1 -way ANOVA and Dunnett post hoc with comparison to vehicle, n=4.

[0143] Figure 4E: Apoptosis induced in MDA-MB-231 cells with compound and primary T cells. AUC analysed by 1-way ANOVA and Dunnett post hoc with comparison to vehicle, n=3.

[0144] Figure 5: Effect of compounds on Activation-associated Clustering of Primary Human CD8+ T-cells.

[0145] Figure 6: Primary human T-cell Cluster Formation 120 h Following Activation and Treatment with compounds. Mean number of clusters present per well at 120 h was analysed by 1-way ANOVA and Dunnett post hoc with comparison to vehicle (n=3).

[0146] Figure 7: Primary human CD8+ T-cell Cluster Size 120 h Following Activation and Treatment with compounds. Mean cluster size was analysed by 1-way ANOVA and Dunnett post hoc with comparison to vehicle (n=3).

[0147] Figure 8:

[0148] Figure 8A: Primary human CD8+ T-cell Confluence 120 h Following Activation and Treatment with compounds. Mean confluence was analysed by 1-way ANOVA and Dunnett post hoc with comparison to vehicle (n=3).

[0149] Figure 8B: Primary human T-cell Cluster Formation 120 h Following Activation and Treatment with compounds. Mean confluence of cells in clusters relative to total cell confluence at 120 h was analysed by 1-way ANOVA and Dunnett post hoc with comparison to vehicle (n=3).

[0150] Figure 9: Potentiation of cytolysis of cancer cells by lymphocytes. Rate of apoptosis of TNBC cells induced by NK92 cells is increased by pre-treatment of NK92 cells with a compound of the present invention.

[0151] Figure 10: Apoptotic fraction of target MDA-MB-231 population following incubation with cyclophilin inhibitors.

[0152] A. Apoptosis Timecourse with Incubation with Inhibitor Alone (AUC analysis);

[0153] B. Apoptosis Timecourse with Incubation with NK92 Cells and Inhibitor (AUC analysis);

[0154] C. Apoptosis with Incubation with Inhibitor Alone at 72 h Timepoint;

[0155] D. Apoptosis with Incubation with NK92 Cells and Inhibitor at 48 h Timepoint. 1-way ANOVA was applied with Dunnett’s test post-hoc.

[0156] Values are the mean ± SEM, n=4, * p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001

[0157] Figure 11 : Images of NK92 cells treated with cyclophilin inhibitors. Cells were treated with 10 pM cyclophilin inhibitor. Scale bars are 400 pm.

[0158] Figure 12: Apoptotic fraction of target MDA-MB-231 population following knockdown of NKTR and incubation with Compound 3.

[0159] A. Apoptosis Timecourse with NK92 Cells Transfected with Control Nontargeting Plasmid and Plasmids with NKTR-targeting shRNA and Compound 3.

[0160] B. Apoptosis with NK92 Cells Transfected with Control Non-targeting Plasmid and Plasmids with NKTR-targeting shRNA and Compound 3 at 72 h Timepoint. 1-way ANOVA was applied with Tukey’s test post- hoc.

[0161] Values are the mean ± SD for 6 replicates

[0162] Definitions

[0163] The invention is described using the following definitions unless otherwise indicated.

[0164] Throughout the specification, unless the context demands otherwise, the terms ‘comprise’ or ‘include’, or variations such as ‘comprises’ or ‘comprising’, ‘includes’ or ‘including’ will be understood to imply the includes of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0165] As used herein, the articles “a” and “an” refer to one or to more than one (for example to at least one) of the grammatical object of the article.

[0166] “About” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements.

[0167] The terms, "disease", "disorder", and "condition" may be used interchangeably here to refer to a cyclophilin mediated medical or pathological condition. The term "cyclophilin mediated condition", as used herein, means any disease state or other deleterious condition in which cyclophilins are known to play a role. The term "cyclophilin mediated condition" or "disease" also means those diseases or conditions that are alleviated by treatment with a cyclophilin inhibitor.

[0168] Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects or embodiments.

[0169] As used herein, “modulators” refers to compounds that bind to a cyclophilin and either inhibit and / or induce and / or enhance its activity. For example, inhibiting a particular cyclophilin’s PPIase activity but also enhancing the same cyclophilin’s function as an adaptor protein. Additionally or alternatively, inhibiting a particular cyclophilin’s PPIase activity while concurrently enhancing another cyclophilin isoform’s activity.

[0170] The wavy line as used herein indicates a point of attachment to the rest of the compound.

[0171] The term “C1-6” includes alkyls containing 6, 5, 4, 3, 2, or 1 carbon atoms.

[0172] Lines drawn into the ring systems, such as, for example: indicate that the indicated line (bond) may be attached to any one of the substitutable ring carbon atoms.

[0173] The term "alkyl” as used herein, refers to an aliphatic hydrocarbon group having one of its hydrogen atoms replaced with a bond. An alkyl group may be straight or branched and contain from about 1 to about 10 carbon atoms. In different embodiments, an alkyl group contains from 1 to 6 carbon atoms (C1-6 alkyl) or from about 1 to about 3 carbon atoms (C1-3 alkyl). Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, n-hexyl, isohexyl and neohexyl. In one embodiment, an alkyl group is linear. In another embodiment, an alkyl group is branched. Unless otherwise indicated, an alkyl group is unsubstituted. The term "alkoxy" represents a linear or branched alkyl group of indicated number of carbon atoms attached through an oxygen bridge. Ci-ealkoxy, for example, includes methoxy, ethoxy, propoxy, isopropoxy, and the like.

[0174] "Aminoalkyl" refers to an alkyl group as described above in which one hydrogen atom has been replaced by an amino, monoalkylamino or dialkylamino group. Examples include CH2NH2, CH2CH2NHCH3 and CH(N(CH3)2)CH3.

[0175] The term "halogen" (or “halo”) refers to fluorine, chlorine, bromine and iodine (alternatively referred to as fluoro (F), chloro (Cl), bromo (Br), and iodo (I)).

[0176] Except where noted, the term “aryl” is intended to mean any stable monocyclic or bicyclic carbon ring of up to 12 atoms in each ring, wherein at least one ring is aromatic. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl and indanyl. In one embodiment, an aryl group contains from about 6 to about 10 carbon atoms. In one embodiment, an aryl group can be optionally fused to a cycloalkyl or cycloalkanoyl. Non-limiting examples of aryl groups include phenyl and naphthyl. In one embodiment, an aryl group is phenyl. Unless otherwise indicated, an aryl group is unsubstituted.

[0177] “Carboxy” refers to the functional group -C(O)OR, for example: ethylcarboxy cyclopropycarboxy

[0178] “Celite®” (Fluka) diatomite is diatomaceous earth and can be referred to as "celite". The term “carbocycle” (and variations thereof such as “carbocyclic” or “carbocyclyl”) as used herein, unless otherwise indicated, refers to (i) a C3 to Cs monocyclic, saturated or unsaturated ring or (ii) a C7 to C12 bicyclic saturated or unsaturated ring system. Each ring in (ii) is either independent of, or fused to, the other ring, and each ring is saturated or unsaturated. The carbocycle may be attached to the rest of the molecule at any carbon atom which results in a stable compound.

[0179] “Cycloalkyl” or “C3-12 cycloalkyl” means any univalent radical derived from a monocyclic or bicyclic ring system having 3 to 12 ring carbons atoms; said ring system may be:

[0180] (a) a C3 to a Cs monocyclic, saturated ring,

[0181] (b) a monocyclic saturated ring fused to a benzene or a partially unsaturated ring, or

[0182] (c) a bicyclic saturated ring. Here, the point of attachment for a “cycloalkyl” to the rest of the molecule is on the saturated ring.

[0183] For a bicyclic system, with either (b) or (c), the rings are fused across two adjacent ring carbon atoms (e.g., decalin), or are bridged groups (e.g. norbornane). Additional examples within the above meaning include, but are not limited to univalent radicals of cyclopropane, cyclobutane, cyclopentane, cyclohexane, decalin, bicyclo[2.2.2]octane and 3a,5,6,7-tetrahydro-4H- indene.

[0184] The term “C3-8 cycloalkyl” (or “C3-C8 cycloalkyl”) means a cyclic ring of an alkane having three to eight total carbon atoms (i.e. , cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl). The terms “C3-7 cycloalkyl”, “C3-6 cycloalkyl”, “C5-7 cycloalkyl” and the like have analogous meanings. Unless expressly stated to the contrary, an “unsaturated” ring is a partially or fully unsaturated ring. For example, an “unsaturated monocyclic Ce carbocycle” refers to cyclohexene, cyclohexadiene, and benzene.

[0185] Unless expressly stated to the contrary, all ranges cited herein are inclusive. For example, a heterocycle described as containing from "1 to 4 heteroatoms" means the heterocycle can contain 1 , 2, 3 or 4 heteroatoms.

[0186] When any variable occurs more than one time in any constituent or in any formula depicting and describing compounds of the invention, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and / or variables are permissible only if such combinations result in stable compounds.

[0187] The term "substituted" (e.g., as in “aryl which is optionally substituted with one or more substituents...”) includes mono-and poly-substitution by a named substituent to the extent such single and multiple substitution (including multiple substitution at the same site) is chemically allowed.

[0188] The term "oxy" means an oxygen (0) atom. The term "thio" means a sulfur (S) atom. The term "oxo" means “ = 0”. The term “carbonyl” means “C = 0.”

[0189] Structural representations of compounds having substituents terminating with a methyl group may display the terminal methyl group either using the characters “CH3”, e.g. “-CH3” or using a straight line representing the presence of the methyl group, e.g. “ — ”, i.e. have equivalent meanings.

[0190] As used herein, compound diagrams follow the rules of “skeleton diagrams”, “Kekule structures”, or “Lewis-Kekule structures”, e.g. where, unless otherwise stated, the structural representations include the correct number of implicit hydrogen atoms to provide stable compounds with full valence shells. For instance, a Markush group where a position is e.g. “selected from a group consisting of carbon, sulfur and nitrogen”, the example consists of a carbon atom, a sulfur atom and a nitrogen atom, and - mindful of any requirements for aromaticity etc. at that position - includes any implicit hydrogen atoms required to provide stable compounds with full valence shells.

[0191] "Patient" for the purposes of the present invention includes humans and other animals, particularly mammals and other organisms. Thus the methods are applicable to both human therapy and veterinary applications.

[0192] “Mammal” means humans and other mammalian animals.

[0193] “Therapeutically effective amount” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, a system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

[0194] The term “treat”, “treatment” or “treating” refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or the performance of medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure or reduce the extent of or likelihood of occurrence of the infirmity or malady or condition or event in the instance where the patient is afflicted.

[0195] The term "composition", as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s), and the inert ingredient(s) (pharmaceutically acceptable excipients) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the first, second or third aspect, and pharmaceutically acceptable excipients. The term “optionally substituted” means “unsubstituted or substituted,” and therefore, the generic structural formulas described herein encompasses compounds containing the specified optional substituent as well as compounds that do not contain the optional substituent.

[0196] As used herein, the following system will be used to number positions on a ring moiety (whether saturated or unsaturated), as defined by the IIIPAC 1974 (herein incorporated by reference).

[0197] Optical Isomers - Diastereomers - Geometric Isomers - Tautomers

[0198] The independent syntheses of diastereomers or their chromatographic separations may be achieved as known in the art by appropriate modification of the methodology disclosed herein. Their absolute stereochemistry may be determined by the x-ray crystallography of crystalline products or crystalline intermediates which are derivatised, if necessary, with a reagent containing an asymmetric centre of known absolute configuration.

[0199] Diastereomeric mixtures can be separated into their individual diastereomers based on their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography (e.g. chiral HPLC column) and / or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher’s acid chloride), separating the diastereomers and converting (e.g., hydrolysing) the individual diastereomers to the corresponding pure enantiomers. If desired, racemic mixtures of the compounds may be separated so that the individual enantiomers are isolated. The separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography. The coupling reaction is often the formation of salts using an enantiomerically pure acid or base. The diastereomeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue. The racemic mixture of the compounds can also be separated directly by chromatographic methods utilizing chiral stationary phases, which methods are well known in the art. Alternatively, any enantiomer of a compound can be obtained by stereoselective synthesis using optically pure starting materials or reagents of known configuration by methods well known in the art.

[0200] Salts

[0201] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.

[0202] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases including inorganic bases and organic bases. Salts derived from inorganic bases include aluminium, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganese, potassium, sodium, zinc, and the like.

[0203] Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, n-ethyl-morpholine, n-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0204] When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

[0205] It will be understood that, unless otherwise specified, references to the compound of the first, second or third aspect, subsets thereof, embodiments thereof, as well as specific compounds are meant to also include the pharmaceutically acceptable salts.

[0206] Furthermore, some of the crystalline forms for compounds of the present invention may exist as polymorphs and as such all forms are intended to be included in the present invention.

[0207] Prodrugs and Solvates

[0208] Prodrugs and solvates of the compounds of the first, second or third aspect are also contemplated herein. A discussion of prodrugs is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems (1987) 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, (1987) Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press (both of which are incorporated herein by reference).

[0209] The term “prodrug” means a compound (e.g. a drug precursor) that is transformed in vivo to yield a compound of the first, second or third aspect, or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (e.g. by metabolic or chemical processes), such as, for example, through hydrolysis in blood. A discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, “Pro-drugs as Novel Delivery Systems,” Vol.14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987 (both of which are incorporated herein by reference).

[0210] Similarly, if a compound of the first, second or third aspect contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as, for example, (Ci- C6)alkanoyloxymethyl, 1 -((Ci-C6)alkanoyloxy)ethyl, 1 -methyl-1 -((Ci- C6)alkanoyloxy)ethyl, (Ci-C6)alkoxycarbonyloxymethyl, n-(Ci- C6)alkoxycarbonylaminomethyl, succinoyl, (Ci-Ce)alkanoyl, -amino(Ci- C4)alkanyl, arylacyl and -aminoacyl, or -aminoacyl-aminoacyl, where each - aminoacyl group is independently selected from the naturally occurring L- amino acids, P(O)(OH)2, -P(O)(O(Ci-Ce)alkyl)2 or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate), and the like.

[0211] Where a compound of the first, second or third aspect incorporates an amine functional group, a prodrug can be formed by the replacement of a hydrogen atom in the amine group with a group such as, for example, R-carbonyl, RO- carbonyl, NRR’-carbonyl where R and R’ are each independently (Ci- Cio)alkyl, (C3-C7) cycloalkyl, benzyl, or R-carbonyl is a natural -aminoacyl or natural -aminoacyl, — C(OH)C(O)OY1wherein Y1is H, (Ci-Ce)alkyl or benzyl, — C(OY2)Y3wherein Y2is (C1-C4) alkyl and Y3is (Ci -Ce)alkyl, carboxy (Ci-Ce)alkyl, amino(Ci-C4)alkyl or mono-N — or di-N,N-(Ci- C6)alkylaminoalkyl, — C(Y4)Y5wherein Y4is H or methyl and Y5is mono-N- or di-N,N-(Ci-C6)alkylamino morpholino, piperidin-1 -yl or pyrrolidin-1 -yl, and the like.

[0212] One or more compounds of the first, second or third aspect may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms.

[0213] "Solvate" means a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate" encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like. "Hydrate" is a solvate wherein the solvent molecule is H2O.

[0214] One or more compounds of the invention may optionally be converted to a solvate.

[0215] Preparation of solvates is generally known. Thus, for example, M. Caira et al, J. Pharmaceutical Sci., 93(3), 601 -611 (2004) (incorporated herein by reference) describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, hemisolvate, hydrates and the like are described by E. C. van Tonder et al, AAPS PharmSciTech., 5(1 ), article 12 (2004); and A. L. Bingham et al, Chem. Commun., 603-604 (2001 ) (both of which are incorporated herein by reference). A typical, non-limiting, process involves dissolving the inventive compound in desired amounts of the desired solvent (organic or water or mixtures thereof) at a higher than ambient temperature and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods.

[0216] Analytical techniques such as, for example IR spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).

[0217] Labelled Compounds

[0218] In the compounds of the first, second or third aspect, the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number predominantly found in nature. The present invention is meant to include all suitable isotopic variations of the compounds of the first, second or third aspect. For example, different isotopic forms of hydrogen (H) include protium (1H) and deuterium (2H). Protium is the predominant hydrogen isotope found in nature. Enriching for deuterium may afford certain therapeutic advantages, such as increasing in vivo half-life or reducing dosage requirements, or may provide a compound useful as a standard for characterization of biological samples.

[0219] Isotopical ly enriched compounds of the first, second or third aspect can be prepared without undue experimentation by conventional techniques well known to those skilled in the art or by processes analogous to those described in the Schemes and Examples herein using appropriate isotopically-enriched reagents and / or intermediates.

[0220] Use of the Compounds

[0221] Compounds of the Invention have activity for CypA, CypB and / or CypD, preferably selectivity between CypB and / or CypD over CypA, further preferably selectively CypB activity over CypA activity. Compounds of this invention have been tested using the assays described in the Examples and have been determined to be modulators of CypA, CypB and / or CypD, preferably selectivity between CypB and / or CypD over CypA, further preferably selectively CypB activity over CypA activity. Suitable in vitro assays for measuring CypA, CypB and CypD activity and the modulation thereof by compounds are known in the art. For further details of an in vitro assay for measuring CypA, CypB and / or CypD, see the Examples herein. Cell-based assays for measurement of in vitro efficacy in treatment of cancer are known in the art. In addition, assays are described in the Examples provided herein. Suitable in vivo models for cancer are known to those of ordinary skill in the art. See for example, international patent application published as WO 2012 / 037226, Sirait-Fischer et al, Front. Oncol. 2020: 10:1771 , Lelliot et al., Sci Rep. 2019; 9:1225 and Maniati et al., Cell Reports 2020: 30:2 (each of which are herein incorporated by reference) for further details of in vivo models for prostate adenocarcinoma, glioblastoma, lung carcinoma, and melanoma.

[0222] Following the examples disclosed herein, as well as that disclosed in the art, a person of ordinary skill in the art can determine the activity of a compound of this invention.

[0223] Compounds of the first, second or third aspects, or pharmaceutically acceptable salts thereof may be useful for treating various conditions, including hyperproliferative disorders (e.g., cancer), fibrosis (e.g. fibrosis of the kidney, liver, lung or pancreas), cardiac failure, viral infections, chronic and acute inflammatory disorders, spinal cord injury, traumatic brain injury, ischaemia / reperfusion injury, stroke, myocardial infarction, acute pancreatitis, chronic mitochondrial disorders (e.g. congestive heart failure, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, macular degeneration, epilepsy, diabetic retinopathy, liver disease and other diseases due to mitochondrial dysfunction), neurodegenerative disorders, diabetes and metabolic dysfunction associated steatohepatitis (MASH)., particularly suitable types of which are listed below.

[0224] Cancers: - Adrenal glands: neuroblastoma.

[0225] - Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors.

[0226] - Breast: carcinoma (e.g. ductal, lobular, tubular, medullary, mammary secretory, papillary, no special type), inflammatory breast cancer, phyllodes tumor, triple-negative breast cancer.

[0227] - Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma.

[0228] - Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma).

[0229] - Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor [nephroblastoma], lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma). - Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma], granulosa-thecal cell tumors, SertoliLeydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma], fallopian tubes (carcinoma).

[0230] - Hematologic: blood (myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplasia syndrome), Hodgkin's disease, non-Hodgkin's lymphoma [malignant lymphoma],

[0231] - Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblast-oma, angiosarcoma, hepatocellular adenoma, hemangioma.

[0232] - Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adeno-carcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hanlartoma, mesothelioma.

[0233] - Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma). - Skin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis.

[0234] Autoimmune diseases: Hashimoto's thyroiditis, systemic lupus erythematosus (SLE), Goodpasture's syndrome, pemphigus, receptor autoimmune diseases, Basedow's disease (Graves' disease), myasthernia gravis, insulin resistant diseases, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, autoimmune encephalomyelitis, rheumatism, rheumatoid arthritis, scleroderma, mixed connective tissue disease, polymyositis, pernicious anemia, idiopathic Addison's disease, some types of infertility, glomerulonephritis, bullous pemphigus, Sjogren's syndrome, some types of diabetes, adrenergic agent resistance, chronic active hepatitis, primary biliary cirrhosis, endocrine failure, vitiligo, angiitis, post-cardiac surgery syndrome, urticaria, atopic dermatiti and multiple sclerosis, autoimmune polyglandular disease (also known as autoimmune polyglandular syndrome), autoimmune alopecia; pernicious anemia; vitiligo; autoimmune hypopituatarism, and Guillain-Barre syndrome.

[0235] Inflammatory Diseases: asthma, allergic rhinitis, psoriasis, inflammatory arthritis, rheumatoid arthritis, psoriatic arthritis or osteoarthritis, irritable bowel syndrome, ulcerative colitis, Crohn's disease, respiratory allergies (asthma, hay fever, allergic rhinitis) or skin allergies, scleracierma, mycosis fungoides, acute inflammatory responses (such as acute respiratory distress syndrome and ishchemia / reperfusion injury), dermatomyositis, alopecia greata, chronic actinic dermatitis, eczema, Behcet's disease, Pustulosis palmoplanteris, Pyoderma gangrenum, Sezary's syndrome, atopic dermatitis, systemic sclerosis, and morphea.

[0236] Central Nervous System Disorders: multiple sclerosis, schizophrenia.

[0237] Thus, in one embodiment, the invention provides a method of modulating CypA, CypB and / or CypD, particularly selectively modulating CypB and / or CypD over CypA, further preferably selectively modulating CypB over CypA, comprising contacting the Cyp with an effective amount of a compound as disclosed herein.

[0238] In one embodiment, the compounds of the instant invention are selective CypB and / or CypD modulators relative to CypA. The determination of relative selectivity for a given compound of CypB inhibition is defined as the relative ratio of the (CypA IC50 value / CypB IC50 value and / or CypD IC50 value) is at least 1 :2, preferably at least 1 :4.

[0239] In another embodiment, the invention provides a method of treating a CypA, CypB and / or CypD, preferably CypB and / or CypD over CypA, further preferably CypB over CypA modulated disease comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound as disclosed herein.

[0240] In another embodiment, the invention provides a method of treating cancer disease mediated by CypA, CypB and / or CypD, preferably CypB and / or CypD over CypA, further preferably CypB over CypA comprising administering to a mammal in need of such treatment a therapeutically effective amount of a compound as disclosed herein.

[0241] Compounds of the invention are also useful as modulators of CypA, CypB and / or CypD, preferably selective modulators of CypB and / or CypD over CypA, further preferably selective modulators of CypB over CypA in vivo for studying the in vivo role of CypA, CypB and / or CypD, particularly CypB and / or CypD over CypA, especially CypB over CypA in biological processes, including the diseases described herein. Accordingly, the invention also comprises a method of modulating CypA, CypB and / or CypD, particularly selectively modulating CypB and / or CypD over CypA, especially CypB over CypA in vivo comprising administering a compound or composition of the invention to a mammal. Accordingly, another aspect of the present invention provides a method for the treatment or prevention of a CypA, CypB and / or CypD, particularly a CypB and / or CypD mediated condition comprising administering to a mammal in need thereof a therapeutically effective amount of a compound of the first, second or third aspect. In one embodiment such diseases include asthma and rheumatoid arthritis.

[0242] Another aspect of the present invention provides for the use of a compound of the first, second or third aspect in the manufacture of a medicament for the treatment or prevention of a CypA, CypB and / or CypD, particularly CypB and / or CypD mediated diseases or disorders.

[0243] Dose Ranges

[0244] The magnitude of prophylactic or therapeutic dose of a compound of the first, second or third aspect will, of course, vary with the nature and the seventy of the condition to be treated and with the compound of the first, second or third aspect and its route of administration. It will also vary according to a variety of factors including the age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination and response of the individual patient. In general, the daily dose from about 0.001 milligram of active agent per kilogram body weight of a mammal (mg / kg) to about 100 mg / kg, typically, between 0.01 mg to about 10 mg per kg. On the other hand, it may be necessary to use dosages outside these limits in some cases.

[0245] The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the mode of administration. For example, a formulation intended for the oral administration of humans may contain from 0.01 mg to 10 g of active agent compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 99.95 percent of the total composition. Dosage unit forms will generally contain between from about 0.1 mg to about 0.4 g of an active ingredient, typically 0.5 mg, 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 400mg, or 500 mg.

[0246] The final dosage regimen will be determined by the attending physician in view of good medical practice, considering various factors that modify the action of drugs, e.g., the agent's specific activity, the identity and seventy of the disease state, the responsiveness of the patient, the age, condition, body weight, sex, and diet of the patient, and the severity of the disease state. Additional factors that can be considered include time and frequency of administration, drug combinations, reaction sensitivities, and tolerance / response to therapy. Further refinement of the dosage appropriate for treatment involving any of the formulations mentioned herein is done routinely by the skilled practitioner without undue experimentation, especially considering the dosage information and assays disclosed, as well as the pharmacokinetic data observed in human clinical trials. Appropriate dosages can be ascertained through use of established assays for determining concentration of the agent in a body fluid or other sample together with dose response data.

[0247] The frequency of dosing will depend on the pharmacokinetic parameters of the agent and the route of administration. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Accordingly, the pharmaceutical compositions can be administered in a single dose, multiple discrete doses, continuous infusion, sustained release depots, or combinations thereof, as required to maintain desired minimum level of the agent. Short-acting pharmaceutical compositions (i.e. , short half-life) can be administered once a day or more than once a day (e.g., two, three, or four times a day). Long-acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks. Pumps, such as subcutaneous, intraperitoneal, or subdural pumps, can be preferred for continuous infusion. Pharmaceutical Compositions

[0248] Another aspect of the present invention provides pharmaceutical compositions comprising a compound of the first, second or third aspect with a pharmaceutically acceptable carrier. For the treatment of any of the diseases listed herein, compounds of the first, second or third aspect may be administered orally, by inhalation spray, topically, parenterally or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In addition to the treatment of warm-blooded animals such as mice, rats, horses, cattle, sheep, dogs, cats, etc., the compound of the invention is effective in the treatment of humans. The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.

[0249] Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from a group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated by the technique described in the U.S. Patent 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutic tablets for control release.

[0250] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredients are mixed with water-miscible solvents such as propylene glycol, PEGs and ethanol, or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

[0251] Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.

[0252] Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0253] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavouring and colouring agents, may also be present.

[0254] The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavouring agents.

[0255] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavouring and colouring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1 ,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. Cosolvents such as ethanol, propylene glycol or polyethylene glycols may also be used. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Dosage forms for inhaled administration may conveniently be formulated as aerosols or dry powders. For compositions suitable and / or adapted for inhaled administration, it is preferred that the active substance is in a particle-size-reduced form, and more preferably the size-reduced form is obtained or obtainable by micronization.

[0256] In one embodiment the medicinal preparation is adapted for use with a pressurized metered dose inhaler (pMDI) which releases a metered dose of medicine upon each actuation. The formulation for pMDIs can be in the form of solutions or suspensions in halogenated hydrocarbon propellants. The type of propellant being used in pMDIs is being shifted to hydrofluoroalkanes (HFAs), also known as hydrofluorocarbons (HFCs). In particular, 1 , 1 ,1 ,2- tetrafluoroethane (HFA 134a) and 1 ,1 ,1 ,2,3,3,3-heptafluoropropane (HFA 227) are used in several currently marketed pharmaceutical inhalation products. The composition may include other pharmaceutically acceptable excipients for inhalation use such as ethanol, oleic acid, polyvinylpyrrolidone and the like.

[0257] Pressurized MDIs typically have two components. Firstly, there is a canister component in which the drug particles are stored under pressure in a suspension or solution form. Secondly, there is a receptacle component used to hold and actuate the canister. Typically, a canister will contain multiple doses of the formulation, although it is possible to have single dose canisters as well. The canister component typically includes a valve outlet from which the contents of the canister can be discharged. Aerosol medication is dispensed from the pMDI by applying a force on the canister component to push it into the receptacle component thereby opening the valve outlet and causing the medication particles to be conveyed from the valve outlet through the receptacle component and discharged from an outlet of the receptacle. Upon discharge from the canister, the medication particles are "atomized", forming an aerosol. It is intended that the patient coordinate the discharge of aerosolized medication with his or her inhalation, so that the medication particles are entrained in the patient's aspiratory flow and conveyed to the lungs. Typically, pMDIs use propellants to pressurize the contents of the canister and to propel the medication particles out of the outlet of the receptacle component. In pMDIs, the formulation is provided in a liquid or suspension form and resides within the container along with the propellant. The propellant can take a variety of forms. For example, the propellant can comprise a compressed gas or liquefied gas.

[0258] In another embodiment the medicinal preparation is adapted for use with a dry powder inhaler (DPI). The inhalation composition suitable for use in DPIs typically comprises particles of the active ingredient and particles of a pharmaceutically acceptable carrier. The particle size of the active material may vary from about 0.1 Pm to about 10 Pm; however, for effective delivery to the distal lung, at least 95 percent of the active agent particles are 5 Pm or smaller. Each of the active agent can be present in a concentration of 0.01 - 99%. Typically however, each of the active agents is present in a concentration of about 0.05 to 50%, more typically about 0.2 - 20% of the total weight of the composition.

[0259] As noted above, in addition to the active ingredients, the inhalable powder preferably includes pharmaceutically acceptable carrier, which may be composed of any pharmacologically inert material or combination of materials which is acceptable for inhalation. Advantageously, the carrier particles are composed of one or more crystalline sugars; the carrier particles may be composed of one or more sugar alcohols or polyols. Preferably, the carrier particles are particles of dextrose or lactose, especially lactose. In embodiments of the present invention which utilize conventional dry powder inhalers, such as the Handihaler, Rotohaler, Diskhaler, Twisthaler and Turbohaler, the particle size of the carrier particles may range from about 10 microns to about 1000 microns. In certain of these embodiments, the particle size of the carrier particles may range from about 20 microns to about 120 microns. In certain other embodiments, the size of at least 90% by weight of the carrier particles is less than 1000 microns and preferably lies between 60 microns and 1000 microns. The relatively large size of these carrier particles gives good flow and entrainment characteristics. Where present, the amount of carrier particles will generally be up to 95%, for example, up to 90%, advantageously up to 80% and preferably up to 50% by weight based on the total weight of the powder. The amount of any fine excipient material, if present, may be up to 50% and advantageously up to 30%, especially up to 20%, by weight, based on the total weight of the powder. The powder may optionally contain a performance modifier such as L-leucine or another amino acid, and / or metals salts of stearic acid such as magnesium or calcium stearate.

[0260] Compounds of the first, second or third aspect may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ambient temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0261] For topical use, creams, ointments, gels, solutions or suspensions, etc., containing the compound of the first, second or third aspect are employed - for purposes of this application, topical application shall include mouth washes and gargles. Topical formulations may generally be comprised of a pharmaceutical carrier, cosolvent, emulsifier, penetration enhancer, preservative system, and emollient.

[0262] Combinations with Other Drugs

[0263] In certain embodiments, a compound of the first, second or third aspect is combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with one or more other therapeutic agents that has anti- hyperprol iterative, immunomodulatory or anti-inflammatory properties; or that is useful for treating a hyperproliferative disorder (e.g., cancer), an inflammation, a viral infection, cardiovascular disease, aging, metabolic dysfunction-associated steatohepatitis or an immune-response disorder. The other therapeutic agent of the pharmaceutical combination formulation or dosing regimen preferably has complementary activities to the compound of the first, second or third aspect such that they do not adversely affect each other. Such agents are suitably present in combination in amounts that are effective for the purpose intended.

[0264] In one embodiment of the invention, the compound of the first, second or third aspect, or a stereoisomer, tautomer, or pharmaceutically acceptable salt or prodrug thereof, may be co-administered with one or more other therapeutic agents for the treatment and prevention of CypB mediated diseases. Thus in another aspect the present invention provides pharmaceutical compositions for treating CypB mediated diseases comprising a therapeutically effective amount of a compound of the first, second or third aspect and one or more other therapeutic agents.

[0265] In one embodiment of the invention, the compounds of the first, second or third aspect or pharmaceutically acceptable salt or prodrug thereof, may be employed alone or in combination with other therapeutic agents for the treatment of hyperproliferative disorders (e.g., cancer) including standard chemotherapy regimens or immunotherapies, including those that enhance tumour-killing lymphocytes (e.g. ex-vivo activated NK cells, T cells and B cells or genetically modified lymphocytes including CAR NK and CAR T-cell infusion), immune checkpoint inhibitors, or agents which counteract resistance to immune checkpoint inhibition or agents which activate or enhance the anti-cancer ability of lymphocytes or other immune cells with anti-cancer function, or agents which reverse T-cell exhaustion or agents which reprogram immunosuppressive tumour-associated macrophages to non-suppressive phenotypes or agents which reverse immunosuppressive signalling within the tumour microenvironment. These include immune checkpoint inhibitors such as anti-PD-1 , anti-PD-L1 , anti-CTLA-4 monoclonal antibodies such as pembrolizumab, atezolizumab and / or Ipilimumab.

[0266] In another embodiment, for the treatment of the inflammatory diseases rheumatoid arthritis, psoriasis, inflammatory bowel disease, COPD, asthma and allergic rhinitis a compound of the first, second or third aspect may be combined with other therapeutic agents such as: (1) TNF-a inhibitors such as Remicade® and Enbrel®); (2) non-selective COX-l / COX-2 inhibitors (such as piroxicam, diclofenac, propionic acids such as naproxen, flubiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates such as mefenamic acid, indomethacin, sulindac, apazone, pyrazolones such as phenylbutazone, salicylates such as aspirin); (3) COX-2 inhibitors (such as meloxicam, celecoxib, rofecoxib, valdecoxib and etoricoxib); (4) other agents for treatment of rheumatoid arthritis including low dose methotrexate, lefunomide, ciclesonide, hydroxychloroquine, d-penicillamine, auranofin or parenteral or oral gold; (5) leukotriene biosynthesis inhibitor, 5-lipoxygenase (5-LO) inhibitor or 5-lipoxygenase activating protein (FLAP) antagonist such as zileuton; (6) LTD4 receptor antagonist such as zafirlukast, montelukast and pranlukast; (7) PDE4 inhibitor such as roflumilast; (8) antihistaminic H1 receptor antagonists such as cetirizine, loratadine, desloratadine, fexofenadine, astemizole, azelastine, and chlorpheniramine; (9) a1- and a2- adrenoceptor agonist vasoconstrictor sympathomimetic agent, such as propylhexedrine, phenylephrine, phenylpropanolamine, pseudoephedrine, naphazoline hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline hydrochloride, xylometazoline hydrochloride, and ethylnorepinephrine hydrochloride; (10) anticholinergic agents such as ipratropium bromide, tiotropium bromide, oxitropium bromide, aclidinium bromide, glycopyrrolate, pirenzepine, and telenzepine; (11 ) [3-adrenoceptor agonists such as metaproterenol, isoproterenol, isoprenaline, albuterol, salbutamol, formoterol, salmeterol, terbutaline, orciprenaline, bitolterol mesylate, and pirbuterol, or methylxanthanines including theophylline and aminophylline, sodium cromoglycate; (12) insulin-like growth factor type I (IGF-I) mimetic; (13) inhaled glucocorticoid with reduced systemic side effects, such as prednisone, prednisolone, flunisolide, triamcinolone acetonide, beclomethasone dipropionate, budesonide, fluticasone propionate, ciclesonide and mometasone furoate.

[0267] The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. The combined administration includes co-administration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active therapeutic agents simultaneously exert their biological activities.

[0268] An embodiment of the present invention will now be described by way of example only with reference to the accompanying figures.

[0269] Detailed Description of the Invention

[0270] Example 1: Chemicals, Materials, and Methods

[0271] Preparative

[0272] Reagents were purchased from Fluorochem, Sigma Aldrich and Acres and were used as supplied unless otherwise stated. Solvents and chemicals were purified according to standard procedures. Titration of n-butyllithium (n-BuLi) solutions was carried out to ascertain their exact concentration using 1 ,10- phenanthroline as an indicator. Tetrahydrofuran (THF), dichloromethane (DCM) and dimethyl sulfoxide (DMSO), Ethyl Acetate (EtOAc), Dimethyl Formamide (DMF), Diethyl Ether (D2O), Ethanol (EtOH) and Methanol (MeOH) were obtained from the Inert Solvent Purification System (SPS) or purchased as anhydrous solvents from Sigma-Aldrich. All anhydrous reactions were performed in flame-dried flasks under a positive pressure of dry nitrogen unless otherwise stated. Air- and moisture-sensitive compounds were introduced via syringes using standard inert atmosphere techniques. Temperature control during certain procedures was maintained using a Julabo chiller unit. Reactions were monitored by thin layer chromatography (TLC) using E. Merck silica gel plates, Kieselgel 60 F254 with 0.2 mm thickness. Components were visualised by illumination with short-wavelength ultraviolet light and / or staining.

[0273] Chromatography

[0274] Flash column chromatography was performed either manually with E. Merck silica gel 60 (230-400 mesh ASTM), or using either a Biotage Isolera™ or a Teledyne ISCO CombiF / as / ?® NextGen 300+ using Biotage® Sfar or Teledyne ISCO RediSep® silica gel flash columns. Fraction collection was based on UV detection. Analytical reverse phase HPLC analysis was performed using a Waters 600E (100 pL) gradient pump using a 717plus autosampler and a Waters 996 PDA equipped with a Phenomenex Luna C18(2), 5 pm, 250 x 4.6 mm column at a flow rate of 1 mL min’1. Semipreparative reverse phase HPLC was performed using a Waters 600 (225 pL) system using a Waters 486 tuneable absorbance detector recording at 254 nm equipped with a Phenomonex Luna C18(2), 5 pm, 250 x 21.2 mm column at a flow rate of 21 .2 mL min’1. Preparative HPLC was carried using a Waters HPLC comprising of a Waters 2767 Sample Manager, Waters 2545 Binary Gradient Module, Waters Systems Fluidics Organiser, Waters 515 ACD pump, Waters 2998 Photodiode Array Detector, using a Waters XBridge Prep OBD C18, 5 pm, 19 mm x 50mm i.d. column and a flow rate of 20 mL I minute. Compounds were purified using acidic reverse phase HPLC (water / acetonitrile 10.1 % trifluoroacetic acid) using a standard gradient of 5% acetonitrile / 95% water to 100% acetonitrile, or basic reverse phase HPLC (water I acetonitrile 1 0.01 M ammonia solution) using a standard gradient of 10% acetonitrile 190% water to 100% acetonitrile. Fraction collection was based on UV detection.

[0275] Supercritical Fluid Chromatography

[0276] SFC separations were carried out using a Waters system comprising of a fluid delivery model, thermocube (set to 4 °C), autosampler, column oven (set to 40 °C), 2489 UV / visible detector (254 nm), fraction collection module, heat exchanger, and back pressure regulator. Semi-preparative separations were achieved using Chiralpak (AS-H or AD-H) or Chiralcel (OD-H or OJ-H) columns (all 10 x 250 mm, 5 pm). Liquid CO2 was used as the bulk mobile phase.

[0277] Analytical Methods

[0278] 1H and13C NMR nuclear magnetic resonance spectra were recorded in deuteriochloroform (CDCI3) or deuterated DMSO (DMSO-de) at ambient temperature (unless otherwise specified) on a Broker Avance III 400 spectrometer, Broker Avance III 500 (optimised for1H / 13C), Broker Avance III 500 (optimised for1H) or Broker Avance III 600 operating at 400 MHz, 500 MHz, 500 MHz or 600 MHz respectively for1H, and at 75 MHz, 125 MHz, 125 MHz or 150 MHz respectively for13C. All spectra were calibrated at 5 7.26 ppm (residual CHCI3) or 5 2.50 ppm (residual DMSO) for1H spectra, and 5 77.16 ppm (CHCI3) or 5 39.52 ppm (DMSO) for13C spectra. Splitting patterns were designated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad.

[0279] IR absorption spectra were recorded neat on a Shimadzu I RAffinity-1 spectrometer from 4000 cm-1to 400 cm-1. Electrospray (ESI) mass spectra were obtained on a Broker 12 T SolariX, Broker microTOF II or Kratos MS50TC mass spectrometer.

[0280] LCMS samples were analysed osing an Agilent 1200 Series HPLC system and a 6140 single qoadropole mass spectrometer eqoipped with a molti-mode (APCI+ESI) soorce. The reverse phase HPLC was carried oot osing a Phenomenex Lona® C18 (2)-HST colomn (2.5 pm, 50 x 2.0 mm) with monitoring at 254 nm and 220 nm. The colomn oven temperatore was set to 45 °C. The colomn flow rate was set to 1.0 mL / min and the solvents osed were 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B).

[0281] Method A

[0282] The method timetable was 0.00 - 0.50 min (1 % B), 0.50 - 3.50 min (linear gradient from 1 % to 100% B), and 3.50 - 4.25 min (100% B).

[0283] Method B

[0284] The method timetable was 0.00 - 0.50 min (1 % B), 0.50 - 2.00 min (linear gradient from 1% to 100% B), and 2.00 - 3.25 min (100% B).

[0285] Protein Expression

[0286] The plasmids of HisCypA were provided by the Edinburgh Protein Production Facility (EPPF). Protein expression and purification protocols were slightly modified from Wear et al. Febs Open Bio 2017, 7 (4), 533-549 (herein incorporated by reference) for their usage in ITC studies.

[0287] Recombinant Cyclophilins (Cyps) were produced in C41 BL21 (DH3) E. coli cell lines (Lucigen, Middleton, Wl, USA). Briefly, 1 pL of the stock plasmids were added to the competent cells, left on ice for about half an hour and after a two-minute heat shock at 42 °C were incubated by shaking (250 rpm) in SOC media (500 pL) at 37 °C for 45 min. They were left overnight to colonize in agar plates (100 pL / plate) containing carbenicillin (100 pg mol’1). After this a single colony was picked and grown for six h in LB media. Subsequently, 20 % v / v glycerol was added and these glycerol stocks were used in future reference. A 100 mL pre-culture was left overnight in LB media using a glycerol stock and carbenicillin as antibiotics (100 pg mol’1). The cultures were centrifuged for five minutes at 1 ,500 g and new 500 mL cultures were made by transferring the cell pellets, adding carbenicillin and incubating by shaking (250 rpm) until OD600 0.6 - 0.8 at 37 °C and then induced at 30 °C with 0.5 mM IPTG for 4 h. Finally, the cultures were pelleted by centrifugation at 8,000 g for 20 min at 4 °C prior to cell lysis.

[0288] Protein Purification

[0289] All purifications were performed on an AKTA Pure (GE Healthcare) equipment at 4 °C. Prior to purification cell pellets were lysed using protease inhibitors (Roche) in loading buffer (20 mM phosphate, 300 mM NaCI, 20 mM imidazole, pH 7.4) by a double passage on a Constant Systems Cell Disruptor (1.1 kWTS Benchtop) at 22 kpsi followed by one hour centrifugation at 4 °C (55,000 g). A twostep purification protocol was used in all cases, i.e. Immobilized Metal Ion Affinity Chromatography (IMAC) and Size Exclusion Chromatography (SEC) using the HiTrap IMAC FF 5 mL and the HiLoad Superdex 75 pg 16 / 60 columns, respectively. The buffer used in the SEC purification step was similar to the ITC buffer and for the IMAC elution 20 mM phosphate, 300 mM NaCI, 500 mM imidazole, pH 7.4.

[0290] Protein His-Tag was cleaved for further use of the protein in ITC studies. Proteins were desalted to cleavage buffer (100 mM Tris, 100 mM NaCI, pH 7.5) using a HiPrep 26 / 10 desalting column prior to the addition of TEV protease (200 ng TEV 140 pg protein). Samples were left incubating at 30 °C for about 4 h and the cleaved His-tag was removed by IMAC. At the end of each purification the purity of the fractions was tested by using precast gels (Biorad®) in Tris / Glycine / SDS, pH 8.3 buffer.

[0291] The molecular weights of HisCypA and free CypA are 20.893 and 18.070 kDa, respectively. Protein concentration was determined by measuring the absorbance at 280 nm and the extinction coefficients 14440 and 8480 M-1crrr1, respectively.

[0292] Example 2: Synthesis of selected compounds

[0293] Compounds of the present invention have been prepared by the synthetic procedures shown in the following schemes:

[0294] ,

[0295] 3 (52%) (95%)

[0296] Scheme 1 : Exemplary synthetic route to provide an embodiment of the present invention, with yields shown in parenthesis.

[0297]

[0298] Scheme 2: Overview of exemplary synthetic routes to provide some embodiments of the present invention and related compounds, with yields shown in parenthesis.

[0299]

[0300] Scheme 3: Further exemplary synthetic routes to provide embodiments of the present invention, with yields shown in parenthesis.

[0301]

[0302] Scheme 4: base-induced rearrangements of (a) cyanomethyl substituted ureas and (b) oxadiazole substituted ureas, with yields shown in parenthesis. General Procedure A: n-alkylation

[0303] The benzylamine hydrochloride (1 eq) was dissolved in acetonitrile (0.3 M) in a two-necked RBF and K2CO3 (2 eq) was added to the mixture. The reaction was left to stir for 1 h at rt. After this time, the alkyl halide (1 eq) was added and the mixture was stirred at 70°C for 18 h. Upon completion of the reaction, the mixture was evaporated to dryness. The crude material was dissolved in EtOAc and was washed with NaOH (1 M aq), NaHCOs (sat aq) and brine.

[0304] The combined organic layers were dried over MgSCU, filtered and the solvent removed in vacuo to give the crude substituted compound. The final product was purified using flash column chromatography (hexane / EtOAc, 10:1 to 1 :3).

[0305] General Procedure B: Urea formation

[0306] The n-alkylated amine from General Procedure A (1 eq) was dissolved in DCM (0.12 M). Ethyl isocyanatoacetate (1 eq) was added and the mixture was stirred at rt for 2-18 h. Upon completion of the reaction, the mixture was evaporated to dryness. The final product was purified by flash column chromatography (hexane / EtOAc, 10:1 to 1 :5).

[0307] General Procedure C: Ester hydrolysis

[0308] The ethyl ester (1 eq) was mixed with LiOH (1 M aq, 1 eq) in a 2:2:1 ratio mixture of MeOH / THF / H2O (0.13 M) and stirred at rt for 1 h, while monitoring the reaction progression by TLC. Upon completion, the organic solvents were removed in vacuo, and the basic aqueous solution was washed with EtOAc to remove any impurities. The aqueous solution containing the carboxylic acid product was acidified to ~pH 5 using HCI (3 M aq), and the aqueous layer was extracted 3 times with EtOAc. The combined organic layers were dried over MgSO4, filtered and evaporated to dryness to give the final hydrolyzed product. General Procedure D: Nitro group reduction

[0309] The Nitrobenzylamine (1 eq) was placed in a two-necked RBF and dissolved in anhydrous MeOH (0.065 M). Pd / C (10%, 0.05 eq) and ZnBr2 (1 eq) were added, and the flask was flushed with H2 gas 3 times. The reaction was stirred at rt for 18 h. After confirming its completion by TLC, the reaction mixture was passed through Celite plug and the solvent was removed in vacuo and the crude product was used without further purification in the next reaction.

[0310] General Procedure E: Amide formation

[0311] The carboxylic acid (1 eq) was placed in a two-necked RBF and was dissolved in DCM (0.075 M), followed by the addition of the pyrrolidine analogue (2.5 eq). DIPEA (3 eq) was added, and the reaction mixture was stirred for 30 min at rt. The reaction was cooled to 0°C and propanephosphonic acid anhydride (1.5 eq) was added dropwise. The reaction was allowed to gradually warm to rt overnight with stirring. Upon completion of the reaction, the solvent was removed in vacuo and the crude product was purified using preparative HPLC (H2O / MeCN + 0.1 %TFA).

[0312] Characterisation of Exemplary Compounds of the Present Invention and Comparison Compounds

[0313] 1 -[[(1 S,9 / ?,10S)-10-Hydroxy-12-oxa-8-azatricyclo[7.3.1.02,7]trideca-2,4,6- trien-4-yl]methyl]-3-[2-[(2 / ?)-2-(2-methylsulfanylphenyl)pyrrolidin-1-yl]-2- oxo-ethyl]-1-[(2-methyltetrazol-5-yl)methyl]urea

[0314] 1

[0315] The title compound was synthesized applying the general procedure as follows:

[0316] 1-[[(1 S,9 ?,10S)-10-[tert-Butyl(dimethyl)silyl]oxy-12-oxa-8- azatricyclo[7.3.1.02,7]trideca-2,4,6-trien-4-yl]methyl]-3-[2-[(2F?)-2-(2- methylsulfanylphenyl)pyrrolidin-1 -yl]-2-oxo -ethy I]- 1 -[(2-methyltetrazol-5- yl)methyl]urea (121 mg, 0.170 mmol) was placed in a vial which was purged with argon and dry THF (6 mL) added. Tetrabutylammonium fluoride (0.34 mL, 0.34 mmol) was added dropwise and the solution stirred overnight at room temperature. An additional 170 pL TBAF was added and stirring continued for 2 h. The reaction was concentrated directly on to silica and purified by flash column chromatography on the Biotage Isolera (12 g silica, 0 to 5% 2M-NH3 MeOH / DCM) then purified by reverse phase column chromatography (Xbridge C18, 10 - 95% MeCN / H2O with NH4OH modifier) to provide the title compound as colourless frothy solid (30 mg, 0.048 mmol, 29%).

[0317] LCMS (Method B): 2.103 min, 615.0 [M+Na]+.1H NMR (400 MHz, DMSO) 5 7.38 - 7.14 (m, 2.45H), 7.12 - 6.98 (m, 1.62H), 6.98 - 6.92 (m, 1 H), 6.92 - 6.86 (m, 1H), 6.64 - 6.51 (m, 1H), 6.51 - 6.43 (m, 1H), 6.39 - 6.33 (m, 1 H), 5.30 - 5.21 (m, 1H), 4.99 - 4.94 (m, 1 H), 4.59 - 4.44 (m, 3H), 4.34 - 4.24 (m, 5H), 4.02 - 3.94 (m, 1 H), 3.91 - 3.49 (m, 2.43H), 3.41 - 3.33 (m, 2H), 3.29 - 3.25 (m, 1H), 3.17 - 3.09 (m, 1 H), 3.06 - 2.96 (m, 0.44H), 2.56 - 2.52 (m, 3H), 2.48 - 2.28 (m, 1.51H), 2.24 - 2.10 (m, 0.60H), 2.00 - 1.58 (m, 3H), 1.28 - 1.20 (m, 1 H). 1-[(4-Amino-3,5-difluoro-phenyl)methyl]-1-[(2-methyltetrazol-5- yl)methyl]-3-[2-oxo-2-[(2 / ?)-2-(2-methylsulfanylphenyl)pyrrolidin-1- yl]ethyl]urea

[0318] 2

[0319] The title compound was synthesized applying the general procedure as follows:

[0320] Ethyl 2-[[(4-amino-3,5-difluoro-phenyl)methyl-[(2-methyltetrazol-5- yl)methyl]carbamoyl]amino]acetate (61 mg, 0.16 mmol) was reacted according to General Procedure C to give 2-[[(4-amino-3,5-difluoro-phenyl)methyl-[(2- methyltetrazol-5-yl)methyl]carbamoyl] amino]acetic acid as a viscous yellow oil that was used in the next reaction without further purification. (2R)-2-(2- Methylsulfanylphenyl) pyrrolidine (34.8 mg, 0.180 mmol) and the crude 2-[[(4-amino- 3,5-difluoro-phenyl)methyl-[(2-methyltetrazol-5-yl)methyl]carbamoyl] amino]acetic acid (32 mg, 0.090 mmol) were reacted according to General Procedure D to give crude product as a yellow gum (40 mg). The crude material was purified by flash column chromatography on the Biotage Isolera (4 g silica, 0 to 2% NHs-MeOH / DCM) to provide the title compound as beige gum (15 mg, 0.028 mmol, 18%).

[0321] LCMS (Method A): 2.406 min, 531.2 [M+H]+.1H NMR (400 MHz, DMSO) 5 7.38 - 7.14 (m, 2.48H), 7.11 - 6.97 (m, 1.60H), 6.89 - 6.76 (m, 2H), 6.69 - 6.55 (m, 1H), 5.29 - 5.21 (m, 1H), 5.13 - 5.05 (m, 2H), 4.62 - 4.58 (m, 1.17H), 4.58 - 4.48 (m, 0.76H), 4.38 - 4.27 (m, 5H), 3.99 - 3.93 (m, 1 H), 3.89 - 3.64 (m, 1.45H), 3.64 - 3.53 (m, 0.44H), 3.06 - 2.96 (m, 0.44H), 2.56 - 2.52 (m, 3H), 2.42 - 2.29 (m, 0.55H), 2.23 - 2.10 (m, 0.63H), 1.98 - 1.60 (m, 3H). 1-[(4-Aminophenyl)methyl]-1-[(2-methyltetrazol-5-yl)methyl]-3-[2-oxo-2-

[0322] [(2 / ?)-2-(2-methylsulfanylphenyl)pyrrolidin-1-yl]ethyl]urea

[0323] 3

[0324] The title compound was synthesized applying the general procedure as follows:

[0325] 2-[[(4-Aminophenyl)methyl-[(2-methyltetrazol-5-yl)methyl] carbamoyl] amino]acetic acid (47 mg, 0.15 mmol) and (2R)-2-(2-methylsulfanylphenyl)pyrrolidine (0.25 mL, 0.37 mmol) were reacted according to General Procedure E. The crude product was purified by flash column chromatography on the Biotage Isolera (12 g silica, 0 to 5% MeOH / DCM) to provide the title compound as colourless frothy solid (40 mg, 0.081 mmol, 52%).

[0326] LCMS (Method B): 1.892 min, 495.2 [M+H]+.1H NMR (400 MHz, DMSO) 5 7.36 - 7.15 (m, 2.49H), 7.09 - 6.98 (m, 1.66H), 6.93 - 6.88 (m, 2H), 6.56 (t, J = 5.2 Hz, 0.59H), 6.52 - 6.45 (m, 2.41H), 5.24 (m, 1 H), 4.97 (m, 1.75H), 4.60 - 4.40 (m, 2H), 4.33 - 4.20 (m, 5H), 3.97 - 3.93 (m, 1H), 3.90 - 3.48 (m, 2.49H), 3.01 (dd, J = 16.7, 5.1 Hz, 0.44H), 2.53 (m, 3H), 2.39 - 2.27 (m, 0.48H), 2.16 (m, 0.6H), 1.97 - 1.59 (m, 3H).

[0327] 1 -[(4-Amino-3,5-difluoro-phenyl)methyl]-1 -[(1 -methyltri azol-4-yl)methyl]-

[0328] 3-[2-oxo-2-[(2 / ?)-2-(2-methylsulfanylphenyl)pyrrolidin-1-yl]ethyl]urea

[0329] 4

[0330] The title compound was synthesized applying the general procedure as follows:

[0331] Ethyl 2-[[(4-amino-3,5-difluoro-phenyl)methyl-[(1-methyltriazol-4- yl)methyl]carbamoyl]amino]acetate (65 mg, 0.17 mmol) was reacted according to General Procedure C. The residue was used as obtained without further purification or analysis. To a flask containing the crude Li 2-[[(4-amino-3,5-difluoro- phenyl)methyl-[(1-methyltriazol-4-yl)methyl]carbamoyl] amino]acetate (61 mg, 0.17 mmol) was reacted with by (R)-2-(2-methylsulfanylphenyl) pyrrolidine (39 mg, 0.20 mmol) according to General Procedure E to give the crude product as a yellow gum (65 mg). The crude product was purified by flash column chromatography on the Biotage Isolera (12 g silica, 0 to 4% MeOH / DCM) to provide the title compound as an off white frothy solid (47 mg, 0.089 mmol, 52%).

[0332] LCMS (Method A): 2.264 min, 530.2 [M+H]+.1H NMR (400 MHz, DMSO) 5 7.93 - 7.85 (m, 1 H), 7.38 - 7.14 (m, 2.46H), 7.11 - 6.97 (m, 1.62H), 6.86 - 6.73 (m, 2H), 6.66 - 6.50 (m, 1 H), 5.29 - 5.22 (m, 1 H), 5.11 - 5.01 (m, 2H), 4.36 - 4.16 (m, 4H), 4.00 - 3.94 (m, 4H), 3.90 - 3.65 (m, 1.47H), 3.58 (m, 1 H), 3.02 (m, 0.45H), 2.53 (m, 3H), 2.41 - 2.29 (m, 0.54H), 2.17 (m, 0.61 H), 1.98 - 1.60 (m, 3H).

[0333] 1-[(4-Aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2- oxoethyl}-1-[(2-methyl-2H-1,2,3,4-tetrazol-5-yl)cyclopentyl]urea hydrochloride

[0334] 5

[0335] The title compound was synthesized applying the general procedure as follows:

[0336] The carboxylic acid (377 mg, 1.01 mmol) was reacted with 2-(2-bromophenyl) pyrrolidine (457 mg, 2.02 mmol) according to General Procedure E. The crude product was purified with preparative HPLC to afford the amide as a yellow fluffy solid (200 mg, 0.35 mmol, 35%).

[0337] HRMS (ESI) [M+H]+found 581.1984, C27H34O2N879Br (100.0%) requires 581.1983; found 603.1781 , C27H34O2N879Br23Na requires 603.1789. [M+H]+found 583.1964, C27H34O2N881Br (97.5%) requires 583.1964. mp 222-225°C. LC-MS: Rt= 2.5 min.1H NMR (500 MHz, MeOD) 57.64 - 7.56 (1 H, ddd, J = 36.78, 7.93, 4.84), 7.47 (1H, d, J = 8.2 Hz), 7.44 - 7.38 (1 ,4H, m), 7.31 (1 H, d, J = 8.2 Hz), 7.29 - 7.24 (2H, m), 7.15-7.09 (1.6H, m), 5.40 (1H, m), 5.23 (1H, m), 4.71 (2H, s), 4.52 (2H, s), 4.60 (1H, brs), 4.17-4.04 (1.2H, td, J= 16.9, 10.2 Hz), 4.02-3.93 (0.8H, m), 3.86-3.79 (0.8H, m), 3.78-3.70 (1.2H, m), 2.56-2.34 (1.2H, m), 2.30-2.09 (6H, m), 1.94- 1.73 (4.8H, m).

[0338] 1-[(4-Aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2- oxoethyl}-1-[(2-methyl-2H-1,2,3,4-tetrazol-5-yl)cyclohexyl]urea hydrochloride

[0339] 6

[0340] The title compound was synthesized applying the general procedure as follows:

[0341] The nitro acid intermediate (301 mg, 0.722 mmol) was reacted according to General Procedure D to afford crude aniline which was concentrated in vacuo and reacted without further purification (282 mg, 0.722 mmol, 100%).

[0342] The crude acid (282 mg, 0.722 mmol) was reacted with 2-(2-bromophenyl) pyrrolidine (327 mg, 1.44 mmol) according to General Procedure E to afford the final product as a yellow, fluffy solid (229 mg, 0.380 mmol, 53%).

[0343] HRMS (ESI) [M+H]+found 595.2561, C28H35N8O279Br (100.0%) requires 595.2565; found 597.1974, C27H34O2N881Br (97.5%) requires 597.1956. mp 225-228 °C.

[0344] LC-MS: Rt= 2.3 min.1H NMR (601 MHz, MeOD) 57.62 (1 H, dd, J = 44.1 , 7.9 Hz), 7.53-7.34 (3H, m), 7.33-7.22 (3H, m), 7.17 (3H, m), 5.44-5.39 (1H, m), 4.77- 4.68 (3H, m), 4.68-4.61 (2H, m), 4.13 (1.2H, q, J= 16.9 Hz), 4.01 -3.94 (0.8H, m), 3.95-3.82 (0.8H, m), 3.79-3.68 (1.2H, m), 2.57-2.32 (1.2H, m), 2.20-2.12 (2H, m), 2.07-2.02 (0.8H, m), 2.00-1.84 (6H, m), 1.79-1.72 (0.8H, m), 1.57-1.47 (2H, m), 1.37 - 1.29 (1.2H, m). Rotameric ratio 3:2. 1-[(4-Amino-3-chloro-phenyl)methyl]-1-[(2-methyltetrazol-5-yl)methyl]-3-

[0345] [2-oxo-2-[(2 / ?)-2-(2-methylsulfanylphenyl)pyrrolidin-1-yl]ethyl]urea

[0346] 7

[0347] The title compound was synthesized applying the general procedure as follows:

[0348] 2-[[(4-amino-3-chloro-phenyl)methyl-[(2-methyltetrazol-5-yl)methyl]carbamoyl] amino]acetic acid (42 mg, 0.10 mmol) and (2R)-2-(2-methylsulfanylphenyl) pyrrolidine (38 mg, 0.20 mmol) were reacted according to General Procedure E. The organics were combined, filtered through a hydrophobic frit and concentrated in vacuo to afford crude product. Purification by flash column chromatography (silica column, 0% to 10% 2M methanolic ammonia in DCM gradient) followed by evaporation of solvent from the appropriate fractions afforded partially purified product. Further purification by prep. HPLC (basic) followed by evaporation of solvent from the appropriate fractions and drying under vacuum afforded the title compound as a white solid (24 mg, 0.045 mmol, 45%).

[0349] LCMS (Method A) 2.44 min, 529.2, 531. 2 [M+H]+, chloride isotope splitting pattern.1H NMR (400 MHz, DMSO) 5 7.37 - 7.13 (m, 2.5H), 7.10 - 6.96 (m, 2.5H), 6.94 (s, 1 H), 6.71 (dd, J = 8.2, 5.3 Hz, 1 H), 6.62 (t, J = 5.2 Hz, 0.5H), 6.56 (t, J = 5.3 Hz, 0.5H), 5.28 - 5.20 (m, 3H), 4.57 (s, 1 H), 4.54 - 4.44 (m, 1 H), 4.33 - 4.23 (m, 4H), 3.96 (d, J = 5.0 Hz, 1 H), 3.89 - 3.74 (m, 0.5H), 3.74 - 3.65 (m, 0.5H), 3.63 - 3.49 (m, 0.5H), 3.29 (d, J = 0.9 Hz, 3H), 3.00 (dd, J = 16.7, 5.1 Hz, 0.5H), 2.53 (s, 3H), 2.23 - 2.05 (m, 0.5H), 1.97 - 1.59 (m, 2.5H). 1-[(4-Amino-3-chloro-phenyl)methyl]-1-[(2-methyltetrazol-5-yl)methyl]-3-

[0350] [2-oxo-2-[(2 / ?)-2-(2-bromophenyl)pyrrolidin-1-yl]ethyl]urea

[0351] The title compound was synthesized applying the general procedure as follows:

[0352] 2-[[(4-Amino-3-chloro-phenyl)methyl-[(2-methyltetrazol-5-yl)methyl]carbamoyl] amino]acetic acid (83 mg, 0.19 mmol) and (2R)-2-(2-bromophenyl)pyrrolidine (88 mg, 0.39 mmol) were reacted according to General Procedure E to afford give the crude product. Purification by flash column chromatography (silica column, 0% to 5% 2 M methanolic ammonia gradient) followed by prep. HLPC (basic) afforded the title compound (56 mg, 0.100 mmol, 51.2%).

[0353] LCMS (Method B) 1.49 min, 562.0, 564.0 [M+H]+, chloride isotope splitting pattern.1H NMR (400 MHz, CDCI3) 6 7.57-7.51 (m, 1 H), 7.37 - 6.86 (m, 5H), 6.71-6.66 (m, 1 H), 5.87-5.74 (m, 1 H), 5.43-5.21 (m, 1 H), 4.70-4.58 (m, 2H), 4.48 - 4.34 (m, 2H), 4.30- 4.28 (m, 3H), 4.21-4.02 (m, 3H), 3.86 - 3.33 (m, 3H), 2.45 - 2.39 (m, 1 H), 2.02 - 1.80 (m, 3H).

[0354] 1-[(2-Aminopyrimidin-5-yl)methyl]-1-[(5-methylthiazol-2-yl)methyl]-3-[2- oxo-2-[(2 / ?)-2-(2-methylsulfanylphenyl)pyrrolidin-1-yl]ethyl]urea

[0355] The title compound was synthesized applying the general procedure as follows:

[0356] 2-[[[2-[Bis(tert-butoxycarbonyl)amino]pyrimidin-5-yl] methyl-[(5-methylthiazol-2- yl)methyl]carbamoyl]amino] acetic acid (161 mg, 0.300 mmol) and (2 / ?)-2-(2- methylsulfanylphenyhpyrrolidine (116 mg, 0.60 mmol) were reacted according to General Procedure E to give the crude amide (214 mg, 0.300 mmol, 100%) which was used in the subsequent step without purification.

[0357] Crude tert-butyl n-tert-butoxycarbonyl- / V-[5-[[(5-methylthiazol-2-yl)methyl-[[2-oxo-2- [(2R)-2-(2-methylsulfanylphenyl)pyrrolidin-1- yl]ethyl]carbamoyl]amino]methyl]pyrimidin-2-yl]carbamate (214 mg, 0.300 mmol) was dissolved in DCM (5 mL) at room temperature and trifluoroacetic acid (1 mL, 13.0 mmol) added. The reaction was stirred for 1 h at room temperature and concentrated in vacuo. Purification by SCX chromatography (10 g cartridge, washing with DCM and MeOH and eluting with 2 M methanolic ammonia) followed by concentration in vacuo was followed by purification by prep. HPLC (basic). Evaporation of solvent from the appropriate fractions afforded the purified title compound. Purification by SCX chromatography (washing with DCM and MeOH and eluting with 2 M methanolic ammonia) followed by concentration in vacuo afforded the amide (23 mg, 0.05 mmol, 15%).

[0358] LCMS (method A): 1.20 min, 512.2 [M+H]+.1H NMR (400 MHz, CDCI3) 5 8.22 (s, 2H), 7.38 - 7.05 (m, 4H), 6.98-6.89 (m, 1 H), 6.06-5.99 (m, 1 H), 5.56 - 5.23 (m, 1 H), 5.06 (s, 2H), 4.64-4.52 (d, J = 18.0 Hz, 2H), 4.45 - 4.29 (m, 2H), 4.20 - 4.02 (m, 1 H), 3.94 - 3.31 (m, 3H), 2.71 - 2.14 (m, 7H), 2.12 - 1.76 (m, 3H).

[0359] 1 -[(2-Aminopyrimidin-5-yl)methyl]-1 -[(5-methyl-1 ,3,4-thiadiazol-2- yl)methyl]-3-[2-oxo-2-[(2 / ?)-2-(2-methylsulfanylphenyl)pyrrolidin-1- yl]ethyl]urea

[0360] 10

[0361] The title compound was synthesized applying the general procedure as follows:

[0362] Ethyl 2-[[[2-[bis(tert-butoxycarbonyl)amino] pyrimidin-5-yl]methyl-[(5-methyl-1 ,3,4- thiadiazol-2-yl)methyl] carba moyl]amino] acetate (44 mg, 0.078 mmol) was reacted according to General Procedure C. The reaction mixture was partitioned between EtOAc (20 mL) and saturated ammonium chloride solution (5 mL). Organics were dried over Na2SO4, filtered, and concentrated in vacuo to afford crude acid as a pale yellow gum (33 mg, 0.062 mmol, 79% yield) which was used directly in the next reaction.

[0363] The crude acid (33 mg, 0.062 mmol) was reacted with (R)-2-(2- Methylsulfanylphenyl)pyrrolidine (17.8 mg, 0.090 mmol) according to General Procedure E. Purification by flash column chromatography (silica column, 0% to 5% 2 M methanolic ammonia in DCM gradient) followed by evaporation of solvent from the appropriate fractions afforded the amide as a pale yellow gum (26 mg, 0.037 mmol, 59% yield).

[0364] LCMS (method A): 2.73 min, 735.2 [M+Na]+.

[0365] Crude tert-butyl n-[5-[[(5-methyl-1 ,3,4-thiadiazol-2-yl)methyl-[[2-oxo-2-[(2R)-2-(2- methyl-sulfanylphenyl)pyrrolidin-1-yl]ethyl]carbamoyl] amino]methyl]pyrimidin-2- yl]carbamate (120 mg, 0.104 mmol, purity 53%) was dissolved in DCM (2 mL) and trifluoroacetic acid (0.50 mL, 6.5 mmol) added. The reaction was stirred at room temperature for 1 h. The reaction mixture was concentrated in vacuo to afford crude product as a yellow oil. Purification by SCX chromatography (washing alternately with MeOH and DCM, eluting product with 2 M methanolic ammonia) followed by evaporation of solvent afforded crude product. Purification by prep. HPLC (basic). Product containing fractions were combined with product from reaction followed by evaporation of solvent and drying under vacuum to afford the title compound as a white solid (25 mg, 0.05 mmol, 46%).

[0366] LCMS (Method A): 1.94 min, 513.2 [M+H]+.1H NMR (400 MHz, DMSO) 5 8.16 (d, J = 3.5 Hz, 2H), 7.38 - 7.14 (m, 2.5H), 7.11 - 6.93 (m, 2H), 6.84 (t, J = 5.5 Hz, 0.5H), 6.54 (d, J = 8.0 Hz, 2H), 5.30 - 5.19 (m, 1 H), 4.75 - 4.57 (m, 2H), 4.35 - 4.18 (m, 2H), 3.98 (d, J = 5.3 Hz, 1 H), 3.92 - 3.77 (m, 0.5H), 3.70 (q, J = 6.0, 4.2 Hz, 0.5H), 3.64 - 3.49 (m, 0.5H), 3.02 (dd, J = 16.6, 5.2 Hz, 0.5H), 2.63 (s, 3H), 2.54 (s, 1 ,5H), 2.39 - 2.30 (m, 0.5H), 2.17 (tt, J = 12.0, 7.7 Hz, 0.5H), 2.00 - 1.59 (m, 4H). Note - signal due to SMe only integrates to 1.5H, signal is assumed to be split and second peak of 1.5H hidden beneath adjacent DMSO signal. 1 -[(6-Amino-5-chloro-3-pyridyl)methyl]-1 -[(5-methyl-1 ,3,4-thiadiazol-2- yl)methyl]-3-[2-oxo-2-[(2 / ?)-2-(2-methylsulfanylphenyl)pyrrolidin-1- yl]ethyl]urea

[0367] 11

[0368] The title compound was synthesized applying the general procedure as follows:

[0369] Ethyl 2-[[(6-amino-5-chloro-3-pyridyl)methyl-[(5-methyl-1 ,3,4-thiadiazol-2- yl)methyl]carbamoyl]amino]acetate (84 mg, 0.21 mmol) was reacted according to General Procedure C. The reaction mixture concentrated in vacuo to afford the acid as an off-white solid (321 mg) which was used in the next reaction without further purification.

[0370] The crude 2-[[(6-amino-5-chloro-3-pyridyl)methyl-[(5-methyl-1 ,3,4-thiadiazol-2- yl)methyl]-carbamoyl]amino]acetic acid (39 mg, 0.11 mmol) and (2 / ?)-2-(3- methylsulfanylphenyl)pyrrolidine (41 mg, 0.21 mmol) were reacted according to General Procedure E. Purification by flash column chromatography (silica column, 0% to 5% 2 M methanolic ammonia in DCM gradient) was followed by prep. HLPC (basic, followed by acidic) then solvent was removed from the appropriate fractions in vacuo. The afforded material was stirred in MeCN over K2CO3, filtered and concentrated in vacuo to afford the title compound (10 mg, 0.018 mmol, 17%).

[0371] LCMS (Method B) 1.52 min, 546.2, 548.2 [M+H]+, chloride isotope splitting pattern.1H NMR (400 MHz, CDCI3) 6 7.89-7.84 (m, 1 H), 7.43 - 6.89 (m, 5H), 5.89 - 5.70 (m, 1 ,3H), 5.48-5.21 (m, 1 H), 4.92 (m, 2H), 4.83 - 4.66 (m, 2H), 4.38-3.54 (d, J = 8.9 Hz, 5H), 3.35-3.30 (d, J = 3.4 Hz, 0.7H), 2.72-2.70 (m, 3H), 2.53-2.48 (m, 3H), 2.39-1.81 (m, 4H). 1-[(4-Aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2- oxo-ethyl}-1 - [(5-phenyl-1 ,3,4-thiadiazol-2-yl)methyl]urea

[0372] 12

[0373] The title compound was synthesized applying the general procedure as follows:

[0374] 2-({[(4-anilino)methyl][(5-phenyl-1,3,4-thiadiazol-2-yl)methyl]carbamoyl} amino)acetic acid (0.53 g, 1.29 mmol) was reacted according to General Procedure E to afford the title compound as a light yellow solid (250 mg, 0.41 mmol, 32.0%);

[0375] LC-MS: Rt = 3.3 min; Rf (hexane / EtOAc, 1 :1) = 0.54; mp 222-224 °C;1H NMR (500 MHz, DMSO) 5 7.91 (3H, ddt, J = 15.2, 6.1 , 1.9 Hz, 3Ar / 7), 7.68 (0.6H, dd, J = 8.3, l .3 Hz, 0.6Ar / 7), 7.60 (1 H, dd, J = 8.3, 1 .3 Hz, Ar / - / ), 7 58 - 7.52 (4H, m, 4Ar / 7), 7.42 (0.4H, td, J = 7.6, 1.3 Hz, 0.4ArH), 7.35 - 7.22 (4H, m,a 4Ar / 7), 7.19 - 7.13 (2H, m, 2Ar / 7), 6.93 (t, J = 5.5 Hz, 0.4NH2), 6.83 (1 H, t, J = 5.5 Hz, 0.6NH2), 6.80 (1 H, s, N / 7CH2), 5.25 (1 H, ddd, J = 45.8, 8.2, 2.3 Hz, pyrolCH), 4.81 - 4.67 (2H, m, NCH2), 4.44 - 4.36 (2H, m, ArC / 72), 4.09 - 3.94 (1 ,2H, m, 1 ,2NHCH2), 3.94 - 3.88 (0.6H, m, 0.6pyrolC / 72), 3.68 - 3.61 (0.4H, m, J = 9.7, 7.0 Hz, 0.4pyrolCH2), 3.12 - 3.07 (0.4H, m, 0.4NHC / 72), 2.33 - 2.22 (1.2H, m, 1.2pyrolCH2), 1.98 - 1.92 (0.8H, m, 0.8pyrolC / 72), 1.90 - 1.78 (1.2H, m, 1.2pyrolCH2), 1.74 - 1.66 (0.8H, m, 0.8pyrolCH2). Rotameric ratio = 2:3; m / z 605.13 [M+H]+; (ESI) HRMS [M+H]+found 605.1338, C29H3079BrN6O2S requires 605.1339; found 607.1317, C29H3o81BrN602S requires 607.1316; [M+Na]+found 627.1138, C29H2979BrN6O2SNa requires 627.1138; found 629.1120, C29H2981BrN6O2SNa requires 629.1123. 1-[(4-Aminophenyl)methyl]-3-{2-[2-(2-bromophenyl)pyrrolidin-1-yl]-2- oxoethyl}-1 -[(5-phenyl-1 ,3,4-thiadiazol-2-yl)methyl]urea

[0376] 13

[0377] The title compound was synthesized applying the general procedure as follows:

[0378] 2-({[(4-Nitrophenyl)methyl][(5-cyclopropyl-1 ,3,4-thiadiazole)methyl]carbamoyl} amino)acetic acid was reacted according to General Procedure D. The reaction was monitored by TLC to afford the aniline derivative as a thick orange oil (467 mg, 0.82 mmol; 100%); The crude product was directly used according to General Procedure E to afford the title compound (140mg, 0.25 mmol, 30.0%);

[0379] LC-MS: Rt = 2.7 min; Rf (hexane / EtOAc, 1 :1) = 0.65; mp 203-205 °C;1H NMR (601 MHz, CDCI3) 6 7.62 - 7.55 (1 H, m, Ar / - / ), 7 32 - 7.23 (1 .45H, m, 1 ,45Ar / 7), 7.20 - 7.16 (0.55H, m, 0.55ArH), 7.12 - 7.03 (2.45H, m, 2.45ArH), 6.97 - 6.94 (0.45H, m, 0.45Ar / 7), 6.72 (2H, dd, J = 20.6, 7.9 Hz, 2Ar / 7), 5.87 (0.9H, br s, 0.45NH2), 5.70 (1.1 H, br s, 0.55N / 72), 5.45 (0.45H, d, J = 8.1 Hz, 0.45pyrCH), 5.26 (0.55H, d, J = 8.1 Hz, 0.55pyrC / 7), 4.85 - 4.74 (2H, m, HetArCH2), 4.42 - 4.31 (2H, m, ArC / 72), 4.18 - 4.09 (1 .55H, m, 0.78NHCH2), 3.86 - 3.70 (2H, m, pyrCW2), 3.66 - 3.61 (0.45H, m, 0.22NHC / 72), 3.15 (1 H, qd, J = 7.2, 4.3 Hz, cyclopropCH), 2.47 - 2.32 (2H, m, pyrC / 72), 2.04 - 1.92 (2H, m, pyrCW2), 1.24 (2H, tt, J = 7.2, 4.3 Hz, cyclopropCW2), 1.09 (2H, tt, J = 7.2, 4.3 Hz, cyclopropC / 72). Rotameric ratio 0.55:0.45; m / z 569.13 [M+H]+; ESI-HRMS [M+H]+found 569.1336, C26H3o79BrN602S requires 569.1334, found 571.1341 , C26H3o81BrN602S requires 571.1338; [M+Na]+found 591.1171 , C26H2979BrNeO2SNa requires 591.1168, found 593.1181 C26H2981BrNeO2SNa requires 591.1175. 1 -[[(1 S,9 / ?,10S)-10-Hydroxy-12-oxa-8-azatricyclo[7.3.1.02,7]trideca-2,4,6- trien-4-yl]methyl]-3-[2-[(2S)-2-(2-methylsulfanylphenyl)pyrrolidin-1-yl]-2- oxo-ethyl]-1-[(2-methyltetrazol-5-yl)methyl]urea

[0380] 17

[0381] The title compound was synthesized applying the general procedure as follows:

[0382] The crude 1-[[(1S,9R,10S)-10-[ferf-butyl(dimethyl) silyl]oxy-12-oxa-8- azatricyclo[7.3.1 ,02,7]trideca-2,4,6 -trien-4-yl]methyl]-3-[2-[(2S)-2-(2-methylsulfanyl phenyl)pyrrolidin-1-yl]-2-oxo-ethyl]-1-[(2-methyl tetrazol-5-yl)methyl]urea (127 mg, 0.180 mmol) was placed in a vial which was purged with argon and dry THF (4 mL) added. Tetrabutylammonium fluoride (0.36 mL, 0.36 mmol) was added dropwise and the solution stirred overnight at room temperature. The reaction was concentrated directly on to silica and purified by flash column chromatography on the Biotage Isolera (12 g silica, 0 to 5% 2M-NH3 MeOH / DCM) then purified by reverse phase column chromatography (Xbridge C18, 10 - 95% MeCN / H2O with NH4OH modifier) to provide the title compound as colourless gummy solid (47 mg, 0.076 mmol, 43%).

[0383] LCMS (Method A): 2.108 min, 615.0 [M+Na]+.1H NMR (400 MHz, DMSO) 57.37- 7.13 (m, 2.45H), 7.10-6.96 (m, 1.65H), 6.96-6.90 (m, 1H), 6.90-6.85 (m, 1H), 6.63-6.51 (m, 1H), 6.49-6.41 (m, 1H), 6.38-6.31 (m, 1H), 5.29-5.20 (m, 1H), 4.98-4.92 (m, 1H), 4.58-4.42 (m, 3H), 4.32-4.19 (m, 5H), 3.98-3.93 (m, 1H), 3.89-3.49 (m, 2.46H), 3.41-3.32 (m, 2H), 3.28-3.23 (m, 1H), 3.16-3.07 (m, 1H), 3.06-2.96 (m, 0.46H), 2.55-2.51 (m, 3H), 2.48-2.28 (m, 1.56H), 2.23-2.09 (m, 0.69H), 1.98- 1.59 (m, 3H), 1.27-1.17 (m, 1H). 1-[(4-Amino-3,5-difluoro-phenyl)methyl]-3-[2-oxo-2-[(2 / ?)-2-(2- methylsulfanylphenyl) pyrrolidine-1 -yl]ethyl]imidazolidine-2, 4-dione

[0384] 18

[0385] The title compound was synthesized applying the general procedure as follows:

[0386] Ethyl 2-[[(4-amino-3,5-difluoro-phenyl)methyl-(cyano- methyl)carbamoyl]amino]acetate (66.0 mg, 0.20 mmol) was dissolved in MeOH (3 mL) and lithium hydroxide monohydrate (17.0 mg, 0.40 mmol) dissolved in water (0.5 mL) was added dropwise. The solution was stirred at room temperature for ~ 1.5 hours before acidifying to pH 5 with HCI (1 N aq.). The aqueous layer was extracted with EtOAc (x3) giving an insoluble precipitate before being acidified to pH 2. The EtOAc and aqueous layers were mixed for a second time dissolving all solids and the phases and separated. The aqueous layer was concentrated to dryness to give -95 mg of a colourless gum. The crude material was purified on acidic HPLC to give the hydantoin as a beige solid (22 mg, 0.074 mmol, 37%).

[0387] The hydantoin intermediate (22 mg, 0.074 mmol) and (2R)-2-(2-methylsulfanyl- phenyl)pyrrolidine (14 mg, 0.070 mmol) were reacted according to General Procedure E. The crude product was purified by reverse phase column chromatography (Xbridge C18, 10 - 95% MeCN / H2O with NH4OH modifier) to give the hydantoin as a beige solid (7.0 mg, 0.015 mmol, 20%).

[0388] LCMS (Method A): 2.458 min, 475.2 [M+H]+.1H NMR (400 MHz, DMSO) 5 7.39 - 7.18 (m, 2.4H), 7.15 - 7.06 (m, 1 H), 7.05 - 6.98 (m, 0.6H), 6.86 - 6.74 (m, 2H), 5.36 - 5.20 (m, 1H), 5.19 - 5.14 (m, 2H), 4.45 - 4.34 (m, 1.2H), 4.34 - 4.24 (m, 2H), 4.17 - 4.08 (m, 0.4H), 4.01 - 3.84 (m, 2.4H), 3.77 - 3.63 (m, 1H), 3.60 - 3.48 (m, 0.4H), 3.42 - 3.33 (m, 0.4H), 2.56 - 2.53 (m, 1.2H), 2.49 - 2.48 (m, 1.6H), 2.46 - 2.29 (m, 0.4H), 2.29 - 2.15 (m, 0.6H), 2.03 - 1.58 (m, 3H). 1-[(4-Amino-3,5-difluoro-phenyl)methyl]-3-[2-oxo-2-[(2 / ?)-2-(2- methylsulfanyl phenyl)pyrrolidin-1 -y I] ethyl] -1 -prop-2-ynyl-urea

[0389] 19

[0390] The title compound was synthesized applying the general procedure as follows:

[0391] Ethyl 2-[[(4-amino-3,5-difluoro-phenyl)methyl-prop-2-ynyl-carbamoyl]amino]acetate (68 mg, 0.21 mmol) was reacted according to General Procedure C. The residue was used without further purification or analysis. The crude lithium salt of 2-[[(4- amino-3,5-difluoro-phenyl)methyl-prop-2-ynyl-carbamoyl]amino]acetate (63 mg, 0.21 mmol) and (2R)-2-(2-methylsulfanylphenyl)pyrrolidine (61 mg, 0.31 mmol) were reacted according to General Procedure E to give crude product as a yellow gum (104 mg). The crude product was purified by flash column chromatography on the Biotage Isolera (12 g silica, 0 to 3% MeOH / DCM) then by reverse phase column chromatography (Xbridge C18, 10 - 95% MeCN / FW with NH4OH modifier) to provide the title compound as colorless froth (26.5 mg, 0.056 mmol, 27%).

[0392] LCMS (Method A): 2.503 min, 473.2 [M+H]+.1H NMR (400 MHz, DMSO) 5 7.38 - 7.15 (m, 2.41 H), 7.12 - 6.98 (m, 1.62H), 6.86 - 6.76 (m, 2H), 6.64 - 6.49 (m, 1H), 5.30 - 5.21 (m, 1H), 5.11 - 5.06 (m, 2H), 4.34 (s, 1.17H), 4.29 (s, 0.77H), 4.00 - 3.79 (m, 4H), 3.79 - 3.66 (m, 0.66H), 3.64 - 3.53 (m, 1H), 3.18 - 3.11 (m, 1H), 3.05 - 2.95 (m, 0.46H), 2.54 (s, 3H), 2.41 - 2.10 (m, 1H), 2.00 - 1.58 (m, 3H).

[0393] N-[[1-[(4-amino-3-chloro-phenyl)methyl]-2-oxo-3-[2-oxo-2-[(2 / ?)-2-(2- methylsulfanyl phenyl)pyrrolidin-1 -yl]ethyl]imidazolidin-4- ylidene]amino]acetamide 20

[0394] The title compound was synthesized applying the general procedure as follows:

[0395] Ethyl 2-[[(4-amino-3-chloro-phenyl)methyl-[(5-methyl-1 ,3,4-oxadiazol-2- yl)methyl]carbamoyl]amino]acetate (235 mg, 0.520 mmol) was reacted according to General Procedure C to give 2-[5-(Acetylhydrazono)-3-[(4-amino-3-chloro- phenyl)methyl] -2-oxo-imidazolidin-1-yl]acetic acid as an off-white solid (307 mg, 0.520 mmol, 99%).

[0396] LCMS (method A) 1.57 min, 354.0, 356.0 [M+H]+, chloride isotope splitting pattern.

[0397] 2-[5-(Acetylhydrazono)-3-[(4-amino-3-chloro-phenyl)methyl]-2-oxo-imidazolidin-1- yl]acetic acid (100 mg, 0.170 mmol) and (2F?)-2-(3-methylsulfanylphenyl)pyrrolidine (49 mg, 0.25 mmol) were reacted according to General Procedure E. Purification by flash column chromatography (silica column, 0% to 5% 2 M methanolic ammonia in DCM gradient) followed by evaporation of solvent from the appropriate fractions afforded partially purified product. Further purification by prep. HPLC (basic) followed by evaporation of solvent from the appropriate fractions and drying under vacuum afforded the title compound as a white solid (33 mg, 0.061 mmol, 36%).

[0398] LCMS (Method A) 2.31 min, 529.2, 531.2 [M+H]+, chloride isotope splitting pattern.1H NMR (400 MHz, DMSO) 5 9.87 - 9.76 (m, 1 H), 7.38 - 7.14 (m, 2.5H), 7.14 - 6.95 (m, 2.5H), 6.91 - 6.84 (m, 1 H), 6.75 (dt, J = 8.2, 1.7 Hz, 1 H), 5.45 - 5.28 (m, 2H), 5.22 (d, J = 7.0 Hz, 0.5H), 4.44 - 4.17 (m, 3H), 4.11 - 3.86 (m, 3H), 3.69 (t, J = 8.6 Hz, 1 H), 3.59 - 3.40 (m, 0.5H), 3.29 (s, 3H), 2.53 (d, J = 3.8 Hz, 2H), 2.48 (d, J = 1 .6 Hz, 1 H), 2.18 (t, J = 9.6 Hz, 0.5H), 2.00 - 1.88 (m, 0.5H), 1.87 - 1.69 (m, 3.5H), 1.70 - 1.58 (m, 0.5H).

[0399] N-[[1-[(4-amino-3-chloro-phenyl)methyl]-3-[2-[(2 / ?)-2-(2- bromophenyl)pyrrolidin-1-yl]-2-oxo-ethyl]-2-oxo-imidazolidin-4- ylidene]amino]acetamide

[0400] 21

[0401] The title compound was synthesized applying the general procedure as follows:

[0402] 2-[5-(Acetylhydrazono)-3-[(4-amino-3-chloro-phenyl)methyl] -2-oxo-imidazolidin-1- yl]acetic acid (80 mg, 0.14 mmol) and (2R)-2-(2-bromophenyl)pyrrolidine (61 mg, 0.27 mmol) were reacted according to General Procedure E. Purification by flash column chromatography (silica column, 0% to 5 % 2M methanolic ammonia in DCM gradient) followed by prep. HLPC (basic) afforded the title compound (32.6 mg, 0.057 mmol, 42%).

[0403] LCMS (Method B) 1.68 min, 561.0, 563.0 [M+H]+, halide splitting pattern.1H NMR (400 MHz, CDCI3) 6 9.74 (s, 0.4H), 9.51 (s, 0.3H), 8.69 (s, 0.3H), 7.66 - 6.63 (m, 7H), 5.56 - 5.11 (m, 1 H), 4.62 - 3.35 (m, 10H), 2.67 - 2.24 (m, 1 H), 2.17 - 1.53 (m, 6H).

[0404] N-[[1-[(6-amino-5-chloro-3-pyridyl)methyl]-3-[2-[(2 / ?)-2-(2- methylsulfanylphenyl) pyrrolidin-1-yl]-2-oxo-ethyl]-2-oxo-imidazolidin-4- ylidene]amino]acetamide

[0405] 22

[0406] The title compound was synthesized applying the general procedure as follows: 2-[5-(Acetylhydrazono)-3-[(6-amino-5-chloro-3-pyridyl) methyl]-2-oxo-imidazolidin-1- yl]acetic acid (64 mg, 0.18 mmol) and (2R)-2-(2-methylsulfanylphenyl)pyrrolidine (70 mg, 0.36 mmol) were reacted according to General Procedure E. Purification by flash column chromatography (silica column, 0% to 5% 2 M methanolic ammonia in DCM gradient) followed by prep. HLPC purification (basic) and evaporation of solvent from the appropriate fractions afforded the title compound (34 mg, 0.064 mmol, 36%).

[0407] LCMS (Method B) 1.49 min, 530.2, 532.2 [M+H]+, chloride isotope splitting pattern.1H NMR (400 MHz, CDCI3) 6 9.56 (s, 0.27H), 9.04 (s, 0.38H), 8.50 (s, 0.35H), 7.88-7.79 (m, 1 H), 7.47-6.90 (m, 5H), 5.54-5.34 (m, 1 H), 4.96-4.93 (m, 2H), 4.53- 3.56 (m, 8H), 2.55- 2.30 (m, 5H), 2.11-1.67 (m, 5H).

[0408] N-[[1-[(4-amino-3-chloro-phenyl)methyl]-3-[2-[(2 / ?)-2-(2- bromophenyl)pyrrolidin-1-yl]-2-oxo-ethyl]-2-oxo-imidazolidin-4- ylidene]amino]acetamide

[0409] 23

[0410] The title compound was synthesized applying the general procedure as follows:

[0411] 2-[5-(Acetylhydrazono)-3-[(4-amino-3-chloro-phenyl)methyl] -2-oxo-imidazolidin-1- yl]acetic acid (80 mg, 0.14 mmol) and (2R)-2-(2-bromophenyl)pyrrolidine (61 mg, 0.27 mmol) were reacted according to General Procedure E. Purification by flash column chromatography (silica column, 0% to 5 % 2M methanolic ammonia in DCM gradient) followed by prep. HLPC (basic) afforded the title compound (32.6 mg, 0.057 mmol, 42%).

[0412] LCMS (Method B) 1.68 min, 561.0, 563.0 [M+H]+, halide splitting pattern.1H NMR (400 MHz, CDCh) 6 9.74 (s, 0.4H), 9.51 (s, 0.3H), 8.69 (s, 0.3H), 7.66 - 6.63 (m, 7H), 5.56-5.11 (m, 1H), 4.62-3.35 (m, 10H), 2.67-2.24 (m, 1H), 2.17-1.53 (m,

[0413] 6H).

[0414] Comparison Compounds

[0415] The following compounds were tested as part of the examples.

[0416] CsA

[0417] Table 1: Comparison Compounds. Compounds 24 and 25 are compounds 1 and 4 of PCT / EP2019 / 073106, respectively. Example 3: In vitro Biophysical Assays - Binding Constants and Selectivity

[0418] To determine their selectivity profile, binding constants of various compounds of the present invention and comparison compounds were assayed for cyclophilin isoforms CypA, CypB and CypD using SPR and ITC. Table 2: Binding constants of exemplary compounds and comparison compounds to cyclophilin isoforms CypA, CypB and CypD. Measurements made by SPR unless otherwise mentioned. CsA: cyclosporin. Compounds 24 and 25 are first generation trivector cyclophilin inhibitors disclosed in W02020 / 043831 ). Surface Plasmon Resonance

[0419] SPR measurements were performed on a Biacore T200 instrument (Cytiva). Ni2+- nitrilotriacetic acid (NTA) sensor chips, 1-ethyl-3-(3-diaminopropyl) carbodiimide hydrochloride (EDC) and N hydroxy succinimide (NHS) were purchased from Cytiva. Immobilization and covalent stabilization of His-Cyps Pure His-cyclophilins on the NTA sensor chip was performed essentially as described, using 200 nM concentrations of each protein, in Running Buffer (PBS, pH7.4; 0.05 % surfactant P20, 2% v / v ethanol; 50 pM EDTA), at 30 pl min-1with 60 second contact times on the activated NTA surfaces. This gave signals of 1 ,921 Rll for His-CypA, 1932 Rll for His-CypB and 1 ,397 Rll for His-CypD. Specific surface protein activity was assayed by passing saturating amounts of CsA (2 pM) in Running Buffer over these surfaces; values of 94.1%, 95.5% and 95.6% activity were obtained for His-CypA, -B and -D, respectively.

[0420] Kinetic titration experiments

[0421] CsA:

[0422] Single cycle kinetic titration binding experiments were performed in triplicate at 25°C. 3-fold dilution concentration series of CsA, ranging from 2.45 nM to 200 nM, in Running Buffer, were injected over the sensor surface, at 100 pl min-1with a 90 s contact time and a 90 s dissociation time. The sensor surface was regenerated between experiments by dissociating any formed complex in running buffer for at least 1 ,200 seconds. The apparent on-rate ( +) and off-rate (k-) constants and the equilibrium dissociation constant (KD) were calculated from reference corrected sensorgrams by global fitting of a 1 :1 binding model, including a mass transport term, using analysis software (v.2.02, Cytiva) provided with the Biacore T200 instrument.

[0423] Assays of compounds Kinetic titration binding experiments were performed in triplicate at 25°C. 2- fold dilution concentration series of the compounds, ranging from 0.0195 pM to 20 pM, in Running Buffer, were injected over the sensor surface, at 100 pl min-1with a 15 s contact time and a 600 s dissociation time. The sensor surface was regenerated between experiments by dissociating any formed complex in running buffer for at least a further 600 seconds. The apparent on- rate ( +) and off-rate (k-) constants and the equilibrium dissociation constant (KD) were calculated from reference corrected sensorgrams by global fitting of a 1 :1 binding model, including a mass transport term, using analysis software (v.2.02, GE Healthcare) provided with the Biacore T200 instrument.

[0424] Isothermal Titration Calorimetry

[0425] All ITC experiments were carried out at 25°C on a MicroCai Auto iTC200 (GE Healthcare) instrument. The buffer used in the titrations of the compounds was 50 mM phosphate buffer, pH 6.5 and the concentration of DMSO was 2% v / v for all the compounds. Final compound solutions were heated to 65°C and / or sonicated prior to the experiment. Each experiment consisted of an initial injection of 0.4 pL followed by 19 x 2 pL injections to achieve saturation. Control experiments were performed, where each compound was titrated into buffer and when small amount of heat was detected due to heat of dilution, it was subtracted when processing the data using a linear fit method. In all cases the first injection was omitted from the data processing. All data were analysed using the MicroCai PEAQ-ITC Analysis software. Due to the fact that these compounds lie in the low-mid micromolar range and the c value (Wiseman constant) is very small, a fixed stoichiometry to 12 was applied during the non-linear regression of the raw data for fitting the data. Discussion

[0426] The above data indicates that all the compounds of the present invention can bind to CypA, CypB and CypD at therapeutically relevant concentrations. However, it also demonstrates that the compounds of the present invention have a binding profile that is selective for CypB and / or CypD over CypA, particularly CypB over CypA.

[0427] Compound 1 shows higher binding potency than any previously reported small molecule Cyclophilin inhibitor, particularly compared to CsA binding CypB and CypD. This poses a potential advantage for this compound over the existing Cyclophilin inhibitors that non-specifically bind to the whole Cyclophilin family - for instance in the treatment of MASH.

[0428] Compounds 2, 3 and 4 range from the low pM to high nM scale and are selective with a range of 2- to 7-fold binding preference for CypB over CypA and CypD, whereas Compounds 18 and 19 are neither potent nor selective, with Kd ranging at the high pM scale.

[0429] Compounds 5 and 6 appear to be averagely potent in the low pM range, however they lack selectivity among the different Cyp isoforms. On the contrary Compounds 7 to 11 show as to be noticeably selective for CypB with Compound 9 being approximately 80 and 20 times more potent for CypB and over CypA and CypD respectively. However, Compound 7 appears to be not only 10 to 15 times more selective for CypB but also very potent with Kd, lying at about 10 nM.

[0430] The hydantoin compounds (20, 21, 22 and 23) do not appear to bind efficiently to any isoform. Example 4: In vitro Cellular Efficacy Studies - Mode of Action

[0431] To determine whether the compounds could modulate mitochondrial Permeability Transition Pore (mPTP), the EC50 of selected compounds of the present invention was determined in a Calcium Retention Capacity (CRC) assay, as evaluated through Calcium concentration fluorescence visualization techniques in HepG2 cells. For comparison, the S-enantiomer of 1 (compound 17) was also included.

[0432] Table 3: Calcium retention capacity assay on HepG2 cells.

[0433] Calcium Retention Capacity Assay

[0434] The compounds were prepared as a 10 mM stock solution in 100% DMSO. Subsequent dilutions were made in 100% DMSO and then in assay buffer to 10-fold the final concentration.

[0435] HepG2 cells were permeabilized with 100 mM digitonin for 10 min in ice cold buffer containing 1 mM EGTA. Following two wash steps to remove the digitonin, the cells were plated into 96 well black and clear plates at 106cell per well in a 180 mLassay buffer containing 0.5 mM Calcium Green 5N. Compounds dilution were prepared in DMSO to 1000 fold the final concentration, diluted 1 :100 in assay buffer and added to the assay as 20 mL per well. The assay buffer contained 5 mM glutamate and 2.5 mM malate. The cell plate was immediately run on the FLIPR Tetratmwhich added 5 mL of 200 mM (5 mM) calcium chloride every 5 minutes whilst reading the plate every 3 seconds. The area under the curve (AUC) of the raw trace was calculated at each concentration. AUC values were plotted against concentration and EC50 values calculated. Experiments were repeated three times and the EC50 value was determined from a curve fitted to the averaged replicates.

[0436] Discussion

[0437] This demonstrates that the compounds of the present invention affect the mitochondrial Permeability Transition Pore (mPTP). Without wishing to be bound by theory, this is believed to be due to modulation of CypD, to affect a CypD-mediated pathway. This indicates the suitability of the compounds of the present invention in treating chronic mitochondrial disorders such as fibrosis, congestive heart failure, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, macular degeneration, epilepsy, diabetic retinopathy, liver disease and other diseases due to mitochondrial dysfunction.

[0438] Notably, Compound 1 is twice as effective as CsA as for its potency for inhibition of mPTP opening, with EC50 values at the higher nM range. In contrast, compound 17 does not show significant potency in this assay.

[0439] The particularly low EC50 value for Compound 1 (240 nM) suggests it would be particularly suitable in the treatment of fibrosis, as inhibition of CypD prevents opening of the MPTP pore that causes cell death under Calcium overload and oxidative stress observed during fibrosis.

[0440] Correlation between CypD Kd and EC50 values supports the evidence that the mode of action of Compound 1 is consistent with binding to CypD to inhibit mPTP opening under cellular stress conditions (Figure 2). Example 5: Effect of the Compounds on TNBC Cells

[0441] To determine the effect of the compounds on the proliferation of cancer cells, a series of functional in vitro assays were performed against three TNBC cell lines representing 3 TNBC subtypes which demonstrate higher mortality (Yin et al., Breast Cancer Res 22(1 ): 61 , herein incorporated by reference).

[0442] MDA-MB-231 , HCC1806 and BT549 cells were seeded at a density of 1000 cells per well in GelTrex-coated 384-well cell culture plates, in alphaMEM (without nucleosides) cell culture medium with 10% heat-inactivated foetal bovine serum supplemented with 2000 U / mL IL-2, 2.5 pM NucView 488 and 10 pM test compound or vehicle. Cells were imaged with a 10X objective every 3 hours for 120 hours using an IncuCyte ZOOM microscope from Essen Bioscience.

[0443] Using the IncuCyte ZOOM software, custom image analysis procedures were developed and applied for each cell line to determine cell number (red nuclei) and number of apoptotic cells (NucView 488-positive green nuclei) over the time course of the experiment. Apoptotic fraction of the population (number of nuclei with co-localized red and green fluorescence divided by total number of red nuclei in the population) and fold increase in number of red nuclei were determined at various time points.

[0444] As expected, CsA consistently decreased proliferation of the TNBC lines at higher concentrations.

[0445] In comparison, the effect of tri-vector ligands was variable; Compound 25 (compound 4 of PCT / EP2019 / 073106) slightly decreased the proliferation of MDA-MB-231 cells (mesenchymal stem-like subtype) while Compound 1 slightly decreased proliferation of HCC1806 cells (basal-like 2 subtype), see Figure 3. Discussion

[0446] This indicates that compounds of the present invention have direct antiproliferative activity on various types of TNBC cell lines, indicating their suitability for treating TNBC cancer directly, particularly in treating certain patient populations with cancers of those cell subtypes.

[0447] Example 6: Enhancement of Lymphocyte Cytolytic Activity by Compounds of the Present Invention

[0448] As CsA is known to inhibit T cell proliferation and activation via calcineurin inhibition, an assay was performed to determine the effect of the compounds of the present invention on the ability of lymphocytes to cause cytolysis in a TNBC cell line.

[0449] NK cells are heterogeneous in their cell killing activity; some NK cells only kill once before deactivating, whereas some kill 3-8 cells (serial killing) before deactivating - introducing variability. Thus, to establish whether the compounds of the present invention affected the ability of lymphocytes to cause cytolysis, an NK cell to TNBC cell ratio of 1:4 was used.

[0450] The TNBC cell line MDA-MB-231was chosen, as previous experiments demonstrated that it was more resistant to cytolysis by NK92 cells (a human natural killer cell line) compared to the HCC1806 and BT549 cell lines.

[0451] MDA-MB-231 cells were seeded at a density of 1000 cells per well in GelTrex-coated 384-well cell culture plates in alphaMEM (without nucleosides) cell culture medium with 10% heat-inactivated foetal bovine serum, supplemented with 2000 U / mL IL-2, 2.5 pM NucView 488 and 10 pM test compound or vehicle. To some of these wells was also added NK92 cells at a density of 250 cells per well. The cells were imaged with a 10X objective every 3 hours for 120 hours using an IncuCyte ZOOM microscope from Essen Bioscience. Using the IncuCyte ZOOM software, custom image analysis procedures were developed and applied for each cell line to determine cell number (red nuclei) and number of apoptotic cells (NucView 488-positive green nuclei) over the time course of the experiment. Apoptotic fraction of the population (number of nuclei with co-localized red and green fluorescence divided by total number of red nuclei in the population) and fold increase in number of red nuclei were determined at various time points.

[0452] As expected, CsA, was able to directly induce apoptosis of MDA-MB-231 cells (Figure 4A), but it decreased the ability of NK92 cells to kill MDA-MB- 231 cancer cells, with this effect beginning after 3 hours (Figure 4B). This effect likely results from non-specific toxicity of CsA toward NK92 cells.

[0453] Figure 3A demonstrated that Compound 25 is able to decrease the proliferation of MDA-MB-231 , and Figure 4A confirms this, showing that Compound 25 significantly and directly increases apoptosis in the MDA-MB- 231 cell line.

[0454] In comparison, Figure 4A demonstrates that Compound 1 is not able to significantly and directly increase apoptosis in the MDA-MB-231 cell line.

[0455] Surprisingly however, Figure 4B demonstrates that both Compound 25 and Compound 1 significantly increase apoptosis of MDA-MB-231 , when in the presence of NK92 cells.

[0456] This demonstrates that compounds of the present invention are able to enhance the ability of NK92 cells to cause apoptosis of the MDA-MB-231 TNBC cell line, with the effect beginning after about 24 hours (Figure 4B). After 36 hours of treatment with Compound 1, NK92 cytolysis of MDA-MB- 231 cells was approximately double that of control vehicle-treated NK92 cells. After 72 hours, it was evident that the enhancement of NK92 cytolysis of MDA-MB-231 cells by Compound 1 results in a significant decrease in the MDA-MB-231 population (Figure 4C). This suggests that the increased apoptosis of MDA-MB-231 cells observed with Compound 1 results predominantly from enhancement of NK92 cytolytic ability, rather than direct activity on the MDA-MB-231 cells.

[0457] Notably, Compound 17 is the S enantiomer of Compound 1, yet Compound 17 did not significantly enhance NK92 killing ability.

[0458] In light of Figure 3A and Figure 4A, it is possible that the effect observed for compound 25 in Figure 4B results from a combination of its direct effect on MDA-MB-231 cells, combined with a previously unknown enhancement of NK92 cytolysis of these cells.

[0459] When primary human NK cells using a 4: 1 ratio of NK to TNBC cells were employed in the assay (Figure 4D), using DMEM cell culture medium with 10% heat-inactivated foetal bovine serum, supplemented with 2000U / mL IL-2 and 10 pM compound or vehicle, all compounds tested displayed significantly enhanced primary NK cell-induced apoptosis of MDA-MB-231 cells, with the effect beginning after 3 hours of treatment.

[0460] Compound 25 demonstrated greatest efficacy in enhancing primary NK killing of MDA-MB-231 cells with the peak effect, which was double that of vehicle- treated control, occurring approximately 18 hours after treatment. However, this effect is likely a combination of a direct effect on MDA-MB-231 and enhancement of primary NK cell cytolytic ability. CsA showed the least enhancement of primary NK killing ability, likely due to toxicity towards the primary NK cells.

[0461] When primary human CD8+T cells using a 4:1 ratio of T cells to TNBC cells, were employed and activated in the assay (Figure 4E), using DMEM cell culture medium with 10% heat-inactivated foetal bovine serum, supplemented with 2000U / mL IL-2, 100 ng / mL CD3 antibody, 2 pg / mL CD28 antibody and 10 pM compound or vehicle, about 36 hours after treatment and activation, enhancement of CD8+T cell-induced apoptosis of MDA-MB-231 cells by Compound 1 was seen. After 5 days, this effect was double that of the untreated, activated, control. This contrasted with the effect with CsA, where the CD8+T cell-induced apoptosis decreased after 3 days. This was likely a result of toxicity toward and inhibition of proliferation of the primary T cells since inhibition of CD8+T cell clustering was observed (Figure 5 and Figure 6).

[0462] Discussion

[0463] Taken together, these results show that the compounds of the present invention display a previously unknown activity - enhancing lymphocyte- induced apoptosis of target cells (particularly cancer cells, especially TNBC cells) and that their mode-of-action is distinct from that of CsA.

[0464] The data provides that these compounds are not only useful in enhancing both NK and CD8+T cell cytolytic function, but likely present a novel mode of action in this therapeutic space - use of such compounds as immunoadjuvants (particularly oncoadjuvants). This activity enables combination of these compounds with existing therapeutics that enhance tumour-killing lymphocytes, to further enhance lymphocyte cytolytic ability and persistence of CAR-lymphocytes. Furthermore, as small chemical molecules, these compounds present a significant advantage in synthesis, stability, and patient administration.

[0465] Example 7: In vitro DMPK studies

[0466] Compounds of the present invention were assessed using a standard LADMET panel of in vitro drug metabolism and pharmacokinetics (DMPK) experiments:

[0467]

[0468]

[0469] Table 4: In vitro DMPK properties for selected compounds of the invention.

[0470] DMPK Toxicity Experiments

[0471] Aqueous solubility

[0472] Aqueous solubility (pM) was determined by comparing the peak area of the principal peak in a calibration standard (200 pM) containing organic solvent (methanol / water, 60 / 40, v / v) with the peak area of the corresponding peak in a buffer sample. In addition, chromatographic purity (%) was defined as the peak area of the principal peak relative to the total integrated peak area in the HPLC chromatogram of the calibration standard. A chromatogram of the calibration standard of each test compound, along with a UV / VIS spectrum with labelled absorbance maxima, was generated.

[0473] Partition Coefficient

[0474] The total amount of compound was determined as the peak area of the principal peak in a calibration standard (100 pM) containing organic solvent (methanol / water, 60 / 40, v / v). The amount of compound in buffer was determined as the combined, volume corrected, and weighted areas of the corresponding peaks in the aqueous phases of three organic-aqueous samples of different composition. An automated weighting system was used to ensure the preferred use of raw data from those samples with well quantifiable peak signals. The amount of compound in organic was calculated by subtraction. Subsequently, Log D was calculated as the Log of the amount of compound in the organic phase divided by the amount of compound in the aqueous phase.

[0475] Protein Binding

[0476] The peak areas of the test compound in the buffer and test samples were used to calculate percent binding and recovery.

[0477] Permeability

[0478] The apparent permeability coefficient (Papp) of the test compound was calculated as follows: where VR is the volume of the receiver chamber. CR.end is the concentration of the test compound in the receiver chamber at the end time point, At is the incubation time and A is the surface area of the cell monolayer. CD, mid is the calculated mid-point concentration of the test compound in the donor side, which is the mean value of the donor concentration at time 0 minute and the donor concentration at the end time point. CR.mid is the mid-point concentration of the test compound in the receiver side, which is one half of the receiver concentration at the end time point. Concentrations of the test compound were expressed as peak areas of the test compound.

[0479] Cytochrome P450 Inhibition (HPLC-UV / VIS and HPLC-MS / MS detection)

[0480] Peak areas corresponding to the metabolite of each substrate were recorded. The percent of control activity was then calculated by comparing the peak area obtained in the presence of the test compound to that obtained in the absence of the test compound. Subsequently, the percent inhibition was calculated by subtracting the percent control activity from 100 for each compound. IC50 values (concentration causing a half-maximal inhibition of control values) were determined by non-linear regression analysis of the concentration-response curve using Hill equation curve fitting.

[0481] Intrinsic Clearance (microsomes, S9, cryopreserved hepatocytes)

[0482] Metabolic stability, expressed as percent of the parent compound remaining, was calculated by comparing the peak area of the compound at the time point relative to that at time-0. The half-life (ti / 2) was estimated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming the first-order kinetics. The apparent intrinsic clearance (CLnt, in pL / min / pmol, pL / min / mg or pL / min / Mcell) was calculated according to the following formula:

[0483] 0.693

[0484] Bacterial Cytotoxicity

[0485] Bacterial cytotoxicity tests were carried out using Reverted Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537) that were incubated 96 hours at 37 Celsius. The results for cytotoxicity are expressed as percent of control growth (ODeso). Compounds with growth of less than 60 % of control are flagged and considered cytotoxic. None of the compounds tested showed evidence of cytotoxicity.

[0486] Ames Tests

[0487] Compounds were incubated for 96 hours at 37 °C at 4 different concentrations (5 mM, 10 mM, 50 mM, 100 mM) using Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537) in the absence and presence of rat liver S9 microsomes. Wells that displayed bacteria growth due to the reversion of the histidine mutation (as judged by the ratio of OD430 / OD570 being greater than 1 .0) are counted and recorded as positive counts. The significance of the positive counts between the treatment (in the presence of test compound) and the control (in the absence of test compound) are calculated using the one-tailed Fisher's exact test. Three significance levels are reported as follows:

[0488] Weak positive, if 0.01 < p < 0.05, denoted as "+"

[0489] Strong positive, if 0.001 < p < 0.01 , denoted as "++"

[0490] Very strong positive, if p < 0.001 , denoted as "+++"

[0491] Control experiments were carried out with 2-aminoanthracene (10 mM), 9- aminoacridine (10 mM), quercetin (30 mM) and streptozocitin (2.5 mM). None of the tested cyclophilin inhibitors showed significant differences from background rate.

[0492] Discussion

[0493] Compounds 7 and 8 have the most promising DMPK, with a high solubility profile, good lipophilicity and acceptable intestinal drug permeability and intrinsic clearance as well as score negative in the Ames test.

[0494] Compounds 1, 2 and 7 also show favourable solubility, lipophilicity and intestinal cell permeability. Intrinsic clearance of Compound 7 in rat cryoprotected hepatocytes was relatively low, providing a long half-life.

[0495] Compound 1 shows exceptionally promising potency for CypB. Compound 1 shows acceptable clearance values and no evidence of strong drug-drug interactions.

[0496] To conclude, it appears that compound 7, strongly potent, significantly selective for CypB and with promising DMPK properties appears as the best all-rounder candidate of these series of compounds.

[0497] The AMES mutagenicity tests on 4 strains in the absence (-) and presence (+) of S9 (TA98 - / + S9), (TA100 - / + S9), (TA1535 - / + S9), (TA1537 - / + S9) for compounds 7 to 9 confirm lack of genotoxicity. Without wishing to be bound by theory, this is believed to be due to the replacement of the unsubstituted aniline moiety.

[0498] Example 8: Effect of TCEs on Activation-associated Clustering of Primary Human CD8+T-cells

[0499] CsA is known to inhibit T cell proliferation and activation via calcineurin inhibition. Since the compounds of the present invention increase the ability of T-cells to kill MDA-MB-231 cells, they are not considered to act in this manner. However, to elucidate this, the effect of CsA was compared with the compounds of the present invention on T cell clustering and proliferation, post activation in culture. T cell clustering in culture is a known phenomenon postactivation in culture in vitro.

[0500] Primary human T cells were seeded in GelTrex-coated 384-well cell culture plates at 4000 cells per well in DMEM cell culture medium with 10% heat- inactivated foetal bovine serum, supplemented with 2000U / mL IL-2, 100 ng / mL CD3 antibody, 2 pg / mL CD28 antibody and 10 pM compound or vehicle. The cells were imaged with a 10X objective every 3 hours for 120 hours using an IncuCyte ZOOM microscope from Essen Bioscience. Using the IncuCyte ZOOM software, custom image analysis procedures were developed and applied to identify aggregates of cells 600 pm2or greater in size. These aggregates, which consisted of 5 or more cells, were analysed as ‘T cell clusters’.

[0501] Figure 5: Aggregates of cells 600 pm2or greater in size (5 or more cells) were analysed as ‘clusters’. These clusters are highlighted by yellow masking in the images of this figure.

[0502] Figure 6: Quantification of the mean number of T cell clusters formed in culture over time revealed that CsA significantly reduces the number of activated CD8+ T cell clusters which form after activation. Compound 1 causes a slight (but insignificant) reduction in the number of T-cell clusters formed.

[0503] Figure 7: Quantification of the area occupied by T cells within clusters as a proportion of the total area occupied by T cells, using confluence as a measure of occupied area, revealed that CsA significantly reduces the proportion of CD8+ T cells in clusters which form after activation. Compound 1 causes a slight (but insignificant) reduction in the proportion of CD8+ T cells in clusters.

[0504] Figure 8A: Confluence of primary CD8+ T cells generally increased with time indicating that T cell proliferation occurred. However, when confluence of T cells at 120 h was compared, it was observed that CsA-treated CD8+ T cells showed no increase in confluence over time, resulting in significantly reduced confluence following activation compared to vehicle-treated control.

[0505] Compound 1 causes a slight (but insignificant) reduction in confluence.

[0506] Figure 8B: Size of primary CD8+ T cells clusters generally increased with time. However, when size of T cell clusters at 120 h was compared, it was observed that CsA-treated CD8+ T cells showed significantly reduced size of CD8+ T clusters following activation compared to vehicle-treated control. Compound 1 caused no significant reduction in size of clusters.

[0507] Discussion

[0508] Activation of T-cells in culture is generally associated with proliferation of T- cells and the formation of clusters of T-cells. However, despite reduced cluster formation, 1 clearly does not inhibit T-cell proliferation given its enhancement of T cell-induced TNBC killing. This aligns with the disclosure of NA Zumwalde et al., J Immunology 2013 Oct 1 ; 191 (7): 3681-3693 (herein incorporated by reference) where reduced I-CAM1 expression is associated with reduced clustering but conversely with increased cytotoxic ability of T cells.

[0509] Without wishing to be bound by theory, in light of the above results, it is believed that the compounds of the present invention may be acting in a similar manner to that taught in NA Zumwalde et al.

[0510] Example 9: Pre-Activation of Lymphocytes by Compounds of the Present Invention

[0511] MDA-MB-231 cells were seeded at a density of 1000 cells per well in GelTrex-coated 384-well cell culture plates, in AlphaMEM (without nucleosides) cell culture medium with 10% foetal bovine serum, supplemented with 2000 U / mL IL-2, 2.5 pM NucView 488 and 10 pM Compound 1 or vehicle. To some of these wells was also added NK92 cells which had been pre-treated with 10 pM Compound 1 or vehicle for 24 hours, at a density of 250 cells per well. The cells were imaged with a 10X objective every 3 hours for 120 hours using an IncuCyte ZOOM microscope from Essen Bioscience.

[0512] Using the IncuCyte ZOOM software, custom image analysis procedures were developed and applied for each cell line to determine cell number (red nuclei) and number of apoptotic cells (NucView 488-positive green nuclei) over the time course of the experiment. Apoptotic fraction of the population (number of nuclei with co-localized red and green fluorescence divided by total number of red nuclei in the population) and fold increase in number of red nuclei were determined at various time points, as shown in Figure 9.

[0513] These results show that NK92 cells pre-treated with Compound 1 for 24 hours prior to being incubated with TNBC (MDA-MB-231) cells in the presence of Compound 1 for 72 hours, showed enhanced ability to induce apoptosis of target TNBC (MDA-MB-231) cells, compared to NK92 cells pre-treated with vehicle prior to being incubated with TNBC (MDA-MB-231 ) cells in the presence to Compound 1 for 72 hours.

[0514] Example 10: Biophysical Assays - Binding Constants and Selectivity Profiles To determine the selectivity profile of various compounds of the present invention and comparison compounds, surface plasmon resonance was used to assay their binding constants against cyclophilin isoform NKTR.

[0515] His-NKTR was prepared from the pET28a-LIC vector obtained from Addgene (Plasmid #25597). His-NKTR was expressed, purified and used in SPR experiments following the protocol described in Example 3. Gel densitometry of final preparations indicates purity for both proteins of > 98%. Resulting Kd values are listed in Table 5. Kd values for isoforms CypA, CypB, CypD were provided previously in Table 2.

[0516] Table 5: Kinetic Affinity Constants, KD Values are the mean ± SD, n = 3.

[0517] * Some evidence of heterogeneity in data for concentrations > 0.5 pM.

[0518] ** Kinetic constants were not uniquely determined, although KD should be considered robust due to steady-state data for each concentration being well defined.

[0519] Example 11 : Cell Killing Assays - Ko-Linked Enhancement of Natural Killer (NK) Cell Killing

[0520] To determine their efficacy in promoting NK cell-induced killing of target tumour cells, various compounds of the present invention and comparison compounds were assayed for their ability to boost NK92-cell induced apoptosis of target MDA-MB-231 cells at 10 pM concentration. As Alisporivir (Ali) is a non-immunosuppressive cyclophilin inhibitor, its effects were compared to that of the compounds of the present invention.

[0521] MDA-MB-231 cells expressing nuclear-restricted red fluorescent protein mKate2, were seeded at a density of 1000 cells per well in GelTrex-coated 384-well cell culture plates in alphaMEM (without nucleosides) cell culture medium with 10% heat-inactivated foetal bovine serum, supplemented with 2000 U / mL IL-2, 2.5 pM NucView 488 and 10 pM test compound or vehicle. To some of these wells was also added NK92 cells at a density of 250 cells per well. The cells were imaged with a 10X objective every 3 hours for 120 hours using an IncuCyte S3 microscope from Sartorious.

[0522] Using the IncuCyte software, custom image analysis procedures were developed and applied for each cell line to determine cell number (red nuclei) and number of apoptotic cells (NucView 488-positive green nuclei) over the time course of the experiment. Apoptotic fraction of the population (number of nuclei with co-localized red and green fluorescence divided by total number of red nuclei in the population) was determined at various time points.

[0523] Figure 10A and Figure 10C demonstrate that in the absence of NK92 cells, none of the tested compounds of the present invention induced significant apoptosis of the MDA-MB-231 cells.

[0524] In contrast, Figure 10B and Figure 10D show that in the presence of NK92 cells, all of the tested compounds of the present invention increase apoptosis of the MDA-MB-231 cells in comparison to the vehicle-treated control (veh).

[0525] Of the tested compounds, Compound 3 showed greatest efficacy. It boosted NK92-induced apoptosis of MDA-MB-231 cells more than 5-fold after 48 hours, the timepoint where maximum apoptosis was observed. Notably, Compound 3 demonstrated greatest affinity for NKTR in the SPR assays.

[0526] Compound 1, which boosted NK92-induced apoptosis of MDA-MB-231 cells more than 3-fold, showed a correspondingly lower affinity for NKTR in the SPR assays. In contrast, Compound 17 (provided as a comparison compound to Compound 1) did not significantly boost NK92-induced killing of MDA-MB-231 cells and did not show significant binding to NKTR in the SPR assays. Although Compound 7 showed an affinity for NKTR similar to Compound 3 in the SPR assays, it did not demonstrate a correspondingly high efficacy in the cell killing assay. This may be a result of its higher affinity for CypB; it may preferentially bind CypB compared to NKTR in the NK92 cells.

[0527] Together, this data supports the binding of NKTR as the likely mechanism for the efficacy of the compounds of the present invention in boosting NK cell ability to kill target cancer cells (Figure 10).

[0528] When the effect of the compounds on NK92 cells was examined, the images showed that Alisporivir induced apoptosis of the NK92 cells (Figure 11 ). As Alisporivir also induced significant apoptosis of the target MDA-MB-231 cells in the absence of NK92 cells (Figure 10A and Figure 10C), this suggests that although this macrocyclic, pan-selective cyclophilin inhibitor is nonimmunosuppressive, it still demonstrates general toxicity to immune cells, as previously observed with Cyclosporine A (Figure 4).

[0529] Example 12: Cell Killing Assays - Target-linked Activity Verified by Target Knockdown in NK cells

[0530] To verify that the major target of the compounds of the present invention in natural killer cells is NKTR, modified NK92 cell lines where NKTR expression levels were reduced were used. These NKTR knockdown NK92 cell lines, stably transfected with NKTR-targeting shRNA plasmids (SCBT# sc-78500- SH) were assayed for their ability to boost NK92-induced apoptosis of target MDA-MB-231 cells, compared to a control NK92 cell line, stably transfected with a non-targeting plasmid (SCBT# sc-108060).

[0531] As described above, MDA-MB-231 cells expressing nuclear-restricted red fluorescent protein mKate2 were seeded with the modified NK92 cells in the presence of NucView 488 and target cells undergoing apoptosis were identified by co-localisation of red and green fluorescence in the nuclei. Apoptotic fraction of the population (number of nuclei with co-localized red and green fluorescence divided by total number of red nuclei in the population was determined at various time points.

[0532] Seminal articles on NKTR reported that knockdown of the protein reduced the ability of NK cells to kill their targets. This was also observed in our experiments; when NKTR expression was reduced, NK92-induced apoptosis of target MDA-MB-231 cells was also reduced.

[0533] Furthermore, when MDA-MB-231 cells were cultured with NK92 cells with reduced NKTR expression and incubated with 10 pM Compound 3, although Compound 3 boosted the NK92 killing of MDA-MB-231 cells, this was reduced compared to that of the control NK92 cells - as shown in Figure 12.

[0534] Together, the data demonstrated that when NKTR expression was reduced in the NK92 cell line, the effect of Compound 3 in boosting NK92-induced apoptosis of the target MDA-MB-231 cells was concomitantly reduced - validating NKTR as the target of Compound 3.

[0535] Example 13: Steatosis Assays - Effect on Steatosis in Induced Pluripotent Stem Cell-Derived Hepatocytes

[0536] As shown in Table 2, Compound 7 is particularly selective for cyclophilin B over both cyclophilin A and cyclophilin D.

[0537] As described above, a potential role for selective cyclophilin B inhibitors is in the treatment of metabolic dysfunction-associated steatohepatitis (MASH). As taught in Stauffer WT et al. PLoS One. 2024, 19(3): e0298211 (herein incorporated by reference), a cyclophilin B knockout mouse model prevented the induction of MASH by carbon tetrachloride treatment combined with a Western diet. As such, an assay was developed where compounds of the present invention and comparison compounds were tested for their effect on induced pluripotent stem cell-derived hepatocytes that were stimulated in vitro to a MASH-like phenotype - as reported in Kouridaki M-E et al. J. Med. Chem. 2025, 68, 6815-6831 (herein incorporated by reference).

[0538] Compound 7 significantly reduced steatosis in the above assay. The level of reduction was comparable to that seen in the experiments reported therein for Resmetirom, the only FDA-approved drug for MASH treatment (EC50 = 0.2614 pM for Resmetirom versus 0.2580 pM for Compound 7).

[0539] Discussion

[0540] The enhancement of NK92 cytolysis of TNBC (MDA-MB-231) cells when NK92 cells were pre-treated with Compound 1 for 24 hours was immediate.

[0541] This further supports the theory that the effect of Compound 1 in enhancing NK92 cytolytic ability towards TNBC (MDA-MB-231) cells is due to its direct effect on NK92 cells.

[0542] A possible mechanism for this activity may be engagement of the under- studied cyclophilin family member NKTR in a fashion which supports its activity. NKTR is an IL-2 regulated cell membrane-bound receptor protein found on NK cells and CD8+ T-cells, which possesses a cyclophilin-like PPI- ase domain and is known to mediate lymphocyte killing of tumour cells.

[0543] Preferred compositions, features and embodiments of each aspect of the invention are as for each of the other aspects mutatis mutandis unless context demands otherwise.

[0544] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in the text is not repeated in this text is merely for reasons of conciseness.

[0545] Reference to cited material or information contained in the text should not be understood as a concession that the material or information was part of the common general knowledge or was known in any country.

[0546] Although the invention has been particularly shown and described with reference to particular examples, it will be understood by those skilled in the art that various changes in the form and details may be made therein without departing from the scope of the present invention.

Claims

Claims1 . A compound of formula (I) or a pharmaceutically acceptable salt thereof:WhereinR1is selected from a group consisting of halo, -OCH3, -SCH3, -CF3, - CF2H, cyclopropyl, -Et and isopropyl,A is selected from a group consisting of: a., wherein;R2is selected from a group consisting of -NH2, -OH, -SH, -CF2H and halo, andY1is selected from a group consisting of methine and nitrogen; where Y1is methine, its hydrogen is optionally substituted with fluoro or chloro, and b. A 6-membered unsaturated ring having 0, 1 or 2 nitrogen atoms, where the ring is substituted at the para position with an amine, optionally where the ring is independently substituted with 1 or 2 fluoro or chloro groups.B is:Q is selected from a group consisting of carbon, oxygen, sulfur and nitrogen,Z1is selected from a group consisting of carbon and nitrogen,Z2is selected from a group consisting of carbon and nitrogen,R3selected from a group consisting of -CH3, -CF3, -CF2H and a C0-3- alkyl substituted with a moiety selected from a group consisting of - COOH, -SOOH, -SOOOH, -SOONHCH3, -NHSOOCH3, - CONHSOOCH3, -NHCONHSOOCH3, -SOOCi-3-alkyl, -CONHOH, a 3, 4, 5 or 6-membered saturated ring and a 5 or 6-membered unsaturated ring having 0, 1 , 2, 3 or 4 heteroatoms selected from nitrogen, oxygen or sulfur and optionally substituted with carbonyl, sulfonyl or hydroxy.

2. The compound of claim 1 , wherein R1is selected from a group consisting of -SCHs, Br, -Cl, -CF3,-CF2H and cyclopropyl.

3. The compound of either claim 1 or 2, wherein;A is a 6-membered unsaturated ring having 0, 1 or 2 nitrogen atoms, where the ring is substituted at the para position with an amine, optionally where the ring is independently substituted with 1 or 2 fluoro or chloro groups, andwherein A is not:

4. The compound of either claim 1 or 2, wherein A is:

5. The compound of any one of claims 1-3, wherein A is:, wherein;Y2is selected from a group consisting of methine and nitrogen; where Y2is methine, the hydrogen is optionally substituted with fluoro or chloro,Y3is selected from a group consisting of methine and nitrogen; where Y3is methine, the hydrogen is optionally substituted with fluoro or chloro.

6. The compound of claim 5, wherein when Y3is methine, the hydrogen is substituted with fluoro or chloro.

7. The compound of any one or more of claims 1-6, wherein A is selected from a group consisting of:

8. The compound of claim 7, wherein A is:

9. The compound of any one or more of claims 1-8, wherein B isand whereinQ is selected from a group consisting of carbon, sulfur and nitrogen,Z1is selected from a group consisting of carbon and nitrogen,Z2is selected from a group consisting of carbon and nitrogen, andR3selected from a group consisting of -CH3, a 3, 5 or 6-membered saturated ring and a 6-membered unsaturated ring.

10. The compound of any one or more of claims 1 -9, wherein B is selected from a group consisting of:12111 . The compound of claim 1 , wherein the compound is selected from a group consisting of:1221312. A pharmaceutical composition comprising a compound of any one or more of claims 1-11 or a pharmaceutical salt thereof, and a pharmaceutically acceptable carrier.

13. The pharmaceutical composition of claim 12, further comprising one or more other therapeutic agents.

14. A compound of any one or more of claims 1-11 or a pharmaceutical salt thereof, for use in the treatment of a cyclophilin-mediated condition or for modulation of cyclophilin or cyclophilin-like protein induced signalling in cells wherein such signalling modulation is beneficial for the treatment of a condition.

15. The compound for use of claim 14, wherein the condition is selected from a group consisting of hyperproliferative diseases, inflammation, cardiovascular diseases, viral infections, fibrosis of the kidney, liver, lung or pancreas; metabolic dysfunction-associated steatohepatitis, neurodegeneration, dementia, Alzheimer's disease, Parkinson's disease Amyotrophic Lateral Sclerosis (ALS), Dementia, Multiple Sclerosis, or Huntington's disease, rheumatoid arthritis, sepsis, asthma, periodontitis and aging.

16. The compound for use of claim 14, wherein the condition is cancer, optionally triple-negative breast cancer.

17. A compound of any one or more of claims 1-11 or a pharmaceutical salt thereof, for use as an immunoadjuvant in the treatment or prevention of a disease.12318. A compound of any one or more of claims 1-11 or a pharmaceutical salt thereof, for use in the manufacture of a medicament for the treatment of a condition selected from a group consisting of hyperproliferative diseases, inflammation, cardiovascular diseases, viral infections, fibrosis of the kidney, liver, lung or pancreas; metabolic dysfunction-associated steatohepatitis, neurodegeneration, dementia, Alzheimer's disease, Parkinson's disease Amyotrophic Lateral Sclerosis (ALS), Dementia, Multiple Sclerosis, or Huntington's disease, rheumatoid arthritis, sepsis, asthma, periodontitis and aging.

19. A method for the treatment of a cyclophilin-mediated condition or for modulation of cyclophilin or cyclophil in-like protein induced signalling in cells wherein such signalling modulation is beneficial for the treatment of a condition, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one or more of claims 1-11 or a pharmaceutically acceptable salt thereof.

20. A method of treating a condition in a mammal that can be ameliorated by the selective inhibition of cyclophilin, wherein the condition is selected from: hyperproliferative diseases, inflammation, cardiovascular diseases, viral infections, fibrosis of the kidney, liver, lung or pancreas; metabolic dysfunction-associated steatohepatitis, neurodegeneration, dementia, Alzheimer's disease, Parkinson's disease Amyotrophic Lateral Sclerosis (ALS), Dementia, Multiple Sclerosis, or Huntington's disease, rheumatoid arthritis, sepsis, asthma, periodontitis and aging, the method comprising administering to the mammal in need of such treatment, a therapeutically effective amount of a compound according to any one or more of claims 1-11 or a pharmaceutically acceptable salt thereof.124

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