Cyclosporin analogs

Abstract

The present application relates to cyclosporin analogs and their use in medical applications.

Description

Technical Field

[0001] The present invention relates to novel cyclosporin analogs and their use in medical applications, in particular in gene therapy and as antiviral compounds. Background Art

[0002] Hematopoietic stem cell (HSC) gene therapy can now treat inherited hematopoietic diseases, including immune deficiencies and metabolic disorders, which are often fatal and have limited long-term drug-based treatment options. Gene therapy requires HSC isolation and ex vivo delivery of a functional copy of the disease gene. The modified HSC is returned to the patient to replenish the hematopoietic system for long-term treatment.

[0003] HSC gene delivery requires HIV-based vectors. The main obstacle is the resistance of HSC to vector infection. The key protective antiviral protein that blocks vector entry and gene delivery in HSC is a known antiviral protein, called IFITM3 (see, e.g., Petrillo et al. Cell Stem Cell 23, 820–832, 2018). As described, for example, in Petrillo et al. (supra) and WO 2015 / 162594, it has been previously demonstrated that naturally occurring cyclosporins CsA and CsH act as transduction enhancers (TEs) by inhibiting IFITM3, thereby enhancing vector infection and gene delivery in these cells.

[0004] Unfortunately, CsH has limited availability, high cost, and poor purity. In contrast, CsA has undesirable features that reduce its efficacy, particularly its inhibition of the well-characterized HIV cofactor cyclophilin A (CypA).

[0005] It would be desirable to provide alternative compounds that can enhance transduction without these limitations. In particular, it would be desirable to provide IFITM3 inhibitors that are easily synthesized, highly potent, and selective to enhance HIV-vector infection, reduce the required vector dose, and overcome patient variability.

[0006] Certain cyclosporin analogs have been disclosed in WO 2021 / 229237. However, further development is needed to tailor the properties of the compounds associated with obtaining high IFITM3 inhibition, and in some cases also reduced or abolished CypA inhibition, and thus obtain compounds that are particularly suitable for use as transduction enhancers in clinical applications.

[0007] At the same time, effective treatment of viral infections remains a major challenge for health care systems around the world. For example, there is currently no known effective treatment for a series of recently emerged coronaviruses, including COVID-19 ("COVID-19"). It has been previously proposed that cyclosporine compounds are potentially useful in treating these conditions (see, for example, de Wilde et al., J Gen Virol. 2011; 92 (Pt 11): 2542–2548). At the same time, it has recently been proposed that IFITM proteins can promote SARS-Cov-2 infection, such as by hijacking these normally antiviral proteins for effective viral infection, so IFITM proteins can be a viable target for viral inhibition. There is still an urgent need for effective antiviral therapies, and the current particularly urgent situation is the need for the treatment of COVID-19. Summary of the invention

[0008] A specific class of cyclosporine analogs (more specifically, analogs of CsA) have been found to be effective as transduction enhancers in HSC gene delivery. Cyclosporine analogs have also been found to be effective as antiviral compounds against viruses in which IFITM proteins promote infection, including but not limited to COVID-19.

[0009] Specifically, the present invention provides a cyclosporin analogue, which is a compound of formula (I):

[0010]

[0011] or a pharmaceutically acceptable salt thereof;

[0012] in:

[0013] R1 represents hydrogen, C1–C4 alkyl or C2–C4 alkenyl;

[0014] R2 stands for

[0015] R3 represents ethyl or isopropyl;

[0016] R4 represents a methyl group or an ethyl group;

[0017] R5 represents –CH2CH(CH3)2, –CH2CH(CH3)CH2CH3, –CH(CH3)CH3, or –CH(CH3)CH2CH3;

[0018] R7 represents a hydrogen atom or a C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 an alkynyl moiety which is unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups, and wherein

[0019] (a) 0, 1, 2 or 3 carbon atoms are selected from C 6-10 Arylene, 5-membered to 10-membered heteroarylene, C 3-7 carbocyclylene and 5- to 10-membered heterocyclylene, and

[0020] (b) at most half of the –CH2– groups are selected from –O–, –S–, –C(O)– and –N(C 1-6 alkyl)-group, wherein:

[0021] (i) the arylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted with one or more halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(R N )2(where each R N independently represent hydrogen atoms or C 1-6 alkyl), –CN, –S(O)2NH2, nitro and sulfonic acid substituents; and

[0022] (ii) 0, 1 or 2 carbon atoms in the carbocyclylene and heterocyclylene are replaced by -C(O)- groups; and

[0023] Ring A represents a monocyclic or bicyclic ring system, which is C 6-10 -arylene or 5- to 10-membered heteroarylene, and is unsubstituted or substituted by one or more halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(R N )2(where each R N independently represent hydrogen atoms or C 1-6 alkyl), –CN, –S(O)2NH2, nitro, and sulfonic acid substituents;

[0024] And among them – R C –R6–N(R 8A )(R 8B ) is defined in part as (X) or (Y):

[0025] (X):

[0026] R C represents a group consisting of –C(O)O–, –OC(O)–, –C(O)N(R N )–、–N(R N )C(O)–,–S(O)2N(R N )–、–N(R N )S(O)2–、–NR N )–C(O)–N(R N )–、–N(R N )–C(S)–N(R N )–, –C(O)CH2–, –CH2C(O)–, –C(CF3)N(R N )–、–N(R N )C(CF3)–, –C(O)NF–, –NFC(O)–, –C(CN)=N–O–, –O–N=C(CN)–, –N(R N )C(O)O–、–OC(O)N(R N )–, 5-membered to 6-membered heteroarylene, C 5-6 A moiety consisting of a carbocyclylene group and a 5- to 6-membered heterocyclylene group, wherein each R N independently represent hydrogen atoms or C 1-6 alkyl, and wherein the phenylene, the heteroarylene, the carbocyclylene and the heterocyclylene are each unsubstituted or substituted by one or more selected from C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(R N )2(where each R N independently represent hydrogen atoms or C 1-6 alkyl), –CN, –S(O)2NH2, nitro, and sulfonic acid substituents;

[0027] R6 represents C 1-6 Alkylene, C 2-6 Alkenylene or C2-6 an alkynylene group, and is unsubstituted or substituted with one or more substituents selected from a halogen atom and a sulfonic acid group; and

[0028] and Or:

[0029] (a) together with the nitrogen atom to which it is attached, form a 5- to 10-membered heteroaryl or a 5- to 10-membered heterocyclic group, wherein the heteroaryl and the heterocyclic group are unsubstituted or substituted by one or more halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(R N )2(where each R N independently represent hydrogen atoms or C 1-6 alkyl), –CN, –S(O)2NH2, nitro and sulfonic acid substituents; or

[0030] (b) Independently represents C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 an alkynyl group, which is unsubstituted or substituted with one or more substituents selected from a halogen atom, a sulfonic acid group and a hydroxyl group;

[0031] (Y):

[0032] Together they form a group of formula (VI)

[0033]

[0034] in

[0035] Represents –C(O)–, –S(O)2–, –N(R N )–C(O)–,–N(R N )–C(S)–, –C(CF3)– or –OC(O)–,

[0036] Ring B contains the The nitrogen atoms and connections A 5- to 10-membered heterocyclylene ring having a nitrogen atom of and R 62 are each an alkylene group, and

[0037] C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 The alkynyl group is unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen atom, a sulfonic acid group and a hydroxyl group.

[0038] The present invention also provides the use of the cyclosporin analogs of the present invention in improving the transduction efficiency of isolated mammalian cell populations, preferably wherein the mammalian cells are human cells. More preferably, the population is selected from human hematopoietic stem cells and / or progenitor cells, induced human hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from human hematopoietic stem cells and / or progenitor cells or induced human hematopoietic stem cells and / or progenitor cells, for example, selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, through vectors derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus.

[0039] The present invention also provides a method for transducing a mammalian cell population, preferably wherein the mammalian cell is a human cell. More preferably, the cell population is selected from human hematopoietic stem cells and / or progenitor cells, induced human hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from human hematopoietic stem cells and / or progenitor cells or induced human hematopoietic stem cells and / or progenitor cells, for example, selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells One or more cells, comprising the following steps: a) contacting the population with a cyclosporine analog of the present invention; and b) transducing the population cells with a vector derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus.

[0040] The present invention also provides a gene therapy method, comprising the following steps: a) transducing a mammalian cell population, preferably wherein the mammalian cells are human cells. More preferably, according to the transduction method of the present invention, the cell population is selected from human hematopoietic stem cells and / or progenitor cells, induced human hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from human hematopoietic stem cells and / or progenitor cells or induced human hematopoietic stem cells and / or progenitor cells, for example, selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells One or more cells; and b) administering the transduced cells to a subject.

[0041] The present invention also provides a mammalian cell population, preferably wherein the mammalian cells are human cells. More preferably, the population is selected from human hematopoietic stem cells and / or progenitor cells prepared by the transduction method of the present invention, induced human hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from human hematopoietic stem cells and / or progenitor cells or induced human hematopoietic stem cells and / or progenitor cells, for example, selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, one or more cells, with And a pharmaceutical composition comprising such mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from the hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells, for example, a population of cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells.

[0042] The present invention also provides a population of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells for use in treatment, such as cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells.

[0043] The present invention also provides the cyclosporin analogs of the present invention for use in mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, and gene therapy.

[0044] The present invention also provides a cyclosporin analog according to the present invention for treating a pathological condition associated with IFITM3 expression (e.g., a viral infection). In addition, the present invention provides a method for treating a pathological condition associated with IFITM3 expression (e.g., a viral infection) in a patient in need thereof, comprising administering to the patient an effective amount of a cyclosporin analog of the present invention.

[0045] The present invention also provides a compound of the formula

[0046]

[0047] The present invention also provides a compound of the formula

[0048] BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Shown are the % infected cells for a range of test compounds when tested in THP-1 cells in the experiments detailed in Example 4.

[0050] Figure 2 Shown are the % infected cells for a range of test compounds when tested in U87 cells in the experiments detailed in Example 4.

[0051] Figure 3 Shown are the % infected cells of IFN treated (closed symbols) and untreated (open symbols) THP-1 cells when treated with BG147 (Panel A) or CsH (Panel B) in the experiments detailed in Example 4.

[0052] Figure 4 Shown are the % of infected human stem cells in the presence of various concentrations of various transduction enhancers in the experiments detailed in Example 5.

[0053] Figure 5 Shown is the % cell viability of human stem cells in the presence of various transduction enhancers at various concentrations in the experiments detailed in Example 5.

[0054] Figure 6Shown are the % of infected human stem cells in the presence of various concentrations of various transduction enhancers in further experiments detailed in Example 5.

[0055] Figure 7 Shown are the mean fluorescence intensities of human stem cells in the presence of different transduction enhancers at different concentrations in the experiments detailed in Example 6.

[0056] Figure 8 Live / dead staining data for various test compounds detailed in Example 6 at various concentrations are shown.

[0057] Fig. 9 Shown are results from: (a) THP-1 cells induced with IFITM3 expression treated with various compounds, protein samples harvested and IFITM3 levels measured by immunoblotting as described in Example 7 (Panel A); and (b) Calu-3 lung epithelial cells pretreated with JW3-158 or an equal volume of DMSO for 4 hours prior to infection with α, δ, or ο isolates of SARS-CoV-2, followed by quantification of the level of viral replication by RT-qPCR as described in Example 7 (Panel B - for each isolate, the left bar shows the DMSO study and the right bar shows the JW3-158 study).

[0058] Fig.10 Shown are the % infected cells of IFNβ treated THP-1 cells when treated with 1.25, 2.5 or 5 μM of various test compounds and GFP vector in the experiments detailed in Example 9 (left, middle and right bars correspond to 1.25, 2.5 and 5 μM, respectively, for each test compound). For comparison, THP-1 cells with and without IFNβ pretreatment were treated with DMSO and are represented by the bar at the far left of the graph.

[0059] Fig.11 Shown are the results of Western blot analysis of THP-1 cells activated with IFN[beta] and treated for 24 hours with 5 [mu]M test compound or DMSO control in the experiment detailed in Example 9.

[0060] Fig.12 Shown are the results of an MTT colorimetric viability assay of THP-1 cells activated by IFNβ in the presence of test compounds (1.25-5 μM: for each test compound, the left, middle and right bars correspond to 1.25, 2.5 and 5 μM, respectively) or DMSO control in the experiments detailed in Example 9. DETAILED DESCRIPTION

[0061] definition

[0062] As used herein, the term "alkyl" includes saturated straight-chain and branched-chain alkyl groups (i.e., monovalent groups derived from an alkane by removing a hydrogen atom). Preferably, unless otherwise specified, an alkyl group is C 1-20 Alkyl, more preferably C 1-15 , and more preferably C 1-12 Alkyl, more preferably C 1-6 Alkyl, and most preferably C 1-4 Alkyl. Unless otherwise indicated, particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl. The term "alkylene" should be interpreted accordingly (i.e., as the divalent counterpart of alkyl, in other words, an alkane from which two hydrogen atoms have been removed).

[0063] As used herein, the term "alkenyl" refers to a group containing one or more carbon-carbon double bonds and may be branched or unbranched (i.e., a monovalent group derived from an alkene by removing a hydrogen atom). Preferably, unless otherwise specified, an alkenyl group is C 2-20 Alkenyl, more preferably C 2-15 Alkenyl, more preferably C 2-12 Alkenyl, or preferably C 2-6 Alkenyl, and most preferably C 2-4 Alkenyl. The term "alkenylene" should be interpreted accordingly (ie, as the divalent counterpart of alkenyl, in other words, an alkene from which two hydrogen atoms are removed).

[0064] As used herein, the term "alkynyl" refers to a carbon chain containing one or more triple bonds, which may be branched or unbranched (i.e., a monovalent group derived from an alkyne by removing a hydrogen atom). Preferably, unless otherwise specified, an alkynyl group is C 2-20 Alkynyl, more preferably C 2-15 Alkynyl, more preferably C 2-12 Alkynyl, or preferably C 2-6 Alkynyl, and most preferably C 2-4 Alkynyl. The term "alkynylene" should be interpreted accordingly (ie, as the divalent counterpart of alkynyl, in other words, the alkyne from which two hydrogen atoms are removed).

[0065] Unless otherwise stated, alkyl, alkenyl or alkynyl are usually unsubstituted. However, when such a group (or its divalent counterpart) is indicated as being unsubstituted or substituted (and again unless otherwise stated), one or more hydrogen atoms are optionally replaced by halogen atoms or sulfonic acid groups. Preferably, the substituted alkyl, alkenyl or alkynyl have 1 to 10 substituents, more preferably 1 to 5 substituents, even more preferably 1,2 or 3 substituents, most preferably 1 or 2 substituents, such as 1 substituent. Preferably, the substituted alkyl, alkenyl or alkynyl have no more than 2 sulfonic acid substituents. Halogen atoms are preferred substituents. However, preferably alkyl, alkenyl or alkynyl are unsubstituted. Haloalkyl refers to an alkyl substituted by one or more halogen atoms.

[0066] wherein alkyl, alkenyl or alkynyl (or its divalent counterpart) is represented as being unsubstituted or substituted with one or more substituents selected from halogen atoms, sulfonic acid groups and hydroxyl groups (e.g., for and ) one or more hydrogen atoms are optionally replaced by halogen atoms, sulfonic acid groups and / or hydroxyl groups. Preferably, the substituted alkyl, alkenyl or alkynyl groups have 1 to 10 substituents, more preferably 1 to 5 substituents, even more preferably 1, 2 or 3 substituents, most preferably 1 or 2 substituents, such as 1 substituent. Preferably, the substituted alkyl, alkenyl or alkynyl groups have no more than 2 sulfonic acid substituents. Halogen atoms and hydroxyl groups are preferred substituents. Halogen atoms are preferred substituents. Hydroxyl groups are also preferred substituents. However, preferably, the alkyl, alkenyl or alkynyl groups are unsubstituted.

[0067] Haloalkyl refers to an alkyl group substituted with one or more halogen atoms.

[0068] The halogen atom used herein is typically a F, Cl, Br or I atom.

[0069] As used herein, C 6-10 Aryl is a monocyclic or polycyclic 6- to 10-membered aromatic hydrocarbon ring system having 6 to 10 carbon atoms. Phenyl and naphthyl are preferred unless otherwise stated. The term "arylene" should be interpreted accordingly (i.e. as the divalent counterpart of aryl), so phenylene and naphthylene are preferred arylene groups unless otherwise stated.

[0070] As used herein, a 5- to 10-membered heteroaryl group is a monocyclic or polycyclic 5- to 10-membered aromatic ring system, such as a 5- to 6-membered ring or a 9- to 10-membered ring system, containing at least one heteroatom, such as 1, 2, 3 or 4 heteroatoms selected from O, S and N. When the ring or ring system contains 4 heteroatoms, these heteroatoms are preferably all nitrogen atoms. The term "heteroarylene" should be interpreted accordingly.

[0071] Examples of monocyclic heteroaryl groups include thienyl, furanyl, pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, Azolyl, iso Azolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and tetrazolyl.

[0072] Examples of polycyclic heteroaryl groups include quinolyl, isoquinolyl, indolinyl, benzothiophenyl, benzofuranyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzo Azolyl, benzyl oxazolyl, benzotriazolyl, indolyl, isoindolyl and indazolyl.

[0073] As used herein, a 5- to 10-membered heterocyclic group is a non-aromatic, saturated or unsaturated, monocyclic or polycyclic C 5-10 A carbocyclic ring system in which one or more, for example 1, 2, 3 or 4, carbon atoms are replaced by a moiety selected from N, O, S(O) and S(O)2. Preferably, unless otherwise indicated, a 5- to 10-membered heterocyclyl is a 5- to 6-membered ring. The term "heterocyclylene" should be interpreted accordingly.

[0074] Examples of heterocyclic groups include azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, Oxazolidinyl, isocyanate oxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl, pyrazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, methylenedioxyphenyl, ethylenedioxyphenyl, thiomorpholinyl, S-oxy-thiomorpholinyl, S,S-dioxy-thiomorpholinyl, 1,3-dioxolanyl 1H-pyrazol-5(4H)-onyl, 1,3,4-thiadiazole-2(3H)-thione, oxypyrrolidinyl, oxythiazolidinyl, oxypyrazolidinyl, succinimidyl and maleimido groups and moieties.

[0075] For the avoidance of doubt, while the above definitions of heteroaryl and heterocyclyl refer to "N" moieties which may be present in the ring, it will be apparent to those skilled in the art of chemistry that if the N atom is attached to each adjacent ring atom by a single bond then it will be protonated (or will carry a substituent as defined below).

[0076] As used herein, C 3-7Carbocyclic radical is a non-aromatic saturated or unsaturated hydrocarbon ring with 3 to 7 carbon atoms. Preferably, unless otherwise indicated, it is a saturated or monounsaturated hydrocarbon ring (i.e. cycloalkyl moiety or cycloalkenyl moiety) with 3 to 7 carbon atoms, more preferably with 5 to 6 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and monounsaturated variants thereof. Particularly preferred carbocyclic radicals, unless otherwise indicated, are cyclopentyl and cyclohexyl. The term "carbocyclic radical" should be interpreted accordingly.

[0077] When specified, 0, 1 or 2 carbon atoms in a carbocyclyl or heterocyclyl group may be replaced by a -C(O)- group. As used herein, a "carbon atom" that is replaced is understood to include the hydrogen atom to which it is attached. When 1 or 2 carbon atoms are replaced, preferably two such carbon atoms are replaced. Preferred such carbocyclyl groups include benzoquinone groups, and preferred such heterocyclyl groups include succinimidyl and maleimido groups.

[0078] Unless otherwise specified, an aryl, heteroaryl, carbocyclyl or heterocyclyl group is typically unsubstituted. However, when such a group is specified as being unsubstituted or substituted, one or more hydrogen atoms may be optionally replaced by a halogen atom or a C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(R N )2(where each R N independently represent hydrogen atoms or C 1-6 Preferably, the substituted aryl, heteroaryl, carbocyclyl or heterocyclyl has 1 to 4 substituents, more preferably 1 to 2 substituents, most preferably 1 substituent. Preferably, the substituted aryl, heteroaryl, carbocyclyl or heterocyclyl has no more than 2 nitro substituents and no more than 2 sulfonic acid substituents.

[0079] As used herein, an alkoxy group is an alkyl group (e.g., C 1-6 Alkyl or C 1-4 alkyl).

[0080] As used herein, an alkylthio group is an alkyl group (e.g., C 1-6 Alkyl or C 1-4 alkyl).

[0081] In some cases, the compounds of the present invention can be provided in the form of pharmaceutically acceptable salts. Essentially any pharmaceutically acceptable salt can be used. Those skilled in the art of preparing compounds for medical applications are familiar with suitable salt compounds of this type. For example, the compounds of the present invention can be in the form of salts with pharmaceutically acceptable bases. Pharmaceutically acceptable bases include but are not limited to alkali metal (e.g., sodium or potassium) and alkaline earth metal (e.g., calcium or magnesium) hydroxides and organic bases such as alkylamines, arylalkylamines or heterocyclic amines.

[0082] Cyclosporine analogs

[0083] Non-limiting preferred embodiments of the cyclosporin analogs of the present invention are described below.

[0084] R1 preferably represents hydrogen.

[0085] R2 preferably represents

[0086] R3 preferably represents ethyl.

[0087] R4 preferably represents methyl.

[0088] R5 preferably represents -CH2CH(CH3)2.

[0089] In R7, hydrogen or C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 The alkynyl moiety is preferably hydrogen or C 1-15 Alkyl, C 2-15 Alkenyl or C 2-15 The alkynyl moiety is more preferably hydrogen or C 1-12 Alkyl, C 2-12 Alkenyl or C 2-12 The alkynyl moiety is more preferably hydrogen or C 1-10 Alkyl or C 2-10 The alkenyl moiety is most preferably hydrogen. In these preferred embodiments, the substituents and preferred choices of (a) and (b) are as follows.

[0090] As for the substituents, it is preferred that the moiety is unsubstituted or substituted with one or more substituents selected from halogen atoms, and it is more preferred that the moiety is unsubstituted. The maximum number of substituents is preferably 5, and more preferably 3.

[0091] With regard to (a), preferably 0, 1 or 2 carbon atoms are replaced, more preferably 0 or 1 carbon atom is replaced. These carbon atoms are preferably selected from phenylene, 5-membered to 6-membered heteroarylene, C 5-6 The carbocyclylene and 5- to 6-membered heterocyclylene groups are preferably substituted with phenylene groups.

[0092] With respect to (b), preferably 0 to 4 -CH2- groups are replaced, more preferably 0 to 3 groups, and most preferably 0, 1 or 2 groups are replaced. For any -CH2- replaced groups, preferably they are replaced by groups selected from -O-, -S- and -C(O)-, more preferably by groups selected from -O- and -C(O)-. For the avoidance of doubt, it is acceptable to replace adjacent -CH2- where it makes chemical sense, for example, to replace -CH2-CH2- with -C(O)-O-, -O-C(O)-, etc. Similarly, it is acceptable to replace a -CH2- terminal methyl group (i.e., -CH3 or -CH2-H) with -OH, for example to replace -CH3. Preferably no more than two adjacent -CH2- groups are replaced (e.g., generally any replacement does not involve the replacement of three or more adjacent / adjacent -CH2- groups).

[0093] Preferably, any arylene, heteroarylene, carbocyclylene and heterocyclylene in (a) is unsubstituted or substituted by one or more substituents selected from halogen atoms and sulfonic acid groups, more preferably unsubstituted. Preferably, 0 carbon atoms in any carbocyclylene and heterocyclylene are replaced by -C(O)- groups.

[0094] Preferably R7 represents hydrogen or C 1-15 Alkyl, C 2-15 Alkenyl or C 2-15 an alkynyl moiety which is unsubstituted or substituted with one or more halogen atoms, and wherein

[0095] (a) 0, 1 or 2 carbon atoms are selected from C 6-10 Arylene, 5-membered to 10-membered heteroarylene, C 3-7 carbocyclylene and 5- to 10-membered heterocyclylene, and

[0096] (b) 0 to 4 –CH2– groups are selected from –O–, –S–, –C(O)– and –N(C 1-6 alkyl)-group, wherein:

[0097] (i) the arylene group, heteroarylene group, carbocyclylene group and heterocyclylene group are unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups; and

[0098] (ii) 0, 1 or 2 carbon atoms in the carbocyclylene and heterocyclylene are replaced by -C(O)- groups.

[0099] More preferably, R7 represents hydrogen or C 1-12 Alkyl, C 2-12 Alkenyl or C 2-12 an alkynyl moiety which is unsubstituted or substituted with one or more halogen atoms, and wherein

[0100] (a) 0, 1 or 2 carbon atoms are selected from phenylene, 5-membered to 6-membered heteroarylene, C 5-6 -carbocyclic and 5- to 6-membered heterocyclic groups, and

[0101] (b) 0 to 3 of the -CH2- groups are replaced by groups selected from -O-, -S- and -C(O)-;

[0102] The phenylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted with one or more halogen atoms.

[0103] More preferably, R7 also represents hydrogen or C 1-10 Alkyl or C 2-10 wherein (a) 0 or 1 carbon atom is replaced by a phenylene group, and (b) 0, 1 or 2 -CH2- groups are replaced by a group selected from -O- and -C(O)- groups.

[0104] R7 most preferably represents hydrogen.

[0105] In a particularly preferred embodiment, R1 represents hydrogen, R2 represents R3 represents an ethyl group, R4 represents a methyl group, R5 represents -CH2CH(CH3)2, and R7 represents hydrogen. It should be understood that in this embodiment, the structure of the resulting cyclosporin analog is very similar to the structure of cyclosporin A ("CsA"), but Partially replaces the methyl group, –CH3, which is located at the same position in CsA.

[0106] The stereochemistry of the vinyl groups connecting the cyclic cyclosporin A nucleus to the group R1 and ring A can be E or Z. Thus, formula (I) encompasses both formulae (Ia) and (Ib):

[0107]

[0108] Moieties of formula (IIa) are currently preferred, and most particularly wherein R1 represents hydrogen, R2 represents Those in which R3 represents ethyl, R4 represents methyl, R5 represents -CH2CH(CH3)2, and R7 represents hydrogen.

[0109] For the avoidance of doubt, regardless of the stereochemistry of the compound (e.g., (IIa) or (IIb) above), wherein the divalent moiety ring A, R C and R6, using the general notation of “–[definition]–”, with the leftmost portion of the definition being the portion closest to the central cyclosporine ring and the rightmost portion of the definition being the portion closest to the terminal For example, for R CDefined as “–a–b–c–”, the group “a” is attached to ring A and the group “c” is attached to R6.

[0110] In the cyclosporin analogs of the present invention, Ring A replaces the methyl group present in cyclosporin A ("CsA") and in its place is attached a cyclic ring extending from the cyclic core of CsA to The structure of the side chain of the vinyl group.

[0111] For the avoidance of doubt, although schematic rings are used in formulas (I), (Ia) and (Ib), and for simplicity, the singular language "ring" is used to identify herein, but ring A represents a monocyclic or bicyclic ring system. Bicyclic ring systems can generally be any of fused, bridged and spirocyclic, but most typically when ring A is a bicyclic ring system, it is a fused bicyclic ring (i.e., it includes a first ring and a second ring, sharing two adjacent ring atoms).

[0112] In the first aspect, it is generally more preferred that Ring A is an unsubstituted or substituted (fused) bicyclic ring system having the general formula (IIa), (IIb) or (IIc)

[0113]

[0114] Wherein B is a monocyclic 5- to 6-membered ring (comprising two carbon atoms of a benzene ring fused thereto). The ring atoms of B may be exclusively carbon atoms, or may contain one or more heteroatoms selected from N, S and O (preferably up to two heteroatoms, preferably wherein the heteroatoms are N atoms).

[0115] Exemplary systems of formula (IIa), (IIb), and (IIc) include (a) naphthylene and (b) 9- to 10-membered heteroarylene, such as, but not limited to, quinolylene, isoquinolylene, or indolinylene. Particularly preferred are naphthylene, such as those of formula (IIIa), (IIIb), or (IIIc):

[0116]

[0117] Particularly preferred is the formula (IIIa).

[0118] Specific examples of quinolinylene and indolinylene include formula (IVa), (IV) and (IVc)

[0119]

[0120] For the avoidance of doubt, in all Formulae (IIa), (IIb), (IIc), (IIIa), (IIIb), (IIIc), (IVa), (IVb) and (IVc), the linkage shown on the right is to R CAlso for the avoidance of doubt, in all Formulae (IIa), (IIb), (IIc), (IIIa), (IIIb), (IIIc), (IVa), (IVb) and (IVc), the ring system is unsubstituted or substituted (i.e., by one or more selected from halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(R N )2(where each R N independently represent hydrogen atoms or C 1-6 alkyl), -CN, -S(O)2NH2, nitro and sulfonic acid groups). In these ring systems, preferred substituents are selected from C 1-4 Alkyl, C 1-4 Haloalkyl and C 1-4 Alkoxy, more preferably C 1-4 Preferably, these ring systems are unsubstituted or substituted by one or two substituents, for example they are unsubstituted.

[0121] In a second aspect, ring A is an unsubstituted or substituted monocyclic ring, wherein the monocyclic ring is usually a phenylene ring or a 5-membered to 6-membered heteroarylene ring. In this aspect, the monocyclic ring is preferably a phenylene ring, and particularly preferably a ring of the general formula (V):

[0122]

[0123] For the avoidance of doubt, such a phenylene ring or a 5- to 6-membered heteroarylene group may be unsubstituted or substituted (i.e., by one or more radicals selected from halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(R N )2(where each R N independently represent hydrogen atoms or C 1-6alkyl), –CN, –S(O)2NH2, nitro and sulfonic acid substituents).

[0124] In this second aspect, Ring A is most preferably a phenylene group of formula (Va)

[0125]

[0126] in and independently selected from hydrogen and halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(R N )2(where each R N independently represent hydrogen atoms or C 1-6 alkyl), –CN, –S(O)2NH2, nitro, and sulfonic acid groups.

[0127] Preferably, in formula (Va), and At least two of them are hydrogen, more preferably at least and is hydrogen. and When (at least) one of R1 and R2 is not hydrogen, then (at least) RV1 is not hydrogen. and When two of them are not hydrogen, then RV1 and RV2 are not hydrogen. Particularly preferred non-hydrogen RV1, RV2, RV3 and RV4 groups are C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –CN and S(O)2NH2, more preferably C 1-4 Alkyl, C 1-4 Haloalkyl and C 1-4 Alkoxy (e.g., methyl, ethyl, propyl, methoxy, ethoxy, propoxy (including -OiPr), and trifluoromethyl), most preferably C 1-4 Alkyl (eg, methyl, ethyl, and propyl).

[0128] Typically (ie, referring to the first and second aspects discussed herein), in ring A, preferably the C 6-10The arylene group or the 5- to 10-membered heteroarylene group is a naphthylene group, a 9- to 10-membered heteroarylene group or a phenylene group. In addition, it is particularly preferred that in ring A, the C 6-10 -arylene or 5- to 10-membered heteroarylene is naphthylene.

[0129] Also generally, when ring A is substituted, preferred substituents for ring A are selected from C 1-4 Alkyl, C 1-4 Haloalkyl and C 1-4 Alkoxy, most preferably C 1-4 Preferably, Ring A is unsubstituted or substituted with one or two substituents.

[0130] in Compounds defined by (X):

[0131] R C It is a linking group that connects ring A to group R6.

[0132] In R C In, R N Any occurrence of is preferably a hydrogen atom or C 1-4 An alkyl group is more preferably a hydrogen atom or a methyl group, and most preferably a hydrogen atom.

[0133] R C Preferably, it represents a group selected from -C(O)O-, -C(O)N(R N )–,–S(O)2N(R N )–、–N(R N )–C(O)–N(R N )–、–N(R N )–C(S)–N(R N )–, –C(O)CH2–, –C(CF3)N(R N )–, –C(O)NF–, –C(CN)=N–O–, –N(R N )C(O)O- and a 5- to 6-membered heteroarylene group.

[0134] When R C When it is a 5- to 6-membered heteroarylene group, preferred such groups are triazolylene (i.e., a divalent group obtainable by removing two hydrogen atoms from triazole), Azolyl (i.e., A divalent group obtained by removing two hydrogen atoms from azole), isocyanate Azolyl (i.e., A divalent group obtained by removing two hydrogen atoms from azole) and an oxadiazole The oxadiazole group (i.e., A divalent group obtained by removing two hydrogen atoms from oxadiazole). Preferred examples of the triazolylene group are 1,2,3-triazolylene, for example Wherein preferably the left side connection is to ring A and the right side connection is to R6, and 1,2,4-triazolylene, for example Wherein preferably the left side connection is to ring A and the right side connection is to R6. Preferred examples of oxazolyl are Wherein preferably the left side connection is to ring A and the right side connection is to R6. Preferred examples of oxadiazole are 1,2,4-oxadiazole. Oxazolyl, such as It is preferred that the connection shown on the left is to ring A and the connection on the right is to R6.

[0135] R C More preferably, it represents a group consisting of -C(O)O-, -C(O)N(R N )– (especially –C(O)NH–), –S(O)2N(R N )– (especially –S(O)2NH–), –N(R N )–C(O)–N(R N )– (especially –NH–C(O)–NH–), –N(R N )–C(S)–N(R N )– (especially –NH–C(S)–NH–), –N(R N )C(O)O— (especially —NH—C(O)—O—) and a triazolylene group (especially 1,2,3-triazolylene, such as the above-mentioned triazolyl group).

[0136] Therefore, the more preferred R C The groups are –C(O)O–, –C(O)NH–, –S(O)2NH–, –NH–C(O)–NH–, –NH–C(S)–NH–, –NH–C(O)–O–, and

[0137] The best choice is R C It is –C(O)O–.

[0138] R6 is a connecting group, which connects R C Connect to part.

[0139] R6 is preferably unsubstituted or substituted by one or more (e.g. up to four) halogen atoms. Most preferably R6 is unsubstituted.

[0140] Preferably R6 represents C 2-4 Alkylene or C 2-4 Alkenylene, and more preferably C 2-4Alkylene, and more preferably -(CH2)2- or -(CH2)3- or -(CH2)4-.

[0141] Most preferably, R6 is ethylene, i.e. -(CH2)2-. Another particularly preferred R6 is -(CH2)4-.

[0142] Group and It is connected to the nitrogen atom and together with the nitrogen atom forms a heterocyclic group or a tertiary amine group.

[0143] In a first aspect of the present invention, and Together with the nitrogen atom to which it is attached, it forms a 5- to 10-membered heteroaryl or a 5- to 10-membered heterocyclic group, wherein the heteroaryl and the heterocyclic group are unsubstituted or substituted by one or more halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(R N )2(where each R N independently represent hydrogen atoms or C 1-6 The heteroaryl and heterocyclic groups are preferably unsubstituted. However, in certain embodiments, it may be desirable for the heteroaryl and heterocyclic groups to include one or more (e.g., one) amino substituents (e.g., -NH2), for example, to obtain a physiological pKa (e.g., between 7.5 and 10).

[0144] In a second aspect of the present invention, and Independently represents C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, and is unsubstituted or substituted with one or more substituents selected from halogen atoms, sulfonic acid groups and hydroxyl groups. For example, in this second aspect of the invention, and Independently represents C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, and is unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups. 1-6 Alkyl, C2-6 Alkenyl and C 2-6 Alkynyl groups are unsubstituted.

[0145] Preferably, in the first aspect, and Together with the nitrogen atom to which it is attached, it forms a 5- to 6-membered heteroaryl group or a 5- to 6-membered heterocyclic group.

[0146] More preferably in the first aspect, and Together with the nitrogen atom to which it is attached, it forms an imidazolyl, pyrazolyl, morpholinyl, piperidinyl or piperazinyl group.

[0147] Most preferably, in the first aspect, and Together with the nitrogen atom to which it is attached, it forms an imidazole group (i.e., of groups).

[0148] Preferably in the second aspect, and Independently represents C 1-4 Alkyl or C 2-4 More preferably in the second aspect, and Independently represents C 1-4 Most preferably in the second aspect, and independently represents methyl or ethyl.

[0149] In the present invention, therefore, it is preferred and or (a) together with the nitrogen atom to which it is attached form a 5- to 6-membered heteroaryl or 5- to 6-membered heterocyclic group; or (b) independently represents C 1-4 Alkyl or C 2-4 More preferably and or (a) together with the nitrogen atom to which it is attached form an imidazolyl, pyrazolyl, morpholinyl, piperidinyl or piperazinyl group; or (b) independently represent C 1-4 Most preferably, and Either: (a) together with the nitrogen atom to which it is attached form an imidazolyl group; or (b) independently represent a methyl or ethyl group - and most preferably and Together with the nitrogen atom to which it is attached, it forms an imidazolyl group.

[0150] Examples of cyclosporin analogs disclosed herein include those of formula (Ia), wherein R1 represents hydrogen, R2 represents R3 represents ethyl, R4 represents methyl, R5 represents –CH2CH(CH3)2, R6 represents ethylene (i.e. –(CH2)2–), R7 represents hydrogen, R C represents –C(O)O–, and and Together with the nitrogen atom to which it is attached, it forms an imidazolyl group. Further examples of cyclosporin analogs disclosed herein are those in which R6 is -(CH2)3- or -(CH2)4-, particularly -(CH2)4-, but not -(CH2)3-. In each of these exemplary cyclosporin analogs disclosed herein, preferred ring A groups include those in which C 6-10 The arylene group or the 5- to 10-membered heteroarylene group is a naphthylene group, a 9- to 10-membered heteroarylene group or a phenylene group. In addition, among these exemplary cyclosporin analogs disclosed herein, it is particularly preferred that, in ring A, the C 6-10 The arylene group or the 5- to 10-membered heteroarylene group is a naphthylene group. Furthermore, in these exemplary cyclosporin analogs disclosed herein, when ring A is substituted, the preferred substituents of ring A are selected from C 1-4 Alkyl, C 1-4 Haloalkyl and C 1-4 Alkoxy, most preferably C 1-4 Alkyl. Preferably, ring A is unsubstituted or substituted with one or two substituents, such as unsubstituted naphthylene (e.g., of formula (IIIa)). Therefore, a specific exemplary compound is "BG147", whose structural formula is shown in the Examples section. Another specific exemplary compound is "JW3-158", whose structural formula is shown in the Examples section. Still further exemplary compounds, whose structural formulas are also shown in the Examples section, are "BG150", "BG181", "BG185", "BG186", "BG190" and "BG149". Still further exemplary compounds, whose structural formulas are also shown in the Examples section, are "VP72", "VP73", "VP74", "VP50", "VP53" and "VP76".

[0151] in Compounds defined by (Y):

[0152] The present disclosure also includes According to the compound defined in (Y), this refers to a compound wherein Together they form a group of formula (VI)

[0153]

[0154] In formula (VI), Represents –C(O)–, –S(O)2–, –N(R N)–C(O)–,–N(R N )—C(S)—, —C(CF 3 )— or —OC(O)—, and preferably represents —C(O)—.

[0155] In formula (VI), ring B is a ring containing The nitrogen atom and the a 5-10 membered heterocyclylene ring having a nitrogen atom of and R 62 Preferably, ring B is a ring containing a The nitrogen atom and the a 5-7 membered heterocyclylene ring having a nitrogen atom of and R 62 Particularly preferred ring B is one containing a bond to The nitrogen atom and the A 6-membered heterocyclylene ring having a nitrogen atom of and R 62 Most preferably, ring B is a ring containing The nitrogen atom and the The nitrogen atom of and R 62 Each is a C2 alkylene group (ie, Ring B is a 1,4-piperazinylene group).

[0156] In formula (VI), C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 Alkynyl, and is unsubstituted or substituted with one or more substituents selected from halogen atoms, sulfonic acid groups, and hydroxyl groups. C 1-4 Alkyl or C 2-4 Alkenyl (and it is unsubstituted or substituted). More preferably C 1-4 Alkyl, which is unsubstituted or substituted. Most preferably is methyl or ethyl, which is unsubstituted or substituted. is unsubstituted or substituted with one or more substituents selected from halogen atoms and hydroxyl groups. is unsubstituted or substituted with hydroxyl groups. Particularly preferred It is hydroxyethyl (–CH2CH2OH).

[0157] A specific exemplary compound of this class is "VP51," the general formula of which is indicated in the Examples section.

[0158] Further disclosures concerning cyclosporine analogs

[0159] As disclosed herein, in the cyclosporine analogs of the present invention, Part is defined according to (X) or (Y). Preferably, however, Part is defined according to (X). In addition, in this preferred aspect, Partially preferably defined according to (X) and and Or:

[0160] (a) together with the nitrogen atom to which it is attached, form a 5- to 10-membered heteroaryl or a 5- to 10-membered heterocyclic group, wherein the heteroaryl and the heterocyclic group are unsubstituted or substituted by one or more halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(R N )2(where each R N independently represent hydrogen atoms or C 1-6 alkyl), –CN, –S(O)2NH2, nitro and sulfonic acid substituents; or

[0161] (b) Independently represents C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 The alkynyl group may be unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen atom and a sulfonic acid group.

[0162] The cyclosporin analogs of the present invention generally have low, minimal, or eliminated ability to bind, inhibit and / or degrade CypA. For example, the cyclosporin analogs of the present invention preferably have a lower ability to bind, inhibit and / or degrade CypA than CsA. In a preferred embodiment, the cyclosporin analogs of the present invention have a lower binding affinity to cyclophilin A (CypA) than cyclosporin A (CsA), preferably less than half of the binding affinity of CsA to CypA, more preferably less than one-fifth of the binding affinity of CsA to CypA, and most preferably less than one-tenth of the binding affinity of CsA to CypA (e.g., when measured by standard binding assays such as surface plasmon resonance SPR). Methods for determining the relative ability of a compound (e.g., a cyclosporin analog of the present invention) to bind, inhibit and / or degrade a protein (e.g., CypA) relative to another compound (e.g., CsA) are well known in the art and can be routinely used by those skilled in the art. A method for determining the ability to bind CypA (e.g., as a K d (Dynamics), K d (affinity), maximum response, K off , and / or K on ) is a non-limiting method.

[0163] The cyclosporin analogs of the present invention are generally IFITM3 inhibitors. For example, in a preferred embodiment, the cyclosporin analogs of the present invention may be more effective IFITM3 inhibitors than CsA. The determination of IFITM3 inhibition, including reference to CsA as a comparator, can be performed using any method known in the art for measuring the ability of a compound to trigger a protein (e.g., in vitro). A suitable non-limiting method is set in Example 2.

[0164] synthesis

[0165] The compounds of the present invention can be prepared by standard methods known in the art. WO 2021 / 229237 (including but not limited to the disclosed working examples) provides representative examples of the synthesis of cyclosporin analogs, which can be easily adapted to provide the compounds of the present invention; the contents of WO 2021 / 229237 are hereby incorporated by reference in their entirety. Other illustrative examples of compound synthesis are provided in the working examples of the present application.

[0166] Particularly useful intermediate compounds, which therefore form further aspects of the invention, are as follows

[0167] (a) Compound "VP75", the synthesis of which is described in Example 8, and which has the following structure:

[0168] and

[0169] (b) Compound "VP_130", the synthesis of which is described in Example 10, and which has the following structure:

[0170]

[0171] These compounds, in particular, are particularly useful in preparing compounds of the present invention wherein:

[0172] R1 represents hydrogen;

[0173] R2 stands for

[0174] R3 represents an ethyl group;

[0175] R4 represents a methyl group;

[0176] R5 represents –CH2CH(CH3)2; and

[0177] R7 represents hydrogen;

[0178] Compound VP75 is particularly useful for preparing compounds further characterized by:

[0179] Ring A represents and

[0180] R C Represents -C(O)O- or -C(O)N-. Compound VP_130 can be similarly used to prepare such compounds as well as other compounds of the present disclosure. Non-limiting illustrative examples of the synthesis and subsequent reactions of these compounds are provided in Examples 8 and 10.

[0181] Gene therapy applications

[0182] The compounds of the present invention can be used for gene therapy, for example, by gene therapy vectors, to improve mammalian cells, human hematopoietic stem cells (HSC) and / or progenitor cells, induced human hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from human hematopoietic stem cells and / or progenitor cells or induced human hematopoietic stem cells and / or progenitor cells, for example, cells selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells One or more cells, transduction efficiency. The present invention includes these uses of the compounds, related pharmaceutical compositions, and methods of treatment. Advantageously, it has been found that the compounds of the present invention are easily synthesized, highly effective IFITM3 inhibitors that can be used to enhance HIV vector infection, reduce the required vector dose, and overcome patient variability. As discussed further elsewhere herein, the compounds may also have reduced binding to CypA compared to CsA and thus overcome limitations associated with the use of previously described CsA and other related cyclosporine analogs in similar applications.

[0183] cell

[0184] Stem cells are able to differentiate into many cell types. Cells that are able to differentiate into all cell types are called totipotent. In mammals, only zygotic and early embryonic cells are totipotent. Stem cells are present in most, if not all, multicellular organisms. They are characterized by the ability to renew themselves by mitotic cell division and to differentiate into various specialized cell types. The two main types of mammalian stem cells are embryonic stem cells isolated from the inner cell mass of the blastocyst, and adult stem cells found in adult tissues. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem and progenitor cells act as the body's repair system, replenishing specialized cells and maintaining the normal renewal of regenerating organs such as blood, skin or intestinal tissue.

[0185] Hematopoietic stem cells (HSCs) are pluripotent stem cells that can be found in, for example, peripheral blood, bone marrow, and umbilical cord blood. HSCs are capable of self-renewal and differentiation into any blood cell lineage. They are able to recolonize the entire immune system, and erythrocyte and bone marrow lineages in all hematopoietic tissues (e.g., bone marrow, spleen, and thymus). They provide lifelong production of all lineages of hematopoietic cells.

[0186] Hematopoietic progenitor cells have the ability to differentiate into specific types of cells. However, in contrast to stem cells, they are already more specific: they are driven to differentiate into their "target" cells. The difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, while progenitor cells can only divide a limited number of times. Hematopoietic progenitor cells can only be strictly distinguished from HSCs by functional in vivo assays (i.e., transplantation and demonstrating whether they can produce all blood lineages over an extended period of time).

[0187] Preferably, the hematopoietic stem and progenitor cells of the present invention include the CD34 cell surface marker (denoted as CD34+).

[0188] Hematopoietic stem cells and / or progenitor cell colonies can be obtained from tissue samples. For example, hematopoietic stem cells and / or progenitor cell colonies can be obtained from peripheral blood (e.g., adult and fetal peripheral blood), umbilical cord blood, bone marrow, liver or spleen. Preferably, these cells are obtained from peripheral blood or bone marrow. They can be obtained after mobilizing cells in vivo by means of growth factor treatment.

[0189] Mobilization can be performed with, for example, G-CSF, prixavor, or a combination thereof. Other agents, such as NSAIDs and dipeptidyl peptidase inhibitors, may also be useful as mobilizing agents.

[0190] Due to the availability of stem cell growth factors such as GM-CSF and G-CSF, most hematopoietic stem cell transplantation procedures are now performed using stem cells collected from peripheral blood rather than from bone marrow. Collection of peripheral blood stem cells provides a larger graft, does not require general anesthesia of the donor to collect the graft, makes the implantation time shorter, and can provide a lower long-term relapse rate. Bone marrow can be collected by standard aspiration methods (steady state or after mobilization), or by using a next-generation harvesting tool (e.g., Marrow Miner). In addition, hematopoietic stem cells and / or progenitor cells can also be derived from induced pluripotent stem cells, and such cells are referred to herein as induced hematopoietic stem cells and / or progenitor cells. Induced pluripotent stem cells, such as induced human pluripotent stem cells, can be produced by directly reprogramming differentiated cells such as fibroblasts (see, e.g., Cell. 126 (4), 663-676, 2006; Cell, 131 (5), 861-872, 2007; Science, 318 (5858), 1917-1920, 2007; and Nat Biotechnol., 26 (1), 101-106, 2008). Induced pluripotent stem cells can be further differentiated to provide induced hematopoietic stem cells and / or progenitor cells. Induced hematopoietic stem cells and / or progenitor cells can be further differentiated into other cells of the hematopoietic lineage.

[0191] HSC usually has low forward scatter and side scatter characteristics by flow cytometry. Some are metabolically quiescent, as demonstrated by rhodamine labeling, which allows the determination of mitochondrial activity. HSC may include certain cell surface markers, such as CD34, CD45, CD133, CD90 and CD49f. They can also be defined as cells lacking expression of CD38 and CD45RA cell surface markers. However, the expression of some of these markers depends on the developmental stage and tissue-specific environment of HSC. Some HSCs, referred to as "side population cells", exclude the Hoechst 33342 dye detected by flow cytometry. Therefore, HSC has descriptive features that allow it to be identified and separated.

[0192] CD38 is the most definitive and useful single negative marker for human HSC. Human HSC may also be negative for lineage markers such as CD2, CD3, CD14, CD16, CD19, CD20, CD24, CD36, CD56, CD66b, CD271, and CD45RA. However, these markers may need to be combined for HSC enrichment. "Negative markers" should be understood as the lack of expression of these markers by human HSC.

[0193] CD34 and CD133 are the most useful positive markers for HSC. Some HSC are also positive for lineage markers such as CD90, CD49f and CD93. However, these markers may need to be combined for HSC enrichment. "Positive markers" should be understood as human HSC expressing these markers.

[0194] Differentiated cells are cells that become more specialized compared to stem cells or progenitor cells. Differentiation occurs during the development of multicellular organisms because organisms become complex systems of tissues and cell types from a single zygote. Differentiation is also a common process for adults: adult stem cells divide and produce fully differentiated daughter cells during tissue repair and normal cell renewal. Differentiation significantly changes the size, shape, membrane potential, metabolic activity and response to signals of cells. These changes are mainly due to the highly controlled modification of gene expression. In other words, differentiated cells are cells with specific structures and perform certain functions due to the developmental process involving the activation and inactivation of specific genes. Here, differentiated cells include differentiated cells of hematopoietic lineages, such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells, T cells, B cells and NK cells. For example, differentiated cells of hematopoietic lineages can be distinguished from stem cells and progenitor cells by detecting cell surface molecules that are not expressed or expressed to a low degree on undifferentiated cells. Examples of suitable human lineage markers include CD33, CD13, CD14, CD15 (myeloid), CD19, CD20, CD22, CD79a (B), CD36, CD71, CD235a (erythroid), CD2, CD3, CD4, CD8 (T), and CD56 (NK).

[0195] An "isolated cell population" is understood to mean a cell population that has been previously removed from the body. An isolated cell population can be cultured and manipulated ex vivo or in vitro using standard techniques known in the art. The isolated cell population can then be reintroduced into a subject. The subject can be the same subject as the subject from which the cells were initially isolated or a different subject. Methods and uses performed on an isolated cell population are ex vivo or in vitro methods and uses.

[0196] Cell populations can be selectively purified from cells that exhibit a particular phenotype or characteristic and other cells that do not exhibit the phenotype or characteristic or exhibit the phenotype or characteristic to a lesser extent. For example, a cell population that expresses a particular marker (such as CD34) can be purified from a starting cell population. Alternatively, or in addition, a cell population that does not express another marker (such as CD38) can be purified.

[0197] By "enriching" a cell population for a certain type of cell, it is understood that the concentration of that type of cell is increased in the population. The concentration of other types of cells may be concomitantly reduced.

[0198] Purification or enrichment may result in a cell population that is substantially pure of other cell types.Purification or enrichment of a cell population that expresses a particular marker (eg, CD34 or CD38) may be achieved by using an agent that binds to that marker, preferably substantially specifically.

[0199] The reagent that binds to the cell marker can be an antibody, such as an anti-CD34 or anti-CD38 antibody. The term "antibody" refers to a complete antibody or antibody fragment that can bind to a selected target, including Fv, ScFv, F(ab') and F(ab')2, monoclonal and polyclonal antibodies, engineered antibodies, including chimeric, CDR-transplanted and humanized antibodies, and artificially selected antibodies produced using phage display or alternative technology. In addition, alternatives to classical antibodies, such as "avipbody", "avimers", "anticalins", "nanoantibodies" and "DARPins" can also be used in the present invention.

[0200] The reagent that is combined with the specific marker can be labeled so that it can be identified by any of the many techniques known in the art. The reagent can be intrinsically labeled, or it can be modified by conjugating the marker thereto. "Conjugation" should be understood as the reagent and the marker being operably connected. This means that the reagent and the label are connected together in a manner that allows both to perform their functions (e.g., to be bound to the marker, to allow fluorescence recognition, or to allow separation when placed in a magnetic field) without hindrance. Suitable conjugation methods are well known in the art and will be easily recognized by the technician.

[0201] The marker can allow, for example, the labeled agent and any cells to which it binds to be purified from their environment (e.g., the agent can be labeled with magnetic beads or affinity tags such as avidin), detected, or both. Detectable labels suitable for use as labels include fluorophores (e.g., green, cherry, cyan, and orange fluorescent proteins) and peptide tags (e.g., His tags, Myc tags, FLAG tags, and HA tags).

[0202] Many techniques for separating the cell colonies expressing specific markers are known in the art. These techniques include separation techniques based on magnetic beads (e.g., separation based on closed loop magnetic beads), flow cytometry, fluorescence activated cell sorting (FACS), affinity tag purification (e.g., using affinity columns or beads, such as biotin columns to separate avidin-labeled reagents) and microscope-based techniques. Separation can also be performed using a combination of different techniques, such as a separation step based on magnetic beads, followed by sorting the resulting cell colonies by flow cytometry for one or more other (positive or negative) markers. Clinical grade separation can be performed, for example, with the Clinimachs system (Miltenyi). This is an example of a separation technique based on closed loop magnetic beads.

[0203] It is also contemplated that dye exclusion properties (eg, side population or rhodamine labeling) or enzyme activity (eg, ALDH activity) can be used to enrich for HSCs.

[0204] The cells of the present invention can be formulated with a pharmaceutically acceptable carrier, diluent or excipient for administration to a subject. Suitable carriers and diluents include isotonic saline solutions, such as phosphate buffered saline, and may contain human serum albumin. Preferably, the B operation of the cell therapy product is performed in accordance with the FACT-JACIE international cell therapy standards.

[0205] Carrier

[0206] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. The vector used for transducing mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated by hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells of the present invention, for example, selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, one or more cells, can be a viral vector. The viral vector can be derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus. These viruses are all lentiviruses. A vector "derived from" a certain type of virus should be understood as a vector that includes at least one component part that can be derived from that type of virus.

[0207] Retroviral vectors may be derived from or may be derived from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abeson murine leukemia virus (A-MLV), avian myelocytoma virus-29 (MC29) and avian erythrocytoma virus (AEV). A detailed list of retroviruses can be found in Coffin, JM et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63.

[0208] Retroviruses can be roughly divided into two categories, "simple" and "complex". Retroviruses can be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are lentiviruses and foamy viruses. A review of these retroviruses is found in Coffin, JM et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63.

[0209] The basic structure of retroviral and lentiviral genomes has many common features, such as the 5'LTR and 3'LTR. Between or within these are located packaging signals to enable the genome to be packaged, primer binding sites, integration sites to enable integration into the host cell genome, and gag, pol, and env genes encoding packaging components - these are polypeptides required for viral particle assembly. Lentiviruses have other features, such as the rev and RRE sequences in HIV, which enable efficient export of the RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of the infected target cell.

[0210] In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration and transcription. The LTRs also act as enhancer-promoter sequences and can control the expression of viral genes.

[0211] The LTR itself is an identical sequence that can be divided into three elements: U3, R, and U5. U3 comes from a unique sequence at the 3' end of the RNA. R comes from a repeated sequence at both ends of the RNA. U5 comes from a unique sequence at the 5' end of the RNA. The sizes of these three elements can vary considerably in different retroviruses.

[0212] In a defective retroviral vector genome, gag, pol and env may be absent or non-functional.

[0213] In a typical retroviral vector, at least a portion of one or more protein coding regions essential for replication may be removed from the virus. This renders the viral vector replication-defective. Portions of the viral genome may also be replaced by a library encoding candidate regulatory portions that are operably linked to regulatory control regions and reporter portions in the vector genome to produce a vector comprising a candidate regulatory portion that is capable of transducing a target host cell and / or integrating its genome into the host genome.

[0214] Lentiviral vectors are part of a larger group of retroviral vectors. A detailed list of lentiviruses can be found in Coffin, JM et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. In brief, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SIV). Examples of non-primate lentiviruses include the prototype "lentivirus" visna / maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and the recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0215] The lentivirus family differs from retroviruses in that lentiviruses have the ability to infect both dividing and non-dividing cells (Lewis, P et al. (1992) EMBO J. 11: 3053–8; Lewis, PF et al. (1994) J. Virol. 68: 510–6). In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells, such as cells that make up, for example, muscle, brain, lung, and liver tissues.

[0216] The lentiviral vector used herein is a vector comprising at least one component that can be derived from a lentivirus. Preferably, the component is involved in the biological mechanism of the vector infecting cells, expressing genes or replicating.

[0217] The lentiviral vector may be a "primate" vector. The lentiviral vector may be a "non-primate" vector (ie, derived from a virus that does not primarily infect primates, especially humans). Examples of non-primate lentiviruses may be any member of the family Lentiviridae that does not naturally infect primates.

[0218] As examples of lentiviral-based vectors, HIV-1 and HIV-2-based vectors are described below.

[0219] HIV-1 vectors contain cis-acting elements that are also found in simple retroviruses. It has been shown that sequences extending into the gag open reading frame are important for the packaging of HIV-1. Therefore, HIV-1 vectors typically contain the relevant portion of gag in which the translation start codon has been mutated. In addition, most HIV-1 vectors also contain a portion of the env gene that includes the RRE. Rev binds to the RRE, which allows full-length or singly spliced ​​mRNA to be transported from the nucleus to the cytoplasm. In the absence of Rev and / or RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, constitutive transport elements from certain simple retroviruses such as the Mason-Pfizer monkey virus can be used to alleviate the need for Rev and RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0220] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1-based vectors, HIV-2 vectors also require RRE for efficient transport of full-length or singly spliced ​​viral RNA.

[0221] In one system, the vector and the auxiliary construct are derived from two different viruses, and the reduced nucleotide homology can reduce the possibility of recombination. In addition to vectors based on primate lentiviruses, vectors based on FIV have also been developed as alternatives to vectors derived from pathogenic HIV-1 genomes. The structures of these vectors are also similar to vectors based on HIV-1.

[0222] Preferably, the viral vector used in the present invention has a minimal viral genome. "Minimal viral genome" should be understood as a viral vector that has been manipulated to remove non-essential elements and retain essential elements to provide the desired functions to infect, transduce and deliver the nucleotide sequence of interest to the target host cell. Further details of this strategy can be found in WO 1998 / 017815.

[0223] Preferably, the plasmid vector used to produce the viral genome in the host cell / packaging cell will have sufficient lentiviral genetic information to allow the RNA genome to be packaged into viral particles capable of infecting target cells but not independently replicating to produce infectious viral particles in the final target cell in the presence of packaging components. Preferably, the vector lacks functional gag-pol and / or env genes and / or other genes essential for replication.

[0224] However, the plasmid vector used to produce the viral genome in the host cell / packaging cell also contains transcriptional regulatory control sequences operably linked to the lentiviral genome to direct the transcription of the genome in the host cell / packaging cell. These regulatory sequences can be natural sequences associated with the transcribed viral sequence (i.e., 5' U3 region), or they can be a heterologous promoter, such as another viral promoter (e.g., CMV promoter).

[0225] The vector may be a self-inactivating (SIN) vector in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be produced and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors. Transcriptional inactivation of the SIN proviral long terminal repeat (LTR) should prevent the mobilization of replication-competent viruses. This should also enable regulation of gene expression from internal promoters by eliminating any cis-acting effects of the LTR.

[0226] The vector may be integration-defective. Integration-defective lentiviral vectors (IDLVs) can be produced, for example, by packaging the vector with a catalytically inactive integrase (e.g., HIV integrase with a D64V mutation in the catalytic site; Naldini, L. et al. (1996) Science 272:263–7; Naldini, L. et al. (1996) Proc. Natl. Acad. Sci. USA 93:11382–8; Leavitt, AD et al. (1996) J. Virol. 70:721–8), or by modifying or deleting essential sequences of the vector LTR (Nightingale, SJ et al. (2006) Mol. Ther. 13:1121–32), or by a combination of the above methods.

[0227] HIV-derived vectors used in the present invention are not particularly limited in terms of HIV strains. Many examples of HIV strain sequences can be found in the HIV sequence database (http: / / www.hiv.lanl.gov / content index). For example, HIV-1 derived vectors can be derived from HIV-1 strains NL4-3, IIIB_LAI or HXB2_LAI (X4-tropical), or BAL (R5-tropical), or any of their chimeras. HIV-2 derived vectors can be derived from, for example, HIV-2 strain ROD.

[0228] As described elsewhere herein, the cyclosporin analogs of the present invention have low, minimal, or ablated ability to bind to, inhibit and / or degrade CypA.

[0229] Many viral vectors used for gene therapy applications recruit CypA to aid efficient infection (e.g., of HSCs) because it protects the capsid from restriction by another antiviral protein called TRIM5. Inhibition of CypA recruitment by cyclosporine analogs therefore risks reducing transduction efficiency to suboptimal levels.

[0230] In one embodiment, a viral vector that is insensitive to CypA or has limited sensitivity (e.g., a viral vector that does not bind CypA) is used. Non-limiting examples of such vectors include HIV capsid mutants that are insensitive to CypA and resistant to TRIM5 restriction such as A92E and G94D (see, e.g., Ylinen et al., Journal of Virology 83(4), 2009, p. 2044–2047).

[0231] However, in a more common embodiment of the present invention, a viral vector sensitive to CypA (e.g., a viral vector that binds CypA) is used. For example, the viral vector may be a vector that is not selected from HIV capsid mutants that are insensitive to CypA and resistant to TRIM5 restriction, such as A92E and G94D.

[0232] Cell transduction

[0233] In one aspect, the present invention provides a use of a cyclosporin analog of the present invention for increasing the transduction efficiency of an isolated population of mammalian cells, human cells, human hematopoietic stem cells and / or progenitor cells, induced human hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from human hematopoietic stem cells and / or progenitor cells or induced human hematopoietic stem cells and / or progenitor cells, such as cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / β T cells, γδ T cells or regulatory T cells, B cells and plasma cells by a vector derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus.

[0234] Improved transduction efficiency refers to an increase in transduction of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells, such as cells selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, in the presence of an agent (e.g., a cyclosporin analog according to the present invention), compared to transduction achieved in the absence of the agent but under substantially the same conditions in other aspects. Thus, increased transduction efficiency can allow for a reduction in the multiplicity of infection (MOI) and / or transduction time required to achieve effective transduction.

[0235] In one embodiment, the percentage of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated by hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells transduced by the vector, the induced hematopoietic stem cells and / or progenitor cells, for example, selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, one or more cells, the percentage increases. In another embodiment, the number of copies of the vector per cell increases. Preferably, both are achieved simultaneously.

[0236] Methods for determining the percentage of cells transduced by a vector are known in the art. Suitable methods include flow cytometry, fluorescence activated cell sorting (FACS) and fluorescence microscopy. The techniques used are preferably those that can be automated and / or high throughput screened.

[0237] For example, a vector containing a reporter gene can be used to transduce a cell colony. The vector can be constructed so that the reporter gene is expressed when the vector transduces the cell. Suitable reporter genes include genes encoding fluorescent proteins, such as green, yellow, cherry, cyan or orange fluorescent proteins. Once the cell colony is transduced by the vector, the number of cells expressing and not expressing the reporter gene can be quantitatively determined using suitable techniques such as FACS. The percentage of cells transduced by the vector can then be calculated.

[0238] Alternatively, quantitative PCR (qPCR) can be used to determine the percentage of cells transduced by a vector that does not contain a reporter gene. For example, a single colony of CD34+ cells can be picked from a semi-solid culture, and qPCR can be performed on each colony to determine the percentage of vector-positive colonies among those analyzed.

[0239] Methods for determining vector copy number are also known in the art. The techniques used are preferably those that can be automated and / or high-throughput screened. Suitable techniques include quantitative PCR (qPCR) and Southern blot-based methods.

[0240] The concentration of the cyclosporin analogs according to the invention applied to mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells, such as cells selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, can be adjusted for different vector systems to optimize the transduction efficiency. Methods for determining transduction efficiency have been described above.

[0241] If administered at too high a concentration, the cyclosporin analogs of the present invention may be toxic to mammalian cells, human cells, hematopoietic stem and / or progenitor cells, induced hematopoietic stem and / or progenitor cells, and / or cells differentiated from hematopoietic stem and / or progenitor cells or induced hematopoietic stem and / or progenitor cells, such as cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / β T cells, γδ T cells or regulatory T cells, B cells and plasma cells. The toxicity of the cyclosporin analogs of the present invention to mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells, such as cells selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, can be determined by quantifying the number of viable cells remaining after exposure to the cyclosporin analogs for a certain period of time. Methods for quantifying the number of viable cells are known in the art. Therefore, those skilled in the art can select the appropriate concentration of the cyclosporin analogs of the present invention to maximize the transduction efficiency while minimizing the toxic effects using the methods described herein.

[0242] For example, the concentration of the cyclosporine analog applied to a population of mammalian cells, human cells, human hematopoietic stem and / or progenitor cells, induced human hematopoietic stem and / or progenitor cells, and / or cells differentiated from human hematopoietic stem and / or progenitor cells or induced human hematopoietic stem and / or progenitor cells, such as cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / β T cells, γδ T cells or regulatory T cells, B cells and plasma cells, can be about 0.1-50 μM, about 1-50 μM, about 5-50 μM, about 10-50 μM, about 5-40 μM, about 10-25 μM, or about 10-15 μM.

[0243] The present invention encompasses the use of the cyclosporin analogs of the present invention. The cyclosporin analogs of the present invention may be those that can increase the transduction efficiency of an isolated population of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells, such as cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / β T cells, γδ T cells or regulatory T cells, B cells and plasma cells by vectors derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CACASNa or visna lentiviruses.

[0244] The cyclosporin analogs of the present invention preferably have low toxicity to mammals, especially humans, and preferably have low toxicity to mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells, for example, cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells.

[0245] Therapeutic applications: gene therapy and transplantation

[0246] The vector used in the present invention preferably includes a target nucleotide (NOI). Preferably, the NOI produces a therapeutic effect and therefore has utility in gene therapy. Suitable NOIs include, but are not limited to, sequences encoding enzymes, cytokines, chemokines, hormones, antibodies, antioxidant molecules, engineered immunoglobulin-like molecules, single-chain antibodies, fusion proteins, immune co-stimulatory molecules, immunomodulatory molecules, antisense RNA, microRNA, shRNA, siRNA, ribozymes, miRNA target sequences, transduction domain negative mutants of target proteins, toxins, conditional toxins, antigens, tumor suppressor proteins, growth factors, transcription factors, membrane proteins, surface receptors, anticancer molecules, vasoactive proteins and peptides, antiviral proteins and ribozymes, and derivatives thereof (e.g., derivatives with associated reporter groups). The NOI may also encode a drug-activating enzyme precursor. An example of an NOI is a β-globin chain, which can be used for gene therapy of thalassemia / sickle cell disease.

[0247] NOIs may also be used to treat other diseases that require non-urgent / selective gene correction of the myeloid lineage, such as chronic granulomatous disease (CGD, such as the gp91 phox transgene), leukocyte adhesion defects, other phagocytic disorders and inherited bone marrow failure syndromes (such as Fanconi anemia) in patients who do not have serious infections, and primary immunodeficiency (SCID). NOIs may also include those used to treat lysosomal storage diseases and immunodeficiencies.

[0248] The present invention also provides a population of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells prepared according to the method of the present invention, for example, a population of one or more cells selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, for use in treatment, for example, for gene therapy. The use can be as part of a transplantation procedure of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / β T cells, γδ T cells or regulatory T cells, B cells and plasma cells.

[0249] Hematopoietic stem cell transplantation (HSCT) can be derived from the transplantation of blood stem cells of bone marrow (in this case, called bone marrow transplantation) or blood. Stem cell transplantation is a medical procedure in the field of hematology and oncology, most often performed on people with blood or bone marrow diseases or certain types of cancer. Many recipients of HSCT are multiple myeloma or leukemia patients, who will not benefit from long-term treatment with chemotherapy, or are already resistant to chemotherapy. Candidates for HSCT include pediatric cases, in which patients have congenital defects, such as severe comprehensive immunodeficiency or congenital neutropenia with defective stem cells, and children or adults with aplastic anemia who also lose stem cells after birth. Other conditions treated with stem cell transplants include sickle cell disease, myelodysplastic syndrome, neuroblastoma, lymphoma, Ewing's sarcoma, desmoplastic small round cell tumors and Hodgkin's disease. Recently, non-myeloablative or so-called "micro-transplantation" procedures have been developed, which only require smaller doses of preparative chemotherapy and radiotherapy. This has allowed HSCT to be performed in the elderly and other patients who would otherwise be considered too frail to undergo conventional treatment regimens.

[0250] In one embodiment, a population of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells prepared according to the method of the present invention, such as cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, is administered as part of an autologous stem cell transplantation procedure.

[0251] In another embodiment, a population of mammalian cells, human cells, hematopoietic stem and / or progenitor cells, induced hematopoietic stem and / or progenitor cells, and / or cells differentiated from hematopoietic stem and / or progenitor cells or induced hematopoietic stem and / or progenitor cells prepared according to the method of the present invention, such as cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, is administered as part of an allogeneic stem cell transplantation procedure.

[0252] An "autologous stem cell transplantation procedure" is understood to mean that the starting cell population (which is then transduced according to the methods of the invention) is obtained from the same subject to which the transduced cell population is administered. Autologous transplantation procedures are advantageous because they avoid problems associated with immune incompatibility and are available to subjects regardless of the availability of a genetically matched donor.

[0253] "Allogeneic stem cell transplantation procedure" is understood as a starting population of cells (which are then transduced according to the method of the invention) obtained from a subject different from the subject to which the transduced cell population is administered. Preferably, the donor is genetically matched to the subject to whom the cells are administered to minimize the risk of immune incompatibility.

[0254] An appropriate dose of the transduced cell population is, for example, therapeutically and / or prophylactically effective. The dose to be administered may depend on the subject and condition to be treated, and can be readily determined by a skilled artisan.

[0255] The products, methods and uses of the present invention may be useful in treating the conditions listed in WO 1998 / 005635. For ease of reference, a portion of this list is now provided: cancer, inflammation or inflammatory diseases, skin conditions, fever, cardiovascular effects, bleeding, coagulation and acute phase reactions, cachexia, anorexia, acute infections, HIV infections, shock states, graft versus host reactions, autoimmune diseases, reperfusion injury, meningitis, migraine and aspirin-dependent antithrombotic; tumor growth, invasion and spread, angiogenesis, metastasis, malignancy, ascites and malignant pleural effusions; cerebral ischemia, ischemic heart disease , osteoarthritis, rheumatoid arthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration, Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn's disease and ulcerative colitis; periodontitis, gingivitis; psoriasis, atopic dermatitis, chronic ulcers, epidermolysis bullosa; corneal ulcers, retinopathy and surgical wound healing; rhinitis, allergic conjunctivitis, eczema, allergic reactions; restenosis, congestive heart failure, endometriosis, atherosclerosis or endosteal sclerosis.

[0256] Additionally, or alternatively, the products, methods and uses of the invention may be useful in treating the conditions listed in WO 1998 / 007859. For ease of reference, a portion of this list is now provided: cytokine and cell proliferation / differentiation activity; immunosuppressant or immunostimulatory activity (e.g., for treating immunodeficiency, including human immunodeficiency virus infection; regulating lymphocyte growth; treating cancer and many autoimmune diseases, and preventing transplant rejection or inducing tumor immunity); regulating hematopoiesis, e.g., for treating bone marrow or lymphatic diseases; promoting the growth of bone, cartilage, tendon, ligament and nerve tissue, e.g., for healing wounds, treating burns, ulcers and periodontal disease, and neurodegeneration; inhibition or activation of follicle stimulating hormone (regulation of fertility); chemotactic / chemokinetic activity (e.g., for moving specific cell types to sites of injury or infection); hemostatic and thrombolytic activity (e.g., for treating hemophilia and stroke); anti-inflammatory activity (for treating, e.g., septic shock or Crohn's disease); as an antimicrobial agent; e.g., a regulator of metabolism or behavior; as an analgesic; treating specific deficiency disorders; treating, e.g., psoriasis, in human or veterinary medicine.

[0257] Additionally, or alternatively, the products, methods and uses of the invention may be useful in treating the conditions listed in WO 1998 / 009985. For ease of reference, a portion of this list is now provided: macrophage inhibitory and / or T cell inhibitory activity, and thus, anti-inflammatory activity; anti-immune activity, i.e., inhibition of cellular and / or humoral immune responses, including responses not related to inflammation; inhibition of the ability of macrophages and T cells to adhere to extracellular matrix components and fibronectin, and upregulation of Fas receptor expression in T cells; inhibition of unwanted immune responses and inflammation, including arthritis (including rheumatoid arthritis), inflammation associated with hypersensitivity reactions, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory diseases, respiratory distress syndrome or other cardiopulmonary diseases, inflammation associated with peptic ulcers, ulcerative colitis and other gastrointestinal diseases, liver fibrosis, cirrhosis or other liver diseases, thyroiditis or other glandular diseases, glomerulonephritis or other kidney and urinary diseases, ear ocular inflammation, such as retinitis or cystoid macular edema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative glaucoma, inflammatory components of ocular trauma, ocular inflammation caused by infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring, such as after glaucoma filtering surgery, immune and / or inflammatory responses to ocular implants and other immune and inflammatory related ophthalmic diseases, inflammation associated with an autoimmune disease or condition or disorder, wherein theimmune and / or inflammatory suppression would be beneficial, Parkinson's disease, complications and / or side effects of Parkinson's disease treatment, AIDS-related dementia complex, HIV-related encephalopathy, Devic's disease, Sydenham's chorea, Alzheimer's disease and other degenerative diseases, central nervous system conditions or disorders, inflammatory component of stroke, post-polio syndrome, immune and inflammatory components of psychiatric illness, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, chronic neuropathy, Guillain-Barré syndrome, Sydenham's chorea, myasthenia gravis, pseudotumor cerebri, Down syndrome, Huntington's disease, amyotrophic lateral sclerosis, inflammatory component of central nervous system compression or central nervous system trauma or central nervous system infection, my Inflammatory components of atrophy and dystrophy, as well as immune and inflammatory related diseases, disorders or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infectious diseases, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplantation complications and / or side effects, inflammatory and / or immune complications and side effects of gene therapy (e.g. due to infection by virus carriers) or inflammation associated with AIDS, suppression or inhibition of humoral and / or cellular immune responses, treatment or improvement of monocytic or leukocytic proliferative diseases, such as leukemia, by reducing the number of monocytes or lymphocytes, for the prevention and / or treatment of transplant rejection during transplantation of natural or artificial cells, tissues and organs (e.g. cornea, bone marrow, organs, lenses, pacemakers, natural or artificial skin tissue).

[0258] Additionally, or alternatively, the products, methods and uses of the invention may be useful in treating beta thalassemia, chronic granulomatous disease, metachromatic leukodystrophy, mucopolysaccharidoses and other lysosomal storage disorders.

[0259] Gene therapy can occur by sustained or transient release of a product encoded by the NOI, such as the product encoded above. For example, hematopoietic progenitor cells generally provide short-term implantation. Therefore, gene therapy by administering transduced hematopoietic progenitor cells can provide non-permanent effects in a subject. For example, the effect may be limited to 1–6 months after administration of transduced hematopoietic progenitor cells. Due to the self-limiting nature of therapeutic intervention, the advantages of this approach will be better safety and tolerability. This hematopoietic progenitor cell gene therapy may be suitable for treating acquired disorders, such as cancer, in which the time-limited expression of the target (potentially toxic) anticancer nucleotide may be sufficient to eradicate the disease. In contrast, HSC is more likely to provide long-term implantation and is therefore more suitable for providing a long-term effect in a subject or an effect that lasts throughout the life of the subject. For example, after administration of transduced HSC, its effect may be limited to 3 months to 30 years. Long-term or sustained effects may be suitable for treating inherited genetic disorders, such as SCID, in which long-term expression of the target nucleotide may be required.

[0260] After gene therapy, the encoded product can be released systemically in the subject, for example, into the circulation. Systemic release can result in an increase in the activity of the encoded product by 1.1, 1.2, 1.5, 2, 5, 10, 25, 50, 100, 250, 500, or 1000 times relative to the activity before gene therapy. The assay for measuring the activity of the encoded product will be apparent to the technician.

[0261] Alternatively, after gene therapy, the encoded product can be released in a targeted manner so that it targets a specific group of tissues and / or organs. For example, the encoded product can target the central nervous system (CNS), heart, face, mouth, eyes, bones, liver, spleen and / or lungs. Targeted release can result in an increase of 1.1, 1.2, 1.5, 2, 5, 10, 25, 50, 100, 250, 500 or 1000 times in the activity of the encoded product in the targeted tissue and / or organ relative to the activity in the same tissue and / or organ before gene therapy. In addition, or as an alternative, relative to the activity in non-targeted tissues and / or organs, targeted release can result in an increase of 1.1, 1.2, 1.5, 2, 5, 10, 25, 50, 100, 250, 500 or 1000 times in the activity of the encoded product in the targeted tissue and / or organ after gene therapy. The assay for measuring the activity of the encoded product will be apparent to the technician.

[0262] Therefore, the present invention provides means for correcting, treating, containing, alleviating and / or preventing the pathological phenotype associated with the above-mentioned indications. Correction can refer to partial, complete correction and overcorrection. Correction can be achieved after about 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 125 days, 150 days, 175 days, 200 days, 250 days, 300 days, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 4 years or 5 years. The effect of correcting, treating, containing, alleviating and / or preventing the phenotype can be temporary. Alternatively, the effect of correcting, treating, containing, alleviating and / or preventing the phenotype can be long-term or continuous.

[0263] Treatment of mammals, especially humans, is preferred. However, both human and veterinary treatments are within the scope of the present invention.

[0264] Reagent test kit

[0265] In another aspect, the present invention provides a kit comprising a cyclosporin analog and / or a cell population of the present invention. The cyclosporin analog and / or a cell population of the present invention may be provided in a suitable container. The kit may also include instructions for use.

[0266] Treatment of viral infections and other pathological conditions associated with IFITM3 expression

[0267] It has been previously disclosed that cyclosporin A CsA has antiviral activity, including anti-coronavirus (see, for example: deWilde et al., J Gen Virol. 2011; 92 (Pt 11): 2542–2548; Carbajo-Lozoya et al., Virus Res. 2014 184: 44–53; Nasiri et al., J Dermatolog Treat. 2020: 1–6; de Wilde et al., Virus Res. 2017 15; 228: 7–13). Recently, it has been proposed that IFITM proteins can actively promote SARS-CoV-2 infection, for example by hijacking these normally antiviral proteins to effectively carry out viral infection. This raises the prospect of treating such viral infections by inhibiting IFITMs, thereby disrupting this hijacking mechanism and reducing the efficiency of viral infection.

[0268] The compounds of the invention are thus useful for treating or preventing a viral infection in a patient. Typically, the patient is a mammal, such as a human or a cat, preferably a human.

[0269] Typically, the viral infection is human immunodeficiency virus 1 (HIV-1), influenza virus, human cytomegalovirus (hCMV), hepatitis C virus (HCV), dengue virus, vaccinia virus (such as smallpox), feline immunodeficiency virus (FIV) or coronavirus (such as COVID-19 or SAR). Preferably, the viral infection is COVID-19, human immunodeficiency virus 1 (HIV-1), influenza virus, human cytomegalovirus (hCMV) or hepatitis C virus (HCV), more preferably COVID-19 or human immunodeficiency virus 1 (HIV-1), and even more preferably COVID-19.

[0270] COVID-19 (i.e., coronavirus disease 2019) is a disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many variants of COVID-19 have been identified and have reached significant epidemic levels in the global population, including α, δ, and ο variants. As shown in the Examples section of the present disclosure, the compounds of the present invention may be particularly useful for treating certain variants of COVID-19 (e.g., δ and / or ο variants, particularly ο variants), which, without being limited by theory, may have evolved to utilize IFITM proteins to effectively survive and / or replicate in infected hosts.

[0271] The compounds of the present invention can be administered to humans in various ways, such as oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, oral or sublingual administration routes. The specific mode of administration and dosage regimen will be selected by the attending physician considering many factors including the age, weight and condition of the patient.

[0272] Compounds are usually used as pharmaceutical compositions, which usually include derivatives of the present invention and pharmaceutically acceptable excipients, diluents or carriers. Therefore, pharmaceutical compositions containing the compounds of this invention are usually formulated with suitable pharmaceutically acceptable excipients, carriers or diluents according to the specific mode of administration used. For example, parenteral preparations are usually injectable fluids, which use pharmaceutically and physiologically acceptable fluids, such as normal saline, balanced salt solutions, etc. as vehicles. On the other hand, oral preparations can be solids, such as tablets or capsules, or liquid solutions or suspensions.

[0273] The compositions may be formulated in unit dosage form, ie, in discrete portions containing a unit dose, or multiples or subunits of a unit dose.

[0274] The amount of the compounds of the invention administered to a patient depends on the activity of the specific compound in question. Other factors include the condition being treated, the nature of the patient being treated, and the severity of the condition being treated. The administration time of the compound should be determined by medical personnel. As a skilled physician will appreciate, and as with any drug, the compound may be toxic at very high doses. For example, the compound may be administered at a dose of 0.01 to 30 mg / kg body weight, such as 0.1 to 10 mg / kg, more preferably 0.1 to 5 mg / kg body weight.

[0275] The compounds of the present invention can be administered alone or in combination with one or more other antiviral agents, preferably one or more agents useful for treating human immunodeficiency virus 1 (HIV-1), influenza virus, human cytomegalovirus (hCMV), hepatitis C virus (HCV), dengue virus, vaccinia virus, feline immunodeficiency virus (FIV) or coronavirus.

[0276] One such antiviral agent is remdesivir.

[0277] Antiviral agents useful for treating HIV-1 include non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside analog reverse transcriptase inhibitors (NRTIs), and nucleotide analog reverse transcriptase inhibitors (NtRTIs). Preferred NRTIs include zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, entecavir, and amitabine, and preferred NNRTIs include efavirenz, nevirapine, delavirdine, elavirine, and rilpivirine. Preferred NtRTIs include tenofovir and adefovir.

[0278] Antiviral agents useful for treating influenza virus include (a) neuraminidase inhibitors, such as oseltamivir and zanamivir, and (b) M2 protein inhibitors, such as amantadine and rimantadine.

[0279] Antiviral agents useful for treating human cytomegalovirus (hCMV) include human cytomegalovirus antibodies and antiviral agents such as ganciclovir, valganciclovir, foscarnet, and cidofovir.

[0280] Antiviral agents useful in treating hepatitis C virus (HCV) include pegylated interferon alpha and ribavirin.

[0281] Antiviral agents used to treat vaccinia virus include cidofovir.

[0282] Antiviral agents useful in treating feline immunodeficiency virus (FIV) include lymphocyte T cell immunomodulators.

[0283] Antiviral agents used to treat coronaviruses including COVID-19 include dexamethasone, remdesivir, pamcyclovir, sotolacimumab, bectelovir, REGEN-COV, banivirumab / etesvirumab, and molopirivir.

[0284] The active ingredients are generally administered as a combined preparation.

[0285] Therefore, the present invention also provides a combination comprising a compound of the present invention and one or more of the other antiviral agents. The combination is generally used to treat or prevent the viral infection in a patient.

[0286] The present invention also provides a compound of the present invention for use in treating or preventing a viral infection in a patient by co-administering it with one or more of the additional antiviral agents. Co-administration may be simultaneous, concurrent, separate or sequential.

[0287] The present invention also provides one or more additional antiviral agents for use in treating or preventing viral infection in a patient by co-administering with the compounds of the present invention. Co-administration may be simultaneous, concurrent, separate or sequential.

[0288] The present invention also provides a product comprising a compound of the present invention and one or more of said other antiviral agents as a combined preparation for simultaneous, concurrent, separate or sequential use in treating or preventing said viral infection in a patient.

[0289] The compounds of the present invention, as IFITM3 inhibitors, also find use more generally in treating pathological conditions associated with IFITM3 expression. The term "associated with IFITM3 expression" is used interchangeably herein with "susceptible to treatment by inhibiting IFITM3." Non-limiting examples of such pathological conditions include viral infections, such as those discussed elsewhere herein, and Alzheimer's disease. However, IFITM3 is known to have a role in regulating innate immune signaling, and the compounds of the present invention may be useful as drugs in any clinical situation where manipulation of IFITM3 favorably affects immunity.

[0290] Example

[0291] Example 1: Representative Synthesis of Certain Exemplary Compounds

[0292] 4-((4R,5R,E)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 2-(1H-imidazol-1-yl)ethyl 5-hydroxy-4-methylpent-1-en-1-yl)benzoate (JW3-158).

[0293]

[0294] To a solution of cyclosporin A (500 mg, 0.42 mmol) in DCM (5 mL) was added 4-vinylbenzoic acid (185 mg, 1.25 mmol) and Hoveyda-Grubbs second generation catalyst (17 mol%). The reaction mixture was stirred in a MW reactor (90° C., 40 min) and then allowed to cool. The solvent was removed under reduced pressure, the material was redissolved in MeOH, the catalyst was removed by passing through a Stratospheres PL Thiol MP solid phase extraction column (Polymer Lab, Varian Inc), and concentrated in vacuo. The crude product (32 mg, 0.024 mmol) was dissolved in DCM (1 mL) and 1-(2-hydroxyethyl)imidazole (4 mg, 0.036 mmol), EDC (12 mg, 0.063 mmol) and 4-pyrrolidinopyridine (0.5 mg, 0.003 mmol) were added. After stirring at room temperature for 15 h, the solvent was removed in vacuo and the crude product was purified by reverse phase C18 (10→100% MeCN, 0.1% FA in H2O, 0.1% FA) to give the final product (13 mg, 0.009 mmol, 38%) as a white solid.

[0295] HRMS (m / z): found 1402.91; for C 73 H119 N 13 O 14 , [MH] + Calculated value: 1402.83.

[0296] 4-((4R,5R,E)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 1-naphthoic acid-2-(1H-imidazol-1-yl)-ethyl ester (BG147).

[0297]

[0298] Methyl 4-bromo-1-naphthoate (530 mg, 1.99 mmol) was dissolved in THF: water = 9: 1 (28 mL), followed by potassium vinyl trifluoroborate (1.34 g, 9.99 mmol), cesium carbonate (1.95 g, 5.99 mmol) and catalyst Pd(dppf)Cl2·DCM (5 mol%). The reaction mixture was stirred at 70°C for 15 hours, warmed to room temperature and diluted with H2O. The organic layer was separated and the aqueous phase was extracted with DCM (×3). The combined organic layers were washed with brine (×3), dried over Mg2SO4, filtered and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (EtOAc 0%→100% in cyclohexane) to obtain methyl 4-vinyl-1-naphthoate (376 mg, 1.78 mmol, 89%) as a yellow oil. Methyl 4-vinyl-1-naphthoate (11 mg, 0.053 mmol) was dissolved in anhydrous DCE, followed by cyclosporin A (32 mg, 0.026 mmol) and Hoveyda-Grubbs second generation catalyst (4 mol%). The reaction mixture was stirred in a MW reactor (70°C, 30 minutes) and then cooled to room temperature. The solvent was removed under reduced pressure, the raw material was redissolved in MeOH, the catalyst was removed by Stratospheres PL Thiol MP solid phase extraction column (PolymerLab, Varian Inc), and vacuum concentrated to give the product as a white solid (36 mg). NaH (21 mg, 0.52 mmol) was added to a two-necked round bottom flask, sealed, and flushed with N2, then anhydrous THF (18 mL) was added thereto with stirring. The mixture was cooled to 0°C and 2-(1H-imidazole-1-yl)ethane-1-ol (118 mg, 1.06 mmol) was added. After stirring for 15 minutes, anhydrous THF solution of the aforementioned crude compound was added and the mixture was stirred at room temperature overnight. Thereafter, the reaction mixture was quenched with saturated ammonium chloride solution (1 mL) and concentrated in vacuo. The crude residue was then dissolved in DCM (20 mL), washed with H2O (3 × 20 mL) and concentrated in vacuo. Purification by reverse phase C18 (0 → 100% MeCN, 0.1% FA + 15% MeOH in H2O, 0.1% FA) gave the final product BG147 (3 mg, 0.002 mmol, 7%, cis / trans mixture) as a colorless solid.

[0299] LCMS (m / z): found 1452.9; for C 77 H 121 N 13 O 14 , [M+H] + Calculated value: 1452.9.

[0300] 1H-NMR (600MHz, MeOD, δppm, J Hz) δ8.90 (d, naphthyl aromatic, 1H), 6.43 (m, alkenyl, 1H), 5.93 (d, alkenyl, 1H), 3.19 (s, NMe, 3H), 3.13 (s, NMe, 3H), 3.05 (s, NMe, 3H), 3.01 (s, NMe, 3H), 2.97 (s, NMe, 3H), 2.90 (s, NMe, 4H), 2.82-2.78 (s, NMe, 4H). (Characteristics of BG147) 1 H-NMR signal)

[0301] ((4R,5R,E)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 1-yl)-5-hydroxy-4-methylpent-1-en-1-yl) trifluoroborate (BG158).

[0302]

[0303] Vinylboronic acid pinacol ester (58 mg, 0.38 mmol) and Hoveyda-Grubbs second generation catalyst (6 mol%) were added to a solution of cyclosporin A (1 g, 0.83 mmol) in DCE (10 mL). The reaction mixture was cooled in a MW reactor (70 ° C, 30 minutes) under N2 atmosphere. The solvent was removed under reduced pressure, the raw material was dissolved in MeOH, the catalyst was removed by Stratospheres PL Thiol MP solid phase extraction column (Polymer Lab, Varian Inc), and vacuum concentrated. The crude product (1.1 g, 0.83 mmol) was dissolved in MeOH (10 mL) and potassium bifluoride solution (4.5 M, 1 mL) was added. The reaction mixture was stirred at room temperature overnight, concentrated under reduced pressure, dissolved in acetone (10 mL×3) and filtered. The filtrate was then precipitated into Et2O (50 mL) to give an orange solid which was collected by filtration, washed with Et2O (50 mL) and concentrated under reduced pressure to give the final product (50 mg, 0.04 mmol, 5%).

[0304] 4-((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 2-(1H-imidazol-1-yl)ethyl 5-hydroxy-4-methylpent-1-en-1-yl)-3,5-dimethoxybenzoate (BG186).

[0305]

[0306] BG158 (6 mg, 0.005 mmol) was dissolved in THF: water = 9: 1 (1.4 mL), followed by 4-bromo-3,5-dimethoxybenzoic acid-2-(1H-imidazol-1-yl)ethyl ester (3 g, 0.01 mmol), cesium carbonate (9 mg, 0.03 mmol) and catalyst Pd(dppf)Cl2·DCM (10 mol%). The reaction mixture was stirred at 90 ° C for 30 minutes under N2 atmosphere, warmed to room temperature, and diluted with H2O. The organic layer was separated and the aqueous phase was extracted with DCM (× 3). The combined organic layers were washed with brine (× 3), dried over Mg2SO4, filtered and concentrated under reduced pressure. The crude product was purified by reverse phase C18 (0→100% MeCN, 0.1% FA + 15% MeOH in H2O, 0.1% FA) to obtain the final product BG186 (0.2 mg, 3%).

[0307] Example 2 - JW3-158 is a more potent transduction enhancer than control compounds CsA, CsH and JW115 in a model system

[0308] HP-1 cells (human monocytic cell line) were treated with 10 ng / ml of type I interferon (IFNβ) overnight to induce IFITM3 expression. The next day, cells were infected with a VSV-G pseudotyped HIV-1 vector encoding GFP (HIV-GFP) at an infection multiplicity (MOI) of 0.2 IU / cell. Cells were infected in the presence of 5 μM compound or an equal volume of DMSO vehicle. After 48 hours, the infection level was determined by counting GFP-positive green cells by flow cytometry.

[0309] Cells not treated with IFN had higher infection, while IFN reduced infection (baseline). Inhibitors rescued IFN infection by inhibiting the antiviral effect of IFITM3. The higher the measured value (rescue fold compared to baseline), the higher the rescue of infection, and the better the inhibition of IFITM3. Compared with the inhibition baseline, the rescue fold of the tested compound measured was 24.6 for CsH, 9.9 for CsA, 29.2 for JW3-158, and 14.2 for JW-115.

[0310] Note that this experiment shows that if the cells are in an antiviral state due to IFN exposure, the molecule improves infection. This is expected to even out differences in transduction efficiency between patients, as variation may be affected by the level of antiviral state of the patient's cells.

[0311] JW-115 is disclosed in WO 2021 / 229237 and has the following structure:

[0312]

[0313] Example 3 - Representative compounds have reduced binding to CypA compared to CsA

[0314] SPR binding assay - Cyp inhibitors

[0315] CypA binding was performed using surface plasmon resonance as described (Warne J Biol Chem. 2016 Feb 26; 291(9):4356–73). Cyclosporin A was used as a positive control.

[0316] process

[0317] Surface plasmon resonance (SPR) using Biacore at 25°C was used to study the binding interactions between CypA and various inhibitors. The SPR binding experiments used a dual flow cell with a blank reference. The sensor chip surface was first activated with a mixture of N-hydroxysuccinimide (NHS) and N-(3-dimethyl-aminopropyl)-N′-ethyl-carbazine diimide hydrochloride (EDC) for 420 s, and the flow rate on the blank flow cell 1 was also 10 μl / min in addition to the flow cell 2.

[0318] Prior to immobilization and covalent attachment to the carboxy-methylated dextran matrix of a Biacore CM5 sensor chip, CypA protein was diluted to a concentration of 50 μg / mL by the addition of 10 mM sodium acetate pH 5. CypA was run on the surface for 420 seconds, followed by quenching of any unreacted surface with ethanolamine for 420 seconds.

[0319] Fixation was performed in the presence of JW47 (Warne J Biol Chem. 2016 Feb 26; 291(9):4356–73). CYP inhibitor compound DMSO stock solution (10 mM concentration) was diluted with HBS-EP+ buffer to give a final DMSO concentration of 2% (eg 5 μl added to 245 μl buffer).

[0320] Kinetic / affinity experiments

[0321] The contact time of the inhibitor with the surface is 120 seconds, followed by a dissociation time of 600 seconds, the latter being extended to 1200 seconds in the case of very strong binders such as CsA. If the dissociation time is sufficient, no regeneration phase is required.

[0322] Solvent correction was performed in each experiment using 8 different DMSO dilutions as reference: 2.8%, 2.6%, 2.4%, 2.2%, 2%, 1.8%, 1.6% and 1.5%.

[0323] A 50 / 50 mixture of buffer and DMSO is required to include wash cycles in the experiment.

[0324] Example

[0325] 50 μL of CypA (0.1 mg / mL) + 40 μL of 10 mM sodium acetate pH 5 + 10 μL of 600 μM JW47 DMSO solution. The mixture was allowed to stand at room temperature for 10 minutes before mounting in the injection holder.

[0326] CYP inhibitor compound DMSO stock solution (10 mM concentration) was diluted with HBS-EP+ buffer to give a final DMSO concentration of 2% (eg 5 μl in 245 μl buffer).

[0327] result

[0328] The results are summarized in the table below.

[0329] Run Order: Sample Meta R Group <![CDATA[K d (Kinetics) μM]]> <![CDATA[K d (Affinity) μM]]> Maximum Response (RU) <![CDATA[K off (s -1 )]]> <![CDATA[K on (M -1 s -1 )]]> 1: CsA (control) - - - ～40 - - 2: JW3-158 H 0.16 1.83 27.0 0.002168 13380 3: BG147 Naphthyl 2.22 5.27 14.0 0.02164 9752 4: BG149 Ethyl 0.1 34.65 4.8 0.001098 11300 5: BG150 methyl 3.84 1.31 18.7 0.04251 11070 6: BG181 Methoxy 35.9 2.66 9.8 0.2282 6356 7: BG185 Isopropyl 259.5 2.92 14.5 0.5843 2252 8: BG186 3,5-Dimethoxy 28.9 4.76 7.6 0.4221 14560 9: BG190 <![CDATA[CF3]]> 28.8 6.07 13.2 0.01382 4799

[0330] The structures of JW3-158, BG147 and BG186 are shown in Example 1. The structures of BG149, BG150, BG181, BG185 and BG190 are shown below.

[0331]

[0332]

[0333] Example 4 - Complementary cell line assay to identify selective IFITM3 inhibitors

[0334] Experiments similar to those in Example 2 were performed in IFN-treated THP-1 cells.

[0335] THP-1 cells (human monocytic cell line) were treated with 10 ng / ml of type 1 interferon (IFNβ) overnight to induce IFITM3 expression. The next day, cells were infected with a VSV-G pseudotyped HIV-1 vector encoding GFP (HIV-GFP) at an infection multiplicity (MOI) of 0.35. Cells were infected in the presence of 3 doses of 1.25, 2.5 and 5 μM of the compound or 0.1% v / v DMSO vehicle. After 48 hours, the infection level was determined by counting GFP-positive green cells by flow cytometry.

[0336] The results are as follows Figure 1As shown. The virus-free bar indicates uninfected (green) cells. The infection of cells not treated with IFN is high (dark gray bars), while the infection of IFN is low (third bar). The inhibitor rescues the infection of IFN by inhibiting the antiviral effect of IFITM3. The higher the bar, the higher the rescue of infection, and the better the inhibition of IFITM3. BG147 completely rescued IFN infection in this experiment, which is better than other molecules. For each compound, three concentrations were tested (the bars from left to right were 1.25 μM, 2.5 μM and 5 μM, respectively).

[0337] Next, experiments were performed in U87 cells. These cells differ from THP-1 in that they express IFITM3 and TRIM5. Therefore, only inhibitors with reduced CypA inhibition could rescue infection. This functionally tested whether the inhibitor had lost CypA inhibition but retained IFITM3 inhibition. The results are shown in Figure 2. Figure 2 shown.

[0338] U87 cells were infected at an MOI of 0.3 in the presence of DMSO (0.1% v / v) or 2.5, 5 or 10 μM inhibitors (left to right bars, respectively, for each compound shown). Inhibition of IFITM3 increased infectivity. Unlike other inhibitors, BG147 produced the greatest infection enhancement at the lowest concentration of 2.5 μM. Other molecules achieved maximum infection rescue at the highest concentration.

[0339] Explanation for differences in drug potency in different cell lines

[0340] Cyclosporine A is a complex molecule that targets multiple pathways in the cell. Specifically, CsA targets cyclophilin A, an enzyme inhibited by CsA, and IFITM3, an antiviral membrane protein that is redirected to a degradation pathway in the lysosome upon addition of CsA. Cyclophilin A (CypA) serves as a cofactor for HIV infection. It serves to protect HIV from the antiviral protein TRIM5. Thus, inhibiting CypA with CsA reduces infection via TRIM5 activity, but only in cells that produce TRIM5. On the other hand, CsA also inhibits IFITM3, which increases HIV infectivity in cells that produce IFITM3. Thus, the effect of CsA depends on the levels of TRIM5 and IFITM3. High TRIM5, low IFITM3, CsA inhibits infection. Low TRIM5, high IFITM3, CsA rescues infection (IFN-treated THP-1 are high IFITM3, low TRIM5, so they allow one to separate the effect of the drug on IFITM3). Most cells produce both proteins to some extent. This is why CsA is not an effective enhancer of transduction and why the results differ between strains, depending on how much CypA and IFITM3 they produce. Stem cells produce TRIM5 and therefore cannot use CsA. Therefore, in this work, we have sought to develop molecules that inhibit IFITM3 instead of CypA, such as BG147.

[0341] Further research

[0342] THP-1 cells (a human monocytic cell line) were treated with 10 ng / ml of type 1 interferon (IFNβ) overnight to induce IFITM3 expression.

[0343] The next day, IFN-treated (filled symbols) and untreated cells (open symbols) were infected with VSV-G pseudotyped HIV-1 vector encoding GFP (HIV-GFP) at a multiplicity of infection (MOI) of 0.4. Cells were infected in the presence of titrated BG147 or cyclosporin H (CsH). After 48 hours, the infection level was determined by counting GFP-positive green cells by flow cytometry.

[0344] The results are as follows Figure 3 In panels A and B, the results for IFN-treated cells are shown as solid symbols, while the results for untreated cells are shown as open symbols. Panel A shows the results for BG147 and panel B shows the results for CsH.

[0345] Both BG147 and CsH rescued infection from the antiviral effects of IFN. BG147 was more effective than CsH.

[0346] Example 5 - Stem Cell Research

[0347] Here the inhibitors are tested for their ability to improve HIV vector transduction in primary human stem cells used for gene therapy. This is the gold standard test because it tests the ability to improve transduction in the same cells that are actually used for gene therapy.

[0348] Human stem cells (HSCs) were infected with a dose of a VSV-G pseudotyped HIV gene therapy vector encoding GFP that infected 40% of the cells. Cells were infected in the presence and absence of various transduction enhancers as follows: Lentiniboost (LB), protamine sulfate (PS), JW3-158, BG147, or CsH at concentrations of 2.5, 1.25, and 0.5 μM. Infected (green) cells were counted by flow cytometry 48 hours after transduction. Results are shown in Figure 2. Figure 4 shown.

[0349] BG147 behaved as an additional inhibitor and was as effective as the prior art transduction enhancer (combination of LB + PS).It was noted that the competitor molecule CsH gave good enhancement, but at higher concentrations, and was toxic at 12.5 μM.

[0350] This first experiment determined the effective concentration range.

[0351] The cell viability measurements of the cells in the above experiments showed that the viability after BG147 and JW3-158 treatment was good and better than that after PS+LB treatment ( Figure 5 ). Note that CsH is toxic at 12.5 μM, as reflected by decreased transduction.

[0352] This experiment was then repeated in cells from a second HSC donor as follows.

[0353] HSCs were infected with a dose of a VSV-G pseudotyped HIV gene therapy vector encoding GFP that infected 40% of the cells. Cells were infected in the presence and absence of various transduction enhancers as follows: Lentiniboost (LB), protamine sulfate (PS), and JW3-158, BG147, or CsH at concentrations of 2.5, 1.25, and 0.5 μM. Infected (green) cells were counted by flow cytometry 48 hours after transduction. Results are shown in Figure 2. Figure 6 shown.

[0354] In this experiment, BG147 was used without other inhibitors and contained a combination of the prior art transduction enhancers LB+PS.

[0355] Importantly, 2.5 μM BG147 was additive with PS and LB, producing the optimal number of transduced (infected) cells. This was expected because PS and LB improve virus attachment to cells, while BG147 improves the process of virus entry, so they work well together.

[0356] process

[0357] Primary CD34+ HSCs were cultured in Stespan II supplemented with 1% Pen / Strep, 100 ng / mL TPO, SCF and FLT3R, and 60 ng / mL IL-3. 6 / mL concentration resuspended and allowed to recover for 24 hours. On the second day, the cells were resuspended and incubated with or without different concentrations and combinations of GFP-lentivirus (MOI 10), Lentibost + protamine sulfate, JW3-158, BG-147 and CsH. In all cases, the final volume was 125 μL, 250,000 cells per condition. The cells were incubated for another 48 hours to allow GFP expression. The cells were then transferred to a FACS tube and centrifuged at 300 × g for 10 minutes. The supernatant was aspirated and then resuspended in 1 mL of FACS buffer and centrifuged again for washing. The cells were then resuspended in 400 μL of FACS buffer containing DAPI. Viability and GFP expression were measured by flow cytometry.

[0358] Example 6 - Further stem cell research

[0359] Here, a different measurement of transgene expression was performed using cells from the same experiment as above (Example 5). Instead of measuring the number of transduced cells (% infection), the mean fluorescence intensity (green color of transgene expressing cells) was measured. The results are shown in Table 1. Figure 7 shown.

[0360] This shows that the greenest cells were in the treatment with 2.5 μM BG147+PS+LB. This shows that infection after this treatment was the highest of all infections and that the cells were greener because they had been infected with more than one HIV GFP virus. This also shows that the maximum number of infected cells was reached, i.e., the remaining 20% ​​could not be infected by HIV GFP regardless of the dose.

[0361] Figure 8 Live / dead staining data from previous experiments were summarized, showing that no treatment significantly killed cells.

[0362] BG147 performed best. Without being limited by theory, it is thought that JW3-158 did not work well because it retained anti-CypA activity, which would be optimally removed or at least further reduced for a molecule to function optimally in stem cells.

[0363] Example 7 - Antiviral activity against COVID-19

[0364] Western blotting

[0365] THP-1 cells (human monocytic cell line) were treated with 10 ng / ul of type I interferon (IFNβ) overnight to induce IFITM3 expression. The next day, cells were treated with 5 μM CsA, CsH, or JW3 (JW3-158) or an equal volume of DMSO vehicle. Protein samples were collected 6 hours later and IFITM3 levels were measured by immunoblotting with actin, which was detected as a loading control. IFN induces IFITM3. Results are shown in Fig. 9 As shown in panel A. The inhibitors caused a loss of IFITM3 protein levels compared to DMSO-treated cells (second lane), with JW3-158 having the most potent effect.

[0366] Calu-3 lung epithelial cells were pretreated with 2.5 μM JW3 (JW3-158) or an equal volume of DMSO for 4 h prior to infection with α, δ, or ο isolates of SARS-CoV-2 at an MOI of 1000 viral E RNA copies / cell. The compounds were present throughout the infection. RNA was harvested 48 h post-infection and viral replication levels were quantified by measuring E copy number by RT-qPCR. Results are shown in Table 1. Fig. 9 The fold changes between DMSO and JW3-158 treated conditions are shown in each variant, with replication of δ and ο being inhibited by JW3-158 to a greater extent than α.

[0367] Example 8 - Further synthetic studies

[0368] Additional work involved: (a) the synthesis of larger batches of BG147; and (b) the synthesis of other compounds of the present disclosure.

[0369] Method flow

[0370]

[0371] Scheme 1. Synthesis of BG147 and its analogs

[0372] Synthesis and characterization of BG147 and its analogs

[0373] 4-vinyl-1-naphthoic acid methyl ester (VP43)

[0374]

[0375] Methyl 4-bromonaphthalene-1-carboxylate (530 mg, 2 mmol) was dissolved in THF: water = 9: 1 (28 mL), followed by potassium vinyl trifluoroborate (1.3 g, 10 mmol), cesium carbonate (1.9 g, 6 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (1: 1) (5 mol%). After stirring overnight at 70°C, the reaction mixture was cooled and concentrated in vacuo. The residue was diluted with CH2Cl2 (10 mL), washed with water (3×10 mL) and brine, dried over MgSO4 and concentrated under reduced pressure. The crude product was purified by silica gel chromatography (EtOAc 0%→100% in cyclohexane) to give VP43 (378 mg, 1.779 mmol, 89%) as a yellow oil.

[0376] 4-Vinyl-1-naphthoic acid (VP57)

[0377]

[0378] VP43 (2.2 g, 10 mmol) was dissolved in THF (12 mL) and treated with 5 M LiOH (25 mL). After stirring overnight at 50° C., THF was removed in vacuo, the residue was acidified to pH 2 with 1 M HCl, extracted with EtOAc (30×3), dried over MgSO , filtered and concentrated under reduced pressure to give VP57 (2 g, 10 mmol, 97%) as a yellow solid.

[0379] 4-((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoic acid (VP47)

[0380]

[0381] VP57 (270 mg, 1.36 mmol) was dissolved in anhydrous DCE (4.5 mL), followed by cyclosporin A (360 mg, 0.299 mmol) and Hoveyda-Grubbs second generation catalyst (15 mol%). The reaction mixture was stirred in a MW reactor under N2 atmosphere (70 °C, 30 min) and then cooled to room temperature. The solvent was removed under reduced pressure, the raw material was redissolved in MeOH, and the catalyst was removed by Stratospheres PL Thiol MP solid phase extraction column (Polymer Lab, Varian Inc). Unreacted CsA was removed by Biotage Isolute PE-AX 10 g (MeOH + 10% NH4OH → 100% MeOH → 5% MeOH in DCM → 5% MeOH + 5% AcOH in DCM). Excess VP57 was removed by reverse phase chromatography (C18, MeCN 10%→100% in H2O+10 mM NH3) to afford VP47 (115 mg, 0.085 mmol, 28%, trans / cis=3 / 1) as an off-white solid.

[0382] 1 H-NMR (500MHz, CDCl3, δppm) δ3.56(s,NMe,3H),3.40(s,NMe,3H),3.27(s,NMe,3H),3.11(s,NMe,3H),3.10(s,NMe,3H),2.72(s,NMe,3H),2.67(s,NMe,3H).

[0383] 13 C-NMR (500MHz, CDCl3, δppm) δ173.92 (C=O), 173.89 (C=O), 173.66 (C=O), 171.71 (C=O), 171.5 0(C=O),171.38(C=O),171.34(C=O),170.63(C=O),170.56(C=O),170.45(C=O),170.37(C=O).

[0384] LCMS (m / z): for C 72 H 115 N 11 O 14 , [MH] + Calculated value: 1358.9; measured value: 1358.4.

[0385] General scheme for the synthesis of BG147 and its ester and amide analogs

[0386] To a solution of VP47 (1 eq) in DMF (1 mL) was added DIPEA (7 eq) and HATU (6 eq). After stirring at room temperature for 10 min, a solution of the appropriate alcohol or amine (10 eq) in DMF (0.5 mL) was added and the reaction mixture was stirred at 50°C overnight. The solvent was then removed under reduced pressure and the crude product was purified by silica gel chromatography (MeOH 0% → 10% in DCM) and reverse phase C4 (MeCN 30% → 100% in H2O + 10 mM NH3) to obtain the final product.

[0387] 4-((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 2-(1H-imidazol-1-yl)ethyl 5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (BG147)

[0388]

[0389] Following the general procedure above and using 2-(1H-imidazol-1-yl)ethan-1-ol as the appropriate alcohol, BG147 was obtained as an off-white solid (17 mg, 0.01 mmol, 20%, trans / cis = 3 / 1).

[0390] 1 H-NMR (600MHz, CDl3, δppm) δ3.55(s,NMe,3H),3.39(s,NMe,3H),3.27(s,NMe,3H),3.10(s,NMe,3H),3.09(s,NMe,3H),2.70(s,NMe,3H),2.66(s,NMe,3H).

[0391] 13 C-NMR (600MHz, CDCl3, δppm) δ173.97(C=O), 173.88(C=O), 173.63(C=O), 173.13(C=O), 172.8 0(C=O),172.16(C=O),171.54(C=O),171.43(C=O),171.26(C=O),170.53(C=O),170.28(C=O).

[0392] HRMS (m / z): for C 77 H 121 N 13 O 14 , [MH] + Calculated value 1452.91560; Measured value 1452.92287

[0393] 4-((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 2-(N-morpholinyl)ethyl 5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (VP50)

[0394]

[0395] Following the general procedure above and using 2-(N-morpholinyl)ethan-1-ol as the appropriate alcohol, VP50 was obtained as an off-white solid (3.0 mg, 0.002 mmol, 17%, trans / cis = 3 / 1).

[0396] 1 H-NMR (600MHz, CDCl3, δppm) δ3.56(s,NMe,3H),3.41(s,NMe,3H),3.28(s,NMe,3H),3.11(s,NMe,3H),3.10(s,NMe,3H),2.70(s,NMe,3H),2.66(s,NMe,3H).

[0397] LCMS (m / z): for C 78 H 126 N 12 O 15 , [MH] + Calculated value: 1471.9; measured value: 1471.6.

[0398] (3S,6S,9S,12R,15S,18S,21S,24S,30S,33S)-30-ethyl-33-((1R,2R)-1-hydroxy-5- (4-(4-(2-Hydroxyethyl)piperazine-1-carbonyl)naphthalene-1- 2-Methylpent-4-en-1-yl)-6,9,18,24-tetraisobutyl-3,21-diisopropyl-1,4,7,10,12,15,19,25,28-nonamethyl- 1,4,7,10,13,16,19,22,25,28,31-Ten Tritriacontane-2,5,8,11,14,17,20,23,26,29,32-undecanone (VP51)

[0399]

[0400] Following the general procedure above and using 2-(piperazin-1-yl)ethan-1-ol as the appropriate amine, VP51 was obtained as an off-white solid (3.0 mg, 0.002 mmol, 18%, trans / cis = 3 / 1).

[0401] 1 H-NMR (600MHz, CDCl3, δppm) δ3.55(s,NMe,3H),3.41(s,NMe,3H),3.27(s,NMe,3H),3.12(s,NMe,3H),3.00(s,NMe,3H),2.71(s,NMe,3H),2.67(s,NMe,3H).

[0402] LCMS (m / z): for C 78 H 127 N 13 O 14 , [MH] +Calculated value: 1471.0; measured value: 1471.2.

[0403] N-(2-(1H-imidazol-1-yl)ethyl)-4-((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R, 26S,29S,32S)-5-ethyl-11,17,26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25, 28,31-nonamethyl-3,6,9,12,15,18,21,24,27,30,33-undecyloxy-1,4,7,10,13,16,19,22, 25,28,31-Undecazacyclotriacontane-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthamide (VP53)

[0404]

[0405] Following the general procedure above and using 2-(1H-imidazol-1-yl)ethan-1-amine as the appropriate amine, VP53 was obtained as a yellow solid (8.0 mg, 0.006 mmol, 47%, trans / cis = 3 / 1).

[0406] 1 H-NMR (600MHz, CDCl3, δppm) δ3.55(s,NMe,3H),3.37(s,NMe,3H),3.28(s,NMe,3H),3.08(s,NMe,3H),3.03(s,NMe,3H),2.71(s,NMe,3H),2.67(s,NMe,3H).

[0407] LCMS (m / z): for C 77 H 122 N 14 O 13 , [MH] + Calculated value: 1451.9; measured value: 1452.2.

[0408] 4-((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 2-(1H-pyrazol-1-yl)ethyl 5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (VP72)

[0409]

[0410] Following the general procedure above and using 2-(1H-pyrazol-1-yl)ethan-1-ol as the appropriate alcohol, VP72 was obtained as a yellow solid (3.0 mg, 0.002 mmol, 14%, trans / cis = 3 / 1).

[0411] 1 H-NMR (500MHz, CDCl3, δppm) δ3.56(s,NMe,3H),3.41(s,NMe,3H),3.27(s,NMe,3H),3.11(s,NMe,3H),3.10(s,NMe,3H),2.71(s,NMe,3H),2.67(s,NMe,3H).

[0412] LCMS (m / z): for C 77 H 121 N13 O 14 , [MH] + Calculated value: 1451.9; measured value: 1453.0.

[0413] 4-((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 3-(1H-pyrazol-1-yl)propyl 5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (VP73)

[0414]

[0415] Following the general procedure above and using 3-(1H-pyrazol-1-yl)propan-1-ol as the appropriate alcohol, VP73 was obtained as a yellow solid (3.0 mg, 0.002 mmol, 14%, trans / cis = 3 / 1).

[0416] 1 H-NMR (500MHz, CDCl3, δppm) δ3.56(s,NMe,3H),3.41(s,NMe,3H),3.28(s,NMe,3H),3.11(s,NMe,3H),3.10(s,NMe,3H),2.71(s,NMe,3H),2.67(s,NMe,3H).

[0417] LCMS (m / z): for C 78 H 123 N 13 O14, [MH] + Calculated value: 1465.9; measured value: 1467.1.

[0418] 4-((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 4-(1H-imidazol-1-yl)butyl 5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (VP74)

[0419]

[0420] Following the general procedure above and using 4-(1H-imidazol-1-yl)butan-1-ol as the appropriate alcohol, VP74 was obtained as a yellow solid (4.0 mg, 0.003 mmol, 18%, trans / cis = 1 / 1).

[0421] 1 H-NMR (500MHz, CDCl3, δppm) δ3.55(s,NMe,3H),3.40(s,NMe,3H),3.28(s,NMe,3H),3.11(s,NMe,3H),3.10(s,NMe,3H),2.71(s,NMe,3H),2.67(s,NMe,3H).

[0422] LCMS (m / z): for C79 H 125 N 13 O 14 , [MH] + Calculated value: 1479.9; measured value: 1481.1.

[0423] 4-((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-undecylazacyclotricarboxane-2- 5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoic acid methyl ester (VP75)

[0424]

[0425] Following the general procedure above and using methanol as the appropriate alcohol, VP75 was obtained as a yellow solid (11.0 mg, 0.008 mmol, 54%, trans / cis = 3 / 1).

[0426] 1 H-NMR (500MHz, CDCl3, δppm) δ3.56(s,NMe,3H),3.40(s,NMe,3H),3.27(s,NMe,3H),3.11(s,NMe,3H),3.10(s,NMe,3H),2.71(s,NMe,3H),2.67(s,NMe,3H).

[0427] LCMS (m / z): for C 73 H 117 N 11 O 14 , [MH] + Calculated value: 1371.9; measured value: 1373.0.

[0428] 4-((4R,5R)-5-((2S,5S,11S,14S,17S,20S,23R,26S,29S,32S)-5-ethyl-11,17, 26,29-tetraisobutyl-14,32-diisopropyl-1,7,10,16,20,23,25,28,31-nonamethyl-3,6,9,12,15,18, 21,24,27,30,33-Undecyloxy-1,4,7,10,13,16,19,22,25,28,31-Undecylazacyclotricyclotriacontane- 2-(2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-N-(2-(N-morpholinyl)ethyl)-1-naphthamide (VP76)

[0429]

[0430] Following the general procedure above and using 2-(N-morpholinyl)ethan-1-amine as the appropriate amine, VP76 was obtained as a white solid (10.0 mg, 0.007 mmol, 46%, trans / cis = 3 / 1).

[0431] 1 H-NMR (500MHz, CDCl3, δppm) δ3.55(s,NMe,3H),3.38(s,NMe,3H),3.27(s,NMe,3H),3.10(s,NMe,3H),3.08(s,NMe,3H),2.72(s,NMe,3H),2.67(s,NMe,3H).

[0432] LCMS (m / z): for C 78 H 127 N 13 O 14 , [MH] + Calculated value: 1470.0; measured value: 1471.3.

[0433] Materials and methods

[0434] Unless otherwise stated, all commercially available solvents and reagents were used as received without further treatment.

[0435] • Solvent evaporation was performed by using a Buchi rotary evaporator R-210 under reduced pressure (40-60°C).

[0436] Microwave-assisted reactions in Biotage Initiator + carried out in a synthesis device.

[0437] The reaction was carried out under anhydrous atmosphere using commercial N2.

[0438] • Thin layer chromatography (TLC) was qualitatively performed on aluminum silica gel plates (Alugram Sil G / UV 254), detected with UV light (λ254 nm) and stained with [(NH4)6MoO4, Ce(SO4)2, H2SO4, H2O].

[0439] Purification was performed on a Biotage Isolera four-stage flash chromatography system. The columns used were: a) Normal phase Biotage Silica gel (5g, 10g, 25g), and b) reverse phase Biotage Snap Bio C4 25g or Biotage SfarC18D-Duo (12 g, 30 g, 60 g). PE-AX 10 g strong base ion exchange resin was used when indicated.

[0440] · 1 H and 13 C NMR spectra were performed in UCL Chemistry NMR equipment and Bruker DRX 500, 600 or 700MHz spectrometers were used. Chemical shifts (δ) are expressed in ppm relative to TMS as an internal standard, and coupling constants (J) are expressed in Hz. J is divided and not repeated. Except when indicated, CDCl and CDOD were used as solvents at room temperature. All assignments were confirmed by 2D spectra (COSY and HSCQ).

[0441] Accurate mass measurements were performed using ASAP-HESI ionization coupled to a Q ExactivePlus mass spectrometer at the UCL Chemistry Mass Spectrometry Facility.

[0442] LC-MS spectroscopy was performed using a single quadrupole LC / MSD XT mass spectrometer with electrospray ionization (ESI) using an analytical C4 column (Symmetry 300, 50×4.6 mm, 3.5 μm) and a C18 column (Kinetex 5 μm 50×4.6 mm). The gradient used was:

[0443] a) 10% → 95% MeCN + 0.1% FA (FA = formic acid) in H2O + 0.1% FA (6.5 min)

[0444] b) 30% → 95% MeCN + 0.1% FA in H2O + 0.1% FA (9 min)

[0445] c) 30% → 95% MeCN in H2O, 10 mM NH3 (9 min)

[0446] Example 9 - Activity studies of other compounds disclosed herein

[0447] Further studies were performed to test the properties of additional compounds of the present disclosure.

[0448] Study 1: THP-1 cells (a human monocytic cell line) were treated with 10 ng / ml of type 1 interferon (IFNβ) overnight to induce IFITM3 expression.

[0449] The next day, IFN-treated cells were infected with VSV-G pseudotyped HIV-1 vector encoding GFP (HIV-GFP) at a multiplicity of infection (MOI) of 0.25. Cells were treated in the presence of a series of control and comparison compounds titrated (1.25–5 μM), as Fig.10 After 48 hours, cells were fixed and infection levels were determined by counting GFP-positive green cells by flow cytometry as indicated. For comparison, THP-1 cells treated with DMSO, with and without IFNβ, are shown with black bars.

[0450] The results are as follows Fig.10 As shown (N=2). High levels of % GFP positive cells at a particular test compound concentration indicate high levels of rescued infection due to the antiviral effect of IFN.

[0451] Study 2 (Western Blot):

[0452] THP-1 cells (human monocytic cell line) were treated with 10 ng / ml of type 1 interferon (IFNβ) overnight to induce IFITM3 expression. The cells were then treated with 5 μM of the indicated test compounds. After 24 hours, the cells were lysed and electrophoresed on SDS PAGE gels. Western blot analysis was performed using anti-actin (control) and anti-IFITM3 antibodies to observe IFITM3 degradation. The results are shown in Fig.11 shown.

[0453] Study 3 (Cell Viability Assay (MTT)):

[0454] MTT colorimetric viability assays of THP1 cells were performed in parallel with infection experiments in the presence of the indicated test compounds. THP-1 cells (a human monocytic cell line) were treated overnight with 10 ng / ml of type 1 interferon (IFNβ) to induce IFITM3 expression. The following day, cells were treated with a series of titrations (1.25–5 μM) of one of the control and comparison compounds, as Fig.12 As shown. Triton X is a control showing cell death. After 48 hours, MTT reagent was added to the treated cells, and after 4 hours at 37°C, the cell crystal metabolites were dissolved and the absorbance at 570 nm was measured using a spectrometer. The results are shown in Fig.12 The error bars represent the standard deviation of three technical replicates from one experiment. N=1. For each test compound, three bars are shown, which correspond in each case from left to right to the results obtained when 1.25, 2.5 and 5 μM of the test compound were used, respectively.

[0455] Example 10 - Synthesis of intermediates useful for preparing target compounds

[0456] VP_130

[0457]

[0458] CsA (500.0 mg 0.4161 eq) was dissolved in DCE (10 mL) followed by Grubbs'-Hoveyda G2 (18.3 mg 0.029 mmol) in a microwave vial and bubbled with ethylene gas. The vial was irradiated at 70 °C for 30 min (MW). The reaction was passed through an SH resin column to remove the catalyst. Rinse with MeOH and concentrate. Crude (490 mg, 0.412 mmol, 92%). The product was used without further purification (purified by LCMS, C4 acetonitrile: water: 0.1% formic acid). [MH] + 1189.

[0459] VP_136

[0460]

[0461] The bromide (80.3 mg, 0.303 mmol, 5.000 eq) was dissolved in THF*: water = 9: 1 (2 mL), followed by VP130 (72.0 mg, 0.061 mmol, 1.000 eq), cesium carbonate (59.2 mg, 0.182 mmol, 3.000 eq), and Pd (dppf) ClCH2Cl2 (2.5 mg, 0.003 mmol, 0.050 eq) (5 mol%). The mixture was stirred in a microwave at 70 ° C for 4 days. The residue was distributed between DCM (10 mL) and water (10 mL), the organic phase was washed with brine, dried over MgSO4 and concentrated under reduced pressure. Purification using acetonitrile / water 0.1% formic acid gave a C4 column product (25 mg, 0.022 mmol, 30%). [MH] + 1373.

[0462] The resulting methyl ester compound VP_136 can be readily converted to the target compound, including but not limited to, by hydrolysis to the corresponding carboxylic acid, i.e., VP47 (e.g., with LiOH), followed by esterification with an appropriate alcohol (see, e.g., the representative example in Example 8 showing a similar reaction using VP47).

Claims

1. A cyclosporine analogue, which is a compound of formula (I): or a pharmaceutically acceptable salt thereof; in: R1 represents hydrogen, C1–C4 alkyl or C2–C4 alkenyl; R2 stands for R3 represents ethyl or isopropyl; R4 represents a methyl group or an ethyl group; R5 represents –CH2CH(CH3)2, –CH2CH(CH3)CH2CH3, –CH(CH3)CH3, or –CH(CH3)CH2CH3; R7 represents a hydrogen atom or a C 1-20 Alkyl, C 2-20 Alkenyl or C 2-20 an alkynyl moiety which is unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups, and wherein (a) 0, 1, 2 or 3 carbon atoms are selected from C 6-10 Arylene, 5-membered to 10-membered heteroarylene, C 3-7 carbocyclylene and 5- to 10-membered heterocyclylene, and (b) at most half of the –CH2– groups are selected from –O–, –S–, –C(O)– and –N(C 1-6 alkyl)-group, wherein: (i) the arylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted with one or more halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(RN)2 (wherein each RN independently represents a hydrogen atom or a C 1-6 alkyl), –CN, –S(O)2NH2, nitro and sulfonic acid substituents; and (ii) 0, 1 or 2 carbon atoms in the carbocyclylene and heterocyclylene are replaced by -C(O)- groups; and Ring A represents a monocyclic or bicyclic ring system, which is C 6-10 -arylene or 5- to 10-membered heteroarylene, and is unsubstituted or substituted by one or more halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(RN)2 (wherein each RN independently represents a hydrogen atom or a C 1-6 alkyl), –CN, –S(O)2NH2, nitro, and sulfonic acid substituents; And among them – R C –R6–N(R8A)(R8B) is defined as (X) or (Y): (X)： R C represents a group selected from –C(O)O–, –OC(O)–, –C(O)N(RN)–, –N(RN)C(O)–, –S(O)2N(RN)–, –N(RN)S(O)2–, –NRN)–C(O)–N(RN)–, –N(RN)–C(S)–N(RN)–, –C(O)CH2–, –CH2C(O)–, –C(CF3)N(RN)–, –N(RN)C(CF3)–, –C(O)NF–, –NFC(O)–, –C(CN)=N–O–, –O–N=C(CN)–, –N(RN)C(O)O–, –OC(O)N(RN–, a 5-membered to 6-membered heteroarylene group, C 5-6 A moiety consisting of a carbocyclylene group and a 5- to 6-membered heterocyclylene group, wherein each RN independently represents a hydrogen atom or a C 1-6 alkyl, and wherein the phenylene, the heteroarylene, the carbocyclylene and the heterocyclylene are each unsubstituted or substituted by one or more selected from C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(RN)2 (wherein each RN independently represents a hydrogen atom or a C 1-6 alkyl), –CN, –S(O)2NH2, nitro, and sulfonic acid substituents; R6 represents C 1-6 Alkylene, C 2-6 Alkenylene or C 2-6 an alkynylene group, and is unsubstituted or substituted with one or more substituents selected from a halogen atom and a sulfonic acid group; and R8A and R8B are either: (a) together with the nitrogen atom to which it is attached, form a 5- to 10-membered heteroaryl or a 5- to 10-membered heterocyclic group, wherein the heteroaryl and the heterocyclic group are unsubstituted or substituted by one or more halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(RN)2 (wherein each RN independently represents a hydrogen atom or a C 1-6 alkyl), –CN, –S(O)2NH2, nitro and sulfonic acid substituents; or (b) Independently represents C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 an alkynyl group, which is unsubstituted or substituted with one or more substituents selected from a halogen atom, a sulfonic acid group and a hydroxyl group; (Y)： –R C –R6–N(R8A)(R8B) together form a group of formula (VI) in R C2 represents –C(O)–, –S(O)2–, –N(RN)–C(O)–, –N(RN)–C(S)–, –C(CF3)– or –OC(O)–, Ring B contains the The nitrogen atom of R8B2 and the 5- to 10-membered heterocyclylene ring, wherein RN2 and R 62 are each an alkylene group, and R8B2 is C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 The alkynyl group is unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen atom, a sulfonic acid group and a hydroxyl group.

2. The cyclosporine analogue according to claim 1, wherein -R C The moiety –R6–N(R8A)(R8B) is defined according to (X), and wherein R8A and R8B are either: (a) together with the nitrogen atom to which it is attached, form a 5- to 10-membered heteroaryl or a 5- to 10-membered heterocyclic group, wherein the heteroaryl and the heterocyclic group are unsubstituted or substituted by one or more halogen atoms and C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, –(C 1-6 alkyl) n C(O)O(C 1-6 alkyl)(wherein n=0 or 1), –(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 Alkylthio, –N(RN)2 (wherein each RN independently represents a hydrogen atom or a C 1-6 alkyl), –CN, –S(O)2NH2, nitro and sulfonic acid substituents; or (b) Independently represents C 1-6 Alkyl, C 2-6 Alkenyl or C 2-6 The alkynyl group may be unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen atom and a sulfonic acid group.

3. The cyclosporine analogue according to claim 1 or 2, wherein In ring A, the C 6-10 The arylene group or the 5- to 10-membered heteroarylene group is a naphthylene group, a 9- to 10-membered heteroarylene group or a phenylene group.

4. The cyclosporine analogue according to claim 3, wherein the C 6-10 The arylene group or the 5- to 10-membered heteroarylene group is naphthylene.

5. The cyclosporine analog according to claim 3, wherein the C 6-10 The arylene group or the 5- to 10-membered heteroarylene group is a 9- to 10-membered heteroarylene group.

6. The cyclosporin analog according to claim 3 or 5, wherein the 9- to 10-membered heteroarylene group is quinolylene, isoquinolylene or indolinylene.

7. The cyclosporine analog according to claim 3, wherein the C 6-10 The arylene group or the 5- to 10-membered heteroarylene group is a phenylene group.

8. A cyclosporin analogue according to any one of the preceding claims, wherein in ring A, the substituent is selected from C 1-4 Alkyl, C 1-4 Haloalkyl and C 1-4 Alkoxy, preferably C 1-4 alkyl.

9. The cyclosporin analogue according to any one of the preceding claims, wherein Ring A is unsubstituted or substituted with one or two of said substituents.

10. The cyclosporine analog according to claim 8, wherein Ring A is unsubstituted.

11. A cyclosporine analogue according to any one of the preceding claims, wherein -R C The –R6–N(R8A)(R8B) moiety is defined as (X), and wherein R C represents a group consisting of –C(O)O–, –C(O)NH–, –S(O)2NH–, –NH–C(O)–NH–, –NH–C(S)–NH–, –NH–C(O)–O–, and The groups that make up the group.

12. The cyclosporine analog according to claim 11, wherein R C It is –C(O)O–.

13. A cyclosporine analogue according to any one of the preceding claims, wherein -R C –R6–N(R8A)(R8B) is defined as (X), and wherein R6 represents C 2-4 Alkylene or C 2-4 Alkenylene, and preferably wherein: (a) R6 is ethylene, -(CH2)2-; or (b) R6 is -(CH2)4-.

14. A cyclosporine analogue according to any one of the preceding claims, wherein -R C The moiety –R6–N(R8A)(R8B) is defined according to (X), and wherein R8A and R8B are either: (a) together with the nitrogen atom to which it is attached, forms a 5- to 6-membered heteroaryl or a 5- to 6-membered heterocyclic group; or (b) Independently represents C 1-4 Alkyl or C 2-4 Alkenyl.

15. The cyclosporin analogue according to claim 14, wherein R8A and R8B together with the nitrogen atom to which they are attached form an imidazole group, preferably of the formula of imidazolyl.

16. A cyclosporine analogue according to any one of the preceding claims, wherein: R1 represents hydrogen; R2 stands for R3 represents an ethyl group; R4 represents a methyl group; R5 represents –CH2CH(CH3)2; and R7 represents hydrogen.

17. A cyclosporin analogue according to any one of the preceding claims, having at least one and preferably both of (a) and (b): (a) the cyclosporine analog has a lower binding affinity to cyclophilin A (CypA) than cyclosporine A (CsA); (b) Cyclosporine analogs are IFITM3 inhibitors.

18. The cyclosporine analog according to claim 1, which is a compound selected from the following compounds, or a pharmaceutically acceptable salt thereof:

19. A cyclosporine analogue according to any one of claims 1 to 18 for use in increasing the transduction efficiency of an isolated population of mammalian cells by a vector derived from one or more of HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus, preferably wherein the mammalian cells are human cells, and more preferably wherein the population of mammalian cells is selected from human hematopoietic stem cells and / or progenitor cells, induced human hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from human hematopoietic stem cells and / or progenitor cells or induced human hematopoietic stem cells and / or progenitor cells, for example selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, and / or progenitor cells. The invention relates to a method for expressing a vector in an animal model of the present invention, wherein the mammalian cell, human cell, hematopoietic stem cell and / or progenitor cell, induced human hematopoietic stem cell and / or progenitor cell, and / or a cell differentiated from a human hematopoietic stem cell and / or progenitor cell or an induced human hematopoietic stem cell and / or progenitor cell, for example, a cell selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, and / or a cell having one or more vector copies per cell, and optionally a vector-transduced mammalian cell, human cell, hematopoietic stem cell and / or progenitor cell, induced human hematopoietic stem cell and / or progenitor cell, and / or a cell differentiated from a human hematopoietic stem cell and / or progenitor cell or induced human hematopoietic stem cell and / or progenitor cell, for example, a cell selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, has an increased percentage and / or an increased number of vector copies per cell.

20. A method for transducing a population of mammalian cells, preferably wherein the mammalian cells are human cells, and more preferably wherein the population of mammalian cells is selected from human hematopoietic stem cells and / or progenitor cells, induced human hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from human hematopoietic stem cells and / or progenitor cells or induced human hematopoietic stem cells and / or progenitor cells, for example, cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / β T cells, γδ T cells or regulatory T cells, B cells and plasma cells, comprising the following steps: (a) contacting the cell population with a cyclosporine analogue according to any one of claims 1 to 18; as well as (b) transducing the cell population with a vector derived from HIV-1, HIV-2, FIV, BIV, EIAV, CAEV or visna lentivirus; The method optionally has at least one of the following further features (i) to (iv): (i) steps (a) and (b) are performed ex vivo or in vitro; (ii) the percentage of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells transduced by the vector, such as cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / β T cells, γδ T cells or regulatory T cells, B cells and plasma cells, is increased and / or the number of vector copies per cell is increased; (iii) a population of mammalian cells, human cells, hematopoietic stem and / or progenitor cells, induced hematopoietic stem and / or progenitor cells, and / or cells differentiated from hematopoietic stem and / or progenitor cells or induced hematopoietic stem and / or progenitor cells, such as cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / β T cells, γδ T cells or regulatory T cells, B cells and plasma cells, which is or has been obtained from mobilized peripheral blood, bone marrow or umbilical cord blood; and (iv) the method comprises a further step of enriching a population of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells, for example, cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / β T cells, γδ T cells or regulatory T cells, B cells and plasma cells.

21. A gene therapy method comprising the following steps: a) transducing a population of mammalian cells according to the method of claim 20, preferably wherein the mammalian cells are human cells, and more preferably wherein the population of mammalian cells is selected from human hematopoietic stem cells and / or progenitor cells, induced human hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from human hematopoietic stem cells and / or progenitor cells or induced human hematopoietic stem cells and / or progenitor cells, for example, cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / β T cells, γδ T cells or regulatory T cells, B cells and plasma cells; as well as b) administering the transduced cells to a subject; And optionally wherein the transduced cells are administered to the subject as part of an autologous stem cell transplant procedure or an allogeneic stem cell transplant procedure.

22. A population of mammalian cells, human cells, human hematopoietic stem cells and / or progenitor cells, induced human hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from human hematopoietic stem cells and / or progenitor cells or induced human hematopoietic stem cells and / or progenitor cells, prepared according to the method of claim 18, for example, a population of one or more cells selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells.

23. A pharmaceutical composition comprising a population of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or cells differentiated from hematopoietic stem cells and / or progenitor cells or induced hematopoietic stem cells and / or progenitor cells according to claim 22, such as cells selected from common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells.

24. A population of mammalian cells, human cells, hematopoietic stem and / or progenitor cells, induced hematopoietic stem and / or progenitor cells, and / or cells differentiated from hematopoietic stem and / or progenitor cells or induced hematopoietic stem and / or progenitor cells according to claim 22, such as cells selected from one or more of common myeloid progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, common lymphoid progenitor cells, natural killer cells, T cells such as α / βT cells, γδT cells or regulatory T cells, B cells and plasma cells, for use in treatment, optionally wherein the population is administered as part of an autologous stem cell transplantation procedure or an allogeneic stem cell transplantation procedure.

25. The cyclosporine analogue according to any one of claims 1 to 18, for use in mammalian cell, human cell, hematopoietic stem cell and / or progenitor cell, induced hematopoietic stem cell and / or progenitor cell, and / or common myeloid progenitor cell, megakaryocyte, erythroblast, mast cell, myeloblast, basophil, neutrophil, eosinophil, monocyte, common lymphoid progenitor cell, natural killer cell, T cell such as α / β T cell, γδ T cell or regulatory T cell, B cell and plasma cell gene therapy.

26. A cyclosporine analogue according to any one of claims 1 to 18, for use in treating a pathological condition associated with IFITM3 expression, preferably wherein the pathological condition is selected from the group consisting of viral infection and Alzheimer's disease, more preferably wherein the pathological condition is coronavirus, and still more preferably wherein the pathological condition is COVID-19.

27. A method for treating a pathological condition associated with IFITM3 expression in a patient in need thereof, the method comprising administering to the patient an effective amount of a cyclosporine analogue according to any one of claims 1 to 18, preferably wherein the pathological condition is selected from the group consisting of a viral infection and Alzheimer's disease, more preferably wherein the pathological condition is a coronavirus, and even more preferably wherein the pathological condition is COVID-19.

28. The compound of the formula 29. The compound of the formula

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