Cyclosporin analog

Abstract

This application relates to cyclosporine analogs and their use in medical applications.

Description

Technical Field

[0001] The present invention relates to novel cyclosporine analogs and their medical uses, specifically their use as gene therapy and antiviral compounds.

Background Art

[0002] Background of the Invention Hematopoietic stem cell (HSC) gene therapy can now treat hereditary hematological diseases such as immunodeficiency and metabolic disorders that are often fatal otherwise and have limited long-term drug-based treatment options. In gene therapy, it is necessary to isolate HSCs and deliver a functional copy of the disease gene ex vivo. The modified HSCs are returned to the patient to replenish the hematopoietic system for long-term treatment.

[0003] HIV-based vectors are required for HSC gene delivery. A major obstacle is the resistance of HSCs to vector infection. The major protective antiviral protein in HSCs that blocks vector entry and gene delivery is a known antiviral protein called IFITM3 (see, for example, Petrillo et al. Cell Stem Cell 23, 820-832, 2018). For example, as described by Petrillo et al. (supra) and WO2015 / 162594, the naturally occurring cyclosporines CsA and CsH have previously been shown to act as transduction enhancers (TEs) by inhibiting IFITM3 and enhancing vector infection and gene delivery in these cells.

[0004] Unfortunately, CsH is limited in availability, costly, and of low purity. Conversely, CsA has undesirable features that reduce its medicinal efficacy, particularly the 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. Particularly desirable is to provide synthetically accessible, highly potent and selective inhibitors of IFITM3 that enhance HIV vector infection, reduce the required vector dose, and eliminate patient variability.

[0006] Certain cyclosporine analogs are disclosed in WO2021 / 229237. However, in relation to obtaining high IFITM3 inhibition, and in some cases combined with a reduction or elimination of CypA inhibition, further development is needed to tune the properties of the compounds and obtain compounds particularly suitable for use as transduction enhancers in clinical applications.

[0007] On the other hand, effective treatment of viral infections remains a major challenge for healthcare systems worldwide. For example, there are currently no known effective treatments for the various coronaviruses that have emerged recently, such as Coronavirus Disease 2019 ("COVID-19"). Cyclosporine compounds have previously been proposed to be potentially useful for treating such conditions (see, for example, de Wilde et al J Gen Virol. 2011;92(Pt 11): 2542-2548). More recently, however, it has been proposed that IFITM proteins may be viable targets for virus inhibition, as they can promote SARS-Cov-2 infection, for example, by hijacking these proteins that are normally antiviral and causing efficient viral infection. The urgent need for effective antiviral therapies remains, particularly as treatment for COVID-19 is a top priority at present. SUMMARY OF THE INVENTION

[0008] Summary of the Invention A specific class of cyclosporine analogs (more specifically, analogs of CsA) has recently been found to be effective as transduction enhancers for HSC gene delivery. Also, cyclosporine analogs have been found to be effective as antiviral compounds against viruses in which the IFITM protein promotes infection (including but not limited to COVID-19).

[0009] Specifically, the present invention provides a cyclosporine analog of formula (I):

[0010]

Chemical formula

[0011] or a pharmaceutically acceptable salt thereof (wherein: R1 represents hydrogen, C1-C4 alkyl or C2-C4 alkenyl; R2 is

[0012]

Chemical formula

[0013] represents; R3 represents ethyl or isopropyl; R4 represents methyl or ethyl; R5 represents -CH2CH(CH3)2, -CH2CH(CH3)CH2CH3, -CH(CH3)CH3, or -CH(CH3)CH2CH3; R7 is a hydrogen atom or a C 1-20 alkyl group, a C 2-20 alkenyl group or a C 2-20 alkynyl group, which part is unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups, where (a) 0, 1, 2 or 3 carbon atoms are C 6-10 arylene, 5- to 10-membered heteroarylene, C 3-7Replaced by a group selected from a carbocyclylene and a 5- to 10-membered heterocyclylene group, and (b) Up to half of the -CH2- groups are replaced by a group selected from -O-, -S-, -C(O)-, and -N(C 1-6 alkyl)- groups (wherein: (i) The arylene, heteroarylene, carbocyclylene, and heterocyclylene groups are unsubstituted or substituted with one or more substituents selected from a halogen atom 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) (where n = 0 or 1), -(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 alkylthiol, -N(R N )2 (wherein each R N independently represents a hydrogen atom or a C 1-6 alkyl group), -CN, -S(O)2NH2, nitro, and sulfonic acid groups; and (ii) 0, 1, or 2 carbon atoms in the carbocyclylene and heterocyclylene groups are replaced by -C(O)- groups; and Ring A represents a monocyclic ring or a bicyclic ring system, a C 6-10 arylene group or a 5- to 10-membered heteroarylene group, unsubstituted or substituted with a halogen atom 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) (where n = 0 or 1), -(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 alkylthiol, -N(R N )2 (wherein each R Nis independently a hydrogen atom or C 1-6 alkyl group), -CN, -S(O)2NH2, nitro and sulfonic acid group, and is substituted with one or more substituents selected from the group consisting of; and moiety -R C -R6-N(R 8A )(R 8B ) is defined according to either (X) or (Y): (X): R C is -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-, -N(R 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 ), phenylene, 5- to 6-membered heteroarylene, C 5-6 carbocyclylene and 5- to 6-membered heterocyclylene, and represents a moiety selected from the group consisting of (wherein each R N is independently a hydrogen atom or C 1-6 alkyl group, and the phenylene, the heteroarylene, the carbocyclylene and the heterocyclylene are each unsubstituted or a halogen atom 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) (where n = 0 or 1), -(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6An alkylthiol, -N(R N )2 (wherein each R N is independently a hydrogen atom or a C 1-6 alkyl group), -CN, -S(O)2NH2, nitro and sulfonic acid group; R6 is a C 1-6 alkylene group, a C 2-6 alkenylene group or a C 2-6 alkynylene group, unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups; and R 8A and R 8B are: (a) together with the nitrogen atom to which they are attached, form a 5- to 10-membered heteroaryl group or a 5- to 10-membered heterocyclyl group, and the heteroaryl group and the heterocyclyl group are unsubstituted or substituted with one or more substituents 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) (where n = 0 or 1), -(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 alkylthiol, -N(R N )2 (wherein each R N is independently a hydrogen atom or a C 1-6 alkyl group), -CN, -S(O)2NH2, nitro and sulfonic acid group; or (b) independently a C 1-6 alkyl group, a C 2-6 alkenyl group or a C 2-6 alkynyl group, unsubstituted or substituted with one or more substituents selected from halogen atoms, sulfonic acid groups and hydroxy groups; and (Y): -R C -R6-N(R 8A )(R 8Bcombine together to form a group of formula (VI)

[0014]

Chemical formula

[0015] where R C2 represents -C(O)-, -S(O)2-, -N(R N )-C(O)-, -N(R N )-C(S)-, -C(CF3)- or -OC(O)-, ring B is a 5- to 10-membered heterocyclylene ring containing both a nitrogen atom bonded to R C2 and a nitrogen atom bonded to R 8B2 , where R N2 and R 62 are each an alkylene group, and R 8B2 is a C 1-6 alkyl group, a C 2-6 alkenyl group or a C 2-6 alkynyl group, unsubstituted or substituted with one or more substituents selected from halogen atoms, sulfonic acid groups and hydroxy groups).

[0016] The present invention further provides the use of the cyclosporine analogs of the present invention for, among other things, enhancing the efficiency of transduction of isolated mammalian cell populations, preferably where the mammalian cells are human cells. More preferably, by vectors derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus, 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, such as myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, one or more cells selected therefrom.

[0017] The present invention also provides a method for transducing a mammalian cell population, preferably 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, such as myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, and comprises the following steps: a) contacting the cyclosporine analog of the present invention with the cell population; and b) transducing the cell population with a vector derived from HIV-1, HIV-2, FIV, BIV, EIAV, CAEV, or visna lentivirus.

[0018] The present invention also provides a method for gene therapy comprising the following steps: a) transducing a mammalian cell population according to the method of transduction of the present invention, preferably 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, such as myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, and is one or more cells selected therefrom; and b) administering the transduced cells to a subject.

[0019] The present invention provides a mammalian cell population, preferably the mammalian cells are human cells. 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, such as myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, and further provides a pharmaceutical composition comprising one or more cells selected from such mammalian cell populations, 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 present invention further provides such mammalian cell populations, 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 therapy, such as myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, one or more cells selected therefrom.

[0020] The present invention further provides a cyclosporine analog 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, for use in gene therapy.

[0021] The present invention further provides a cyclosporine analog of the present invention for use in the treatment of pathological conditions (such as viral infections) associated with IFITM3 expression. Further, the present invention provides a method for treating a pathological condition (e.g., viral infection) associated with IFITM3 expression in a patient in need thereof, the method comprising administering to the patient an effective amount of a cyclosporine analog of the present invention.

[0022] The present invention further provides a compound of the formula

[0023]

Chemical formula

[0024]

[0025] The present invention further provides a compound of the formula

[0026]

Chemical formula

[0027]

Brief Description of the Drawings

[0028]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

[0029] Detailed Description of the Invention Definitions As used herein, the term "alkyl" includes both saturated straight - chain alkyl groups and branched alkyl groups (i.e., as a monovalent group derived by removing one hydrogen atom from an alkane). Preferably, unless otherwise specified, the alkyl group is a C 1-20 alkyl group, more preferably a C 1-15、 even more preferably a C 1-12 alkyl group, even more preferably a C 1-6 alkyl group, and most preferably a C 1-4 alkyl group. Unless otherwise specified, 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 an alkyl group, in other words, as an alkane from which two hydrogen atoms have been removed).

[0030] As used herein, the term "alkenyl" refers to a group containing one or more carbon-carbon double bonds, which may be branched or unbranched (i.e., as a monovalent group derived by removing one hydrogen atom from an alkene). Preferably, unless otherwise specified, the alkenyl group is C 2-20 an alkenyl group, more preferably C 2-15 an alkenyl group, even more preferably C 2-12 an alkenyl group, or preferably C 2-6 an alkenyl group, most preferably C 2-4 an alkenyl group. The term "alkenylene" should be construed accordingly (i.e., as the divalent counterpart of an alkenyl group, in other words, as an alkene from which two hydrogen atoms have been removed).

[0031] 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., as a monovalent group derived by removing one hydrogen atom from an alkyne). Preferably, unless otherwise specified, the alkynyl group is C 2-20 an alkynyl group, more preferably C 2-15 an alkynyl group, even more preferably C 2-12 an alkynyl group, or preferably C 2-6 an alkynyl group, most preferably C 2-4 an alkynyl group. The term "alkynylene" should be construed accordingly (i.e., as the divalent counterpart of an alkynyl group, in other words, as an alkyne from which two hydrogen atoms have been removed).

[0032] Unless otherwise specified, alkyl, alkenyl or alkynyl groups are typically unsubstituted. However, if such groups (or their divalent counterparts) are shown to be unsubstituted or substituted (again, unless otherwise specified), one or more hydrogen atoms may be replaced by halogen atoms or sulfonic acid groups. Preferably, the substituted alkyl, alkenyl or alkynyl group has from 1 to 10 substituents, more preferably from 1 to 5 substituents, even more preferably 1, 2 or 3 substituents and most preferably 1 or 2 substituents, for example 1 substituent. Preferably, the substituted alkyl, alkenyl or alkynyl group bears no more than 2 sulfonic acid substituents. Halogen atoms are preferred substituents. However, preferably the alkyl, alkenyl or alkynyl group is unsubstituted. A haloalkyl group means an alkyl group substituted with one or more halogen atoms.

[0033] If an alkyl, alkenyl or alkynyl group (or its divalent counterpart) is shown to be unsubstituted or substituted with one or more substituents selected from halogen atoms, sulfonic acid groups and hydroxy groups (e.g., for each of R 8A R 8B and R 8B2 ), one or more hydrogen atoms may be replaced by halogen atoms, sulfonic acid groups and / or hydroxy groups. Preferably, such a substituted alkyl, alkenyl or alkynyl group has from 1 to 10 substituents, more preferably from 1 to 5 substituents, even more preferably 1, 2 or 3 substituents and most preferably 1 or 2 substituents, for example 1 substituent. Preferably, such a substituted alkyl, alkenyl or alkynyl group bears no more than 2 sulfonic acid substituents. Halogen atoms and hydroxy groups are preferred substituents. Halogen atoms are preferred substituents. Hydroxy groups are also preferred substituents. However, preferably the alkyl, alkenyl or alkynyl group is unsubstituted.

[0034] A haloalkyl group means an alkyl group substituted with one or more halogen atoms.

[0035] As used herein, a halogen atom is typically an F, Cl, Br, or I atom.

[0036] As used herein, C 6-10 An aryl group is a monocyclic or polycyclic 6- to 10-membered aromatic hydrocarbon ring system having from 6 to 10 carbon atoms. Unless otherwise specified, phenyl and naphthyl are preferred. The term "arylene" should be construed accordingly (i.e., as the divalent counterpart of an aryl group), and thus phenylene and naphthylene are preferred arylenes unless otherwise specified.

[0037] 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- or 6-membered ring or a 9- or 10-membered ring system containing at least one heteroatom selected from O, S, and N, e.g., 1, 2, 3, or 4 heteroatoms. When the ring or ring system contains 4 heteroatoms these are preferably all nitrogen atoms. The term "heteroarylene" should be construed accordingly.

[0038] Examples of monocyclic heteroaryl groups include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, and tetrazolyl groups.

[0039] Examples of polycyclic heteroaryl groups include quinolinyl, isoquinolinyl, indolinyl, benzothienyl, benzofuryl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, benzotriazolyl, indolyl, isoindolyl, and indazolyl groups.

[0040] As used herein, a 5- to 10-membered heterocyclyl group is a non-aromatic, saturated or unsaturated, monocyclic or polycyclic C 5-10 carbocyclic ring system in which one or more, for example 1, 2, 3 or 4, carbon atoms are replaced by moieties selected from N, O, S, S(O) and S(O)2. Preferably, unless otherwise specified, the 5- to 10-membered heterocyclyl group is a 5- to 6-membered ring. The term "heterocyclyene" should be construed accordingly.

[0041] Examples of heterocyclyl groups include azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl, pyrazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, methylenedioxyphenyl, ethylenedioxyphenyl, thiomorpholinyl, S-oxo-thiomorpholinyl, S,S-dioxo-thiomorpholinyl, morpholinyl, 1,3-dioxolanyl, 1,4-dioxolanyl, trioxolanyl, trithianyl, imidazolinyl, pyranyl, pyrazolyl, thioxolanyl, thioxothiazolidinyl, 1H-pyrazol-5-(4H)-onyl, 1,3,4-thiadiazol-2(3H)-thionyl, oxopyrrolidinyl, oxothiazolidinyl, oxopyrazolidinyl, succinimide and maleimide groups and moieties.

[0042] To avoid doubt, the above definitions of heteroaryl and heterocyclyl groups refer to the "N" moieties that can be present within the ring, but as will be apparent to the skilled chemist, the N atom is protonated (or bears a substituent as defined below) when bonded to each of the adjacent ring atoms via a single bond.

[0043] As used herein, C 3-7A carbocyclyl group is a non-aromatic, saturated or unsaturated hydrocarbon ring having from 3 to 7 carbon atoms. Preferably, unless otherwise specified, it is a saturated or mono-unsaturated hydrocarbon ring (i.e., a cycloalkyl moiety or a cycloalkenyl moiety) having from 3 to 7 carbon atoms, more preferably from 5 to 6 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and their mono-unsaturated variants. Unless otherwise specified, particularly preferred carbocyclic groups are cyclopentyl and cyclohexyl. The term "carbocyclylene" should be interpreted accordingly.

[0044] When so specified, 0, 1 or 2 carbon atoms of the carbocyclyl group or heterocyclyl group may be replaced by a -C(O)- group. As used herein, the "carbon atoms" to be replaced are understood to include the hydrogen atoms to which they are attached. When 1 or 2 carbon atoms are replaced, preferably 2 such carbon atoms are replaced. Preferred such carbocyclyl groups include the benzoquinone group, and preferred such heterocyclyl groups include the succinimide and maleimide groups.

[0045] Unless otherwise specified, aryl, heteroaryl, carbocyclyl or heterocyclyl groups are typically unsubstituted. However, when it is indicated that such a group is unsubstituted or substituted, one or more hydrogen atoms are a halogen atom or C 1-6 alkyl, C 1-6 haloalkyl, C 1-6 alkoxy, -(C 1-6 alkyl) n C(O)O(C 1-6 alkyl) (where n = 0 or 1), -(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 alkylthiol, -N(R N )2 (wherein each R Nis independently a hydrogen atom or a C 1-6 alkyl group), -CN, -S(O)2NH2, nitro or sulfonic acid group, and may be substituted. Preferably, the substituted aryl, heteroaryl, carbocyclic or heterocyclic group has from 1 to 4 substituents, more preferably from 1 to 2 substituents and most preferably 1 substituent. Preferably, the substituted aryl, heteroaryl, carbocyclic or heterocyclic group bears no more than 2 nitro substituents and no more than 2 sulfonic acid substituents.

[0046] As used herein, an alkoxy group is alkyl bonded to an oxygen atom (e.g., C 1-6 alkyl or C 1-4 alkyl) group.

[0047] As used herein, an alkylthiol group is alkyl bonded to a sulfur atom (e.g., C 1-6 alkyl or C 1-4 alkyl) group.

[0048] In some cases, the compounds of the invention can be provided in the form of a pharmaceutically acceptable salt. Substantially any salt can be used, provided it is pharmaceutically acceptable. Those skilled in the art of preparing compounds for medical use will be familiar with appropriate such salt compounds. For example, the compounds of the invention may be in the form of a salt with a pharmaceutically acceptable base. Pharmaceutically acceptable bases include, but are not limited to, hydroxides of alkali metals (e.g., sodium or potassium) and alkaline earth metals (e.g., calcium or magnesium), and organic bases such as alkylamines, aralkylamines or heterocyclic amines.

[0049] Cyclosporin analogs Non-limiting preferred embodiments of the cyclosporin analogs of the invention are shown below.

[0050] R1 preferably represents hydrogen.

[0051] R2 preferably represents

[0052] [Chemical formula]

[0053] . R3 preferably represents ethyl.

[0054] R4 preferably represents methyl.

[0055] R5 preferably represents -CH2CH(CH3)2 。

[0056] In R7, the part that is hydrogen or a C 1-20 alkyl group, a C 2-20 alkenyl group or a C 2-20 alkynyl group is preferably hydrogen or a C 1-15 alkyl group, a C 2-15 alkenyl group or a C 2-15 alkynyl group, more preferably hydrogen or a C 1-12 alkyl group, a C 2-12 alkenyl group or a C 2-12 alkynyl group, even more preferably hydrogen or a C 1-10 alkyl group or a C 2-10 alkenyl group, and most preferably hydrogen. In these preferred embodiments, the substituents, as well as the preferred options for (a) and (b), are as follows.

[0057] Regarding the substituents, preferably, the part is unsubstituted or substituted with one or more substituents selected from halogen atoms, and more preferably, the part is unsubstituted. The maximum number of substituents is preferably 5, and more preferably 3.

[0058] (a) Preferably, 0, 1, or 2 carbon atoms are replaced, more preferably 0 or 1 carbon atom is replaced. Such a carbon atom is preferably replaced by a group selected from phenylene, 5- to 6-membered heteroarylene, C 5-6 carbocyclylene, and 5- to 6-membered heterocyclylene groups, and more preferably by phenylene.

[0059] (b) Preferably, 0 to 4 -CH2- groups, more preferably 0 to 3 groups, and most preferably 0, 1, or 2 groups are replaced. For any replaced -CH2- group, it is preferably replaced by a group selected from -O-, -S-, and -C(O)-, and more preferably by a group selected from -O- and -C(O)-. To avoid doubt, when chemically meaningful, replacing adjacent -CH2- is allowed, for example, replacing -CH2-CH2- with -C(O)-O-, -O-C(O)-, etc. is allowed. Similarly, replacing the -CH2- of a terminal methyl group (i.e., CH3 or -CH2-H), for example, replacing -CH3 with -OH is also allowed. Preferably, no more than 2 adjacent -CH2- groups are replaced (e.g., typically, the replacement does not involve the replacement of 3 or more adjacent / adjacent -CH2- groups).

[0060] Preferably, all of the arylene, heteroarylene, carbocyclylene, and heterocyclylene groups in (a) are unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups, and more preferably are unsubstituted. Preferably, 0 carbon atoms in the carbocyclylene and heterocyclylene groups are replaced by -C(O)- groups.

[0061] Preferably, R7 is hydrogen or C 1-15 alkyl group, C 2-15 alkenyl group, or C 2-15 alkynyl group, and that moiety is unsubstituted or substituted with one or more halogen atoms, where (a) 0, 1 or 2 carbon atoms are C 6-10 arylene, 5- to 10-membered heteroarylene, C 3-7 substituted by a group selected from carbocyclylene and 5- to 10-membered heterocyclylene groups, and (b) 0 to 4 -CH2- groups are replaced by a group selected from -O-, -S-, -C(O)- and -N(C 1-6 alkyl)- groups, wherein: (i) said arylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or more substituents selected from halogen atoms and sulfonic acid groups; and (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and heterocyclylene groups are replaced by -C(O)- groups.

[0062] More preferably, R7 is hydrogen or C 1-12 alkyl group, C 2-12 alkenyl group or C 2-12 alkynyl group, and the moiety is unsubstituted or substituted by one or more halogen atoms, where (a) 0, 1 or 2 carbon atoms are replaced by a group selected from phenylene, 5- to 6-membered heteroarylene, C 5-6 carbocyclylene and 5- to 6-membered heterocyclylene groups, and (b) 0 to 3 -CH2- groups are replaced by a group selected from -O-, -S- and -C(O)-; said phenylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or more halogen atoms.

[0063] Even more preferably, R7 is hydrogen or C 1-10 alkyl group or C 2-10 and the moiety, where (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.

[0064] R7 most preferably represents hydrogen.

[0065] In a particularly preferred embodiment, R1 represents hydrogen, R2 represents

[0066]

Chem.

[0067] represents, R3 represents ethyl, R4 represents methyl, R5 represents -CH2CH(CH3)2, and R7 represents hydrogen. In this embodiment, the structure of the resulting cyclosporine analog closely corresponds to the structure of cyclosporine A (“CsA”), but the moiety -[ring A]-R C -R6-N(R 8A )(R 8B ) is understood to be replaced by a methyl group, -CH3, present at the same position in CsA.

[0068] The stereochemistry of the ethenyl group that attaches the cyclic cyclosporine A core to the R1 group and ring A can be either E or Z. Thus, chemical formula (I) encompasses chemical formulas (Ia) and (Ib):

[0069]

Chem.

[0070]

Chem.

[0071] both.

[0072] The moiety of formula (IIa) is presently preferred, most particularly when R1 represents hydrogen and R2 represents

[0073]

Chem.

[0074] represents, R3 represents ethyl, R4 represents methyl, R5 represents -CH2CH(CH3)2, and R7 preferably represents hydrogen.

[0075] To avoid doubt, and regardless of the stereochemistry of the compound (e.g., the above (IIa) or (IIb)), herein for any divalent moiety of ring A, R C and R6, when a specific definition is provided using the notation of the general form “-[definition]-”, the left - hand part of the definition is the one closest to the central cyclosporine ring, and the right - hand part of the definition is the one closest to the terminal -N(R 8A )(R 8B ) group. For example, when the definition of R C is “-a - b - c -”, the group “a” is attached to ring A and the group “c” is attached to R6.

[0076] In the cyclosporine analogs of the present disclosure, ring A replaces the methyl group present in cyclosporine A (“CsA”) and instead has an ethenyl group extending from the cyclic core of CsA linked to the side chain of the structure -R C -R6 - N(R 8A )(R 8B ).

[0077] To avoid doubt, in formulas (I), (Ia) and (Ib) it is shown using a schematic circle and is identified in the specification using the singular language “ring” for brevity, but ring A represents either a monocyclic ring or a bicyclic ring system. Generally, a bicyclic ring system is any of a fused ring, a bridged ring and a spirocyclic ring, but most typically, when ring A is a bicyclic ring system, it is a fused bicyclic ring (i.e., it contains a first ring and a second ring that share two adjacent ring atoms).

[0078] In a first and often more preferred embodiment, ring A has a ring system of the general formula (IIa), (IIb) or (IIc)

[0079]

Chemical formula

[0080] (B is a monocyclic, 5- to 6-membered ring (including two carbon atoms of the fused benzene ring)) and is an unsubstituted or substituted (condensed) bicyclic ring system. The ring atoms of B may be only carbon atoms, or may contain one or more heteroatoms selected from N, S, and O (preferably, there are up to two heteroatoms, and preferably, the heteroatom is an N atom).

[0081] Exemplary systems of general formulas (IIa), (IIb), and (IIc) include, but are not limited to, (a) a naphthylene group and (b) a 9- to 10-membered heteroarylene group such as a quinolinylene group, an isoquinolinylene group, or an indolinylene group. Particularly preferred is the naphthylene group, for example, of formula (IIIa), (IIIb), or (IIIc):

[0082] [Chemical formula]

[0083] and most preferably of formula (IIIa).

[0084] Specific examples of the quinolinylene and indolinylene groups are of formula (IVa), (IV), and (IVc)

[0085] [Chemical formula]

[0086] as may be mentioned.

[0087] To avoid doubt, in all of formulas (IIa), (IIb), (IIc), (IIIa), (IIIb), (IIIc), (IVa), (IVb), and (IVc), the connections shown on the right are R CThis is for. Also, to avoid doubts, in all of formulas (IIa), (IIb), (IIc), (IIIa), (IIIb), (IIIc), (IVa), (IVb) and (IVc), the ring system is unsubstituted or substituted (i.e., a halogen atom 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) (where n = 0 or 1), -(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 alkylthiol, -N(R N )2 (where each R N independently represents a hydrogen atom or a C 1-6 alkyl group), -CN, -S(O)2NH2, nitro and sulfonic acid groups). In these ring systems, preferred substituents are C 1-4 alkyl, C 1-4 haloalkyl and C 1-4 alkoxy groups, more preferably a C 1-4 alkyl group. Preferably, these ring systems are unsubstituted or substituted with one or two substituents, for example, they are unsubstituted.

[0088] In a second aspect, ring A is an unsubstituted or substituted monocyclic ring, and the monocyclic ring is typically a phenylene ring or a 5- to 6-membered heteroarylene group. In this aspect, the monocyclic ring is preferably a phenylene ring, particularly preferably of formula (V)

[0089]

Chemical formula

[0090] is.

[0091] To avoid doubt, such a phenylene ring or 5- to 6-membered heteroarylene group may be unsubstituted or substituted (i.e., by said halogen atom 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) (where n = 0 or 1), -(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 alkylthiol, -N(R N )2 (wherein each R N is independently a hydrogen atom or C 1-6 alkyl group), -CN, -S(O)2NH2, nitro and sulfonic acid group).

[0092] In this second embodiment, ring A is most preferably of formula (Va)

[0093]

Chemical formula

[0094] (wherein R V1 , R V2 , R V3 and R V4 are independently hydrogen and a halogen atom 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) (where n = 0 or 1), -(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 alkylthiol, -N(R N )2 (wherein each R N is independently a hydrogen atom or C1-6 a phenylene group selected from an alkyl group), -CN, -S(O)2NH2, nitro and sulfonic acid groups).

[0095] Preferably, in formula (Va), R V1 , R V2 , R V3 and R V4 at least two of are hydrogen, more preferably at least R V3 and R V4 are hydrogen. Preferably, when R V1 , R V2 , R V3 and R V4 at least one of is other than hydrogen, the other hydrogen is (at least) R V1 . Preferably, when two of R V1 , R V2 , R V3 and R V4 are other than hydrogen, the other hydrogen is R V1 and R V2 . Particularly preferred non-hydrogen R V1 , R V2 , R V3 and R V4 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 groups (e.g., methyl, ethyl, propyl, methoxy, ethoxy, propoxy (-OiPr, etc.), and trifluoromethyl), most preferably C 1-4 alkyl groups (e.g., methyl, ethyl and propyl).

[0096] Generally (i.e., referring to both the first and second aspects described herein), in ring A, the C 6-10 arylene group or 5- to 10-membered heteroarylene group is preferably a naphthylene group, a 9- to 10-membered heteroarylene group or a phenylene group. Further, in ring A, the C 6-10The arylene group or 5- to 10-membered heteroarylene group is particularly preferably a naphthylene group.

[0097] Also generally, when the ring A is substituted, the preferred substituent of the ring A is selected from C 1-4 alkyl, C 1-4 haloalkyl and C 1-4 alkoxy groups, and most preferably C 1-4 alkyl group. Preferably, the ring A is unsubstituted or substituted with 1 or 2 substituents.

[0098] -R C -R6-N(R 8A )(R 8B ) is defined according to (X) compounds:

[0099] R C is a linking group connecting the ring A to the R6 group.

[0100] R C In, any example of R N is preferably a hydrogen atom or a C 1-4 alkyl group, more preferably a hydrogen atom or a methyl group, and most preferably a hydrogen atom.

[0101] R C is preferably selected from the group consisting of -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 5- to 6-membered heteroarylene.

[0102] R CWhen it is a 5- or 6-membered heteroarylene, such preferred groups are triazolylene (i.e., a divalent group obtained by removing two hydrogen atoms from triazole), oxazolylene (i.e., a divalent group obtained by removing two hydrogen atoms from oxazole), isoxazolylene (i.e., a divalent group obtained by removing two hydrogen atoms from isoxazole), and oxadiazolylene (i.e., a divalent group obtained by removing two hydrogen atoms from oxadiazole). Examples of preferred triazolylene are

[0103]

Chem.

[0104] etc., 1,2,3-triazolylene, preferably the connection shown on the left is to ring A, the connection on the right is to R6, and

[0105]

Chem.

[0106] etc., 1,2,4-triazolylene, preferably the connection shown on the left is to ring A, the connection on the right is to R6. 。 Examples of preferred oxazolylene are

[0107]

Chem.

[0108] and preferably the connection shown on the left is to ring A, the connection on the right is to R6. Examples of preferred oxadiazolylene are

[0109]

Chem.

[0110] etc., 1,2,4-oxadiazolylene, preferably the connection shown on the left is to ring A, the connection on the right is to R6.

[0111] R C is more preferably -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 triazolylene (especially 1,2,3-triazolylene as shown above), and represents a group selected from the group consisting of.

[0112] Therefore, more preferred R C groups are -C(O)O-, -C(O)NH-, -S(O)2NH-, -NH-C(O)-NH-, -NH-C(S)-NH-, -NH-C(O)-O- and

[0113]

Chemical formula

[0114] are.

[0115] Most preferred R C is -C(O)O-.

[0116] R6 is R C linked to the moiety -N(R 8A )(R 8B ) by a linker group.

[0117] R6 is preferably either unsubstituted or substituted with one or more (e.g., up to 4) halogen atoms. Most preferably, R6 is unsubstituted.

[0118] Preferably, R6 is a C 2-4 alkylene group or a C 2-4represents an alkenylene group, more preferably a C 2-4 alkylene group, even more preferably -(CH2)2- or -(CH2)3- or -(CH2)4-.

[0119] Most preferably, R6 is ethylene, i.e., -(CH2)2-. Another particularly preferred R6 is -(CH2)4-.

[0120] R 8A and R 8B groups are bonded to a nitrogen atom and together form either a heterocyclic group or a tertiary amine group containing this nitrogen atom.

[0121] In a first aspect of the invention, R 8A and R 8B together with the nitrogen atom to which they are attached form a 5- to 10-membered heteroaryl group or a 5- to 10-membered heterocyclyl group, and the heteroaryl group and the heterocyclyl group are unsubstituted or substituted with one or more substituents selected from a halogen atom 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) (where n = 0 or 1), -(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 alkylthiol, -N(R N )2 (wherein each R N independently represents a hydrogen atom or a C 1-6 alkyl group), -CN, -S(O)2NH2, nitro and sulfonic acid groups. Preferably, the heteroaryl group and the heterocyclyl group are unsubstituted. However, in certain embodiments, the heteroaryl group and the heterocyclyl group may desirably contain one or more (e.g., one) amino substituents (e.g., -NH2) to achieve, for example, a physiological pKa (e.g., between 7.5 and 10).

[0122] In a second embodiment of the present invention, R 8A and R 8B each independently represent a C 1-6 alkyl group, a C 2-6 alkenyl group or a C 2-6 alkynyl group, which is unsubstituted or substituted with one or more substituents selected from halogen atoms, sulfonic acid groups and hydroxy groups. For example, in this second embodiment of the present invention, R 8A and R 8B each independently represent a C 1-6 alkyl group, a C 2-6 alkenyl group or a C 2-6 alkynyl group, which is unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups. Preferably, the C 1-6 alkyl group, the C 2-6 alkenyl group and the C 2-6 alkynyl group are unsubstituted.

[0123] In the first embodiment, preferably, R 8A and R 8B together with the nitrogen atom to which they are attached form a 5- or 6-membered heteroaryl group or a 5- or 6-membered heterocyclyl group.

[0124] In the first embodiment, more preferably, R 8A and R 8B together with the nitrogen atom to which they are attached form an imidazolyl group, a pyrazolyl group, a morpholinyl group, a piperidinyl group or a piperazinyl group.

[0125] In the first embodiment, most preferably, R 8A and R 8B together with the nitrogen atom to which they are attached form an imidazolyl group (i.e., the group of the formula

[0126]

Chemical formula

[0127] ).

[0128] In the second embodiment, preferably R 8A and R 8B independently represent a C 1-4 alkyl group or a C 2-4 alkenyl group. In the second embodiment, more preferably R 8A and R 8B independently represent a C 1-4 alkyl group. In the second embodiment, most preferably R 8A and R 8B independently represent a methyl group or an ethyl group.

[0129] Thus, in the present invention, preferably R 8A and R 8B are: (a) together with the nitrogen atom to which they are attached, form a 5- to 6-membered heteroaryl group or a 5- to 6-membered heterocyclyl group; or (b) independently represent a C 1-4 alkyl group or a C 2-4 alkenyl group. More preferably, R 8A and R 8B are: (a) together with the nitrogen atom to which they are attached, form an imidazolyl group, a pyrazolyl group, a morpholinyl group, a piperidinyl group or a piperazinyl group; or (b) independently represent a C 1-4 alkyl group. Most preferably, R 8A and R 8B are: (a) together with the nitrogen atom to which they are attached, form an imidazolyl group; or (b) independently represent a methyl group or an ethyl group, and optionally R 8A and R 8B together with the nitrogen atom to which they are attached, form an imidazolyl group.

[0130] Exemplary cyclosporine analogs of the present disclosure include those of formula (Ia), where R1 represents hydrogen and R2 is

[0131]

Chemical formula

[0132] 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 R 8A and R 8B together with the nitrogen atom to which they are attached form an imidazolyl group. Further, exemplary cyclosporine analogs of the present disclosure are those in which R6 is -(CH2)3- or -(CH2)4-, particularly -(CH2)4- rather than -(CH2)3-. In each of these exemplary cyclosporine analogs of the present disclosure, preferred ring A groups include C 6-10 an arylene group or a 5- to 10-membered heteroarylene group, a naphthylene group, a 9- to 10-membered heteroarylene group, or a phenylene group. Further, in these exemplary cyclosporine analogs of the present disclosure, particularly preferred is when in ring A the C 6-10 arylene group or 5- to 10-membered heteroarylene group is a naphthylene group. In these exemplary cyclosporine analogs of the present disclosure, moreover, preferred substituents of ring A, when ring A is substituted, are selected from C 1-4 alkyl, C 1-4 , haloalkyl, and C 1-4 alkoxy groups, most preferably selected from C 1-4 alkyl groups. Preferably ring A is unsubstituted or substituted with one or two substituents (such as an unsubstituted naphthylene group (e.g., of formula (IIIa))). Thus, one specific exemplary compound is "BG147" and its formula is shown in the Examples section. Another specific exemplary compound is "JW3-158" and its formula is shown in the Examples section. Further exemplary compounds are "BG150", "BG181", "BG185", "BG186", "BG190", and "BG149" whose formulas are again shown in the Examples section, and further exemplary compounds are "VP72", "VP73", "VP74", "VP50", "VP53", and "VP76" whose formulas are again shown in the Examples section.

[0133] -R C -R6-N(R 8A )(R 8B ) is defined according to (Y): a compound

[0134] The present disclosure also includes a compound in which -R C -R6-N(R 8A )(R 8B ) is defined according to (Y), and -R C -R6-N(R 8A )(R 8B ) together forms a group of formula (VI).

[0135]

Chemical formula

[0136] In formula (VI), R C2 represents -C(O)-, -S(O)2-, -N(R N )(-C(O)-, -N(R N )(-C(S)-, -C(CF3)- or -OC(O)-, preferably represents -C(O)-.

[0137] In formula (VI), ring B is a 5- to 10-membered heterocyclylene ring containing both a nitrogen atom bonded to R C2 and a nitrogen atom bonded to R 8B2 , and R N2 and R 62 are each an alkylene group. Preferably, ring B is a 5- to 7-membered heterocyclylene ring containing both a nitrogen atom bonded to R C2 and a nitrogen atom bonded to R 8B2 , and R N2 and R 62 are each an alkylene group. Particularly preferably, ring B is a 6-membered heterocyclylene ring containing both a nitrogen atom bonded to R C2 and a nitrogen atom bonded to R 8B2 , and R N2 and R 62They are each an alkylene group. Most preferably, ring B is R C2 a heterocyclylene ring containing both the nitrogen atom bonded to R 8B2 and the nitrogen atom bonded to R N2 and R 62 are each a C2 alkylene group (i.e., ring B is a 1,4-piperazinylene group).

[0138] In formula (VI), R 8B2 is a C 1-6 alkyl group, a C 2-6 alkenyl group or a C 2-6 alkynyl group, unsubstituted or substituted with one or more substituents selected from halogen atoms, sulfonic acid groups and hydroxy groups. Preferably, R 8B2 is a C 1-4 alkyl group or a C 2-4 alkenyl group (unsubstituted or substituted). More preferably, R 8B2 is an unsubstituted or substituted C 1-4 alkyl group. Most preferably, R 8B2 is an unsubstituted or substituted methyl group or ethyl group. Preferably, R 8B2 is unsubstituted or substituted with one or more substituents selected from halogen atoms and hydroxy groups. Most preferably, R 8B2 is unsubstituted or substituted with a hydroxy group. Particularly preferably, R 8B2 is hydroxyethyl (-CH2CH2OH).

[0139] A specific exemplary such compound is "VP51", the formula of which is shown in the Examples section.

[0140] Further disclosure regarding the cyclosporine analogs

[0141] As disclosed herein, in the cyclosporine analogs of the present invention, the moiety -R C -R6-N(R 8A )(R 8B ) is defined according to either (X) or (Y). However, preferably, the moiety -R C-R6-N(R 8A )(R 8B ) is defined according to (X). Further, in this preferred embodiment, the moiety -R C -R6-N(R 8A )(R 8B ) is preferably defined according to (X), and R 8A and R 8B are: (a) together with the nitrogen atom to which they are attached, form a 5- to 10-membered heteroaryl group or a 5- to 10-membered heterocyclyl group, said heteroaryl group and said heterocyclyl group being unsubstituted or substituted with one or more substituents 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) (where n = 0 or 1), -(C 1-6 alkyl) n OC(O)(C 1-6 alkyl) (where n = 0 or 1), C 1-6 alkylthiol, -N(R N )2 (where each R N independently represents a hydrogen atom or a C 1-6 alkyl group), -CN, -S(O)2NH2, nitro and sulfonic acid groups; or (b) independently represent a C 1-6 alkyl group, a C 2-6 alkenyl group or a C 2-6 alkynyl group, unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups, either one of which is the case.

[0142] The cyclosporine analogs described in the present invention typically have low or minimal or no ability to bind to, inhibit, and / or degrade CypA. For example, the cyclosporine analogs described in the present invention preferably have a lower ability to bind to, inhibit, and / or degrade CypA than CsA. In a preferred embodiment, the cyclosporine analog of the present invention has a lower binding affinity for cyclophilin A (CypA) than for cyclosporine A (CsA), preferably less than half of the binding affinity of CsA for CypA, more preferably less than one-fifth of the binding affinity of CsA for CypA, and most preferably less than one-tenth of the binding affinity of CsA for CypA (e.g., as measured by a standard binding assay such as surface plasmon resonance (SPR)). Methods for determining the relative ability of a compound (e.g., a cyclosporine analog described in the present invention) to bind to, inhibit, and / or degrade a protein (e.g., CypA) relative to other compounds (e.g., CsA) are well known in the art and can be routinely utilized by those skilled in the art. One non-limiting method for determining the binding ability to CypA (e.g., Kd (kinetics), Kd (affinity), maximum response, Koff, and / or Kon) is shown in Example 3.

[0143] The cyclosporine analogs described in the present invention are typically IFITM3 inhibitors. For example, in a preferred embodiment, the cyclosporine analogs described in the present invention can be more potent IFITM3 inhibitors than CsA. The determination of IFITM3 inhibition, such as by referring to CsA as a comparison, can be performed using any method known in the art for measuring the protein inhibition ability of a compound (e.g., in vitro). One suitable non-limiting method is shown in Example 2.

[0144] Synthesis The compounds of the present invention can be prepared by standard methods well known in the art. Representative examples of the synthesis of cyclosporine analogs, which can be readily adapted to provide the compounds of the present invention, are provided in WO2021 / 229237 (including but not limited to the examples of this publication); the content of WO2021 / 229237 is hereby incorporated by reference in its entirety. Further exemplary embodiments of the synthesis of the compounds are provided in the examples of the present application.

[0145] Particularly useful intermediate compounds, and thus intermediate compounds forming a further aspect of the present invention, are as follows. (a) The compound "VP75" whose synthesis is described in Example 8 and has the following structure:

[0146]

Chemical formula

[0147] ; and (b) The compound "VP_130" whose synthesis is described in Example 10 and has the following structure:

[0148]

Chemical formula

[0149] These compounds are particularly useful, especially for the preparation of the compounds of the present invention, wherein: R1 represents hydrogen; R2 is

[0150]

Chemical formula

[0151] represents; R3 represents ethyl; R4 represents methyl; R5 represents -CH2CH(CH3)2; and R7 represents hydrogen.

[0152] Compound VP75 is particularly useful for preparing compounds that are further characterized specifically as follows: Ring A is

[0153]

Chemical formula

[0154] represents; and R C represents -C(O)O- or -C(O)N-. Compound VP_130 can be used similarly to prepare such compounds as well as other compounds of the present disclosure. Non-limiting, exemplary examples of the synthesis of these compounds and subsequent reactions are provided in Examples 8 and 10.

[0155] Gene therapy applications The compounds of the present invention can be used in gene therapy, such as to enhance the efficiency of transduction of one or more cells selected from mammalian cells, human cells, human hematopoietic stem cells (HSCs) 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common progenitor cells, natural killer cells, α / β T cells, γδ T cells or regulatory T cells such as T cells, B cells and plasma cells, by gene therapy vectors. The present invention encompasses such uses of the compounds, related pharmaceutical compositions, and treatment methods. Advantageously, the compounds of the present invention have been found to be very potent IFITM3 inhibitors that are easy to synthesize and can be utilized for enhancing HIV vector infection, reducing the required dose of the vector, and eliminating variability among patients. As further discussed elsewhere herein, the compounds may also have reduced binding to CypA compared to CsA, thus alleviating the aforementioned limitations associated with the use of CsA and other related cyclosporine analogs in similar applications.

[0156] Cell Stem cells can differentiate into many cell types. Cells that can differentiate into all cell types are known as totipotent. In mammals, only the zygote and early embryonic cells are totipotent. Stem cells are found in most, if not all, multicellular organisms. They are characterized by their ability to self-renew by mitosis and differentiate into various specialized cell types. Mammalian stem cells are broadly classified into two types: embryonic stem cells, which are isolated from the inner cell mass of the blastocyst, and adult stem cells, which are found in adult tissues. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem cells and progenitor cells function as the body's repair system, replenishing specialized cells and maintaining the normal turnover of regenerative organs such as blood, skin, or intestinal tissue.

[0157] Hematopoietic stem cells (HSCs) are multipotent stem cells found in peripheral blood, bone marrow, and umbilical cord blood. HSCs have the ability to self-renew and differentiate into all blood cell lineages. HSCs have the capacity to reconstitute the entire immune system and the erythroid and myeloid lineages of all hematopoietic tissues such as bone marrow, spleen, and thymus. HSCs provide the production of all hematopoietic cell lineages throughout life.

[0158] Hematopoietic progenitor cells have the ability to differentiate into specific types of cells. However, in contrast to stem cells, hematopoietic progenitor cells are more specific: they are induced to differentiate into their "target" cells. The difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, whereas progenitor cells can divide only a limited number of times. Hematopoietic progenitor cells can be strictly distinguished from HSCs only by functional in vivo assays (i.e., transplantation and demonstration of the ability to generate all blood lineages over a long period).

[0159] Preferably, the hematopoietic stem cells and progenitor cells of the present invention comprise the CD34 cell surface marker (referred to as CD34+).

[0160] A population of hematopoietic stem cells and / or progenitor cells may be obtained from a tissue sample. For example, a population of hematopoietic stem cells and / or progenitor cells may be obtained from peripheral blood (e.g., adult and fetal peripheral blood), umbilical cord fluid, bone marrow, liver, or spleen. Preferably, these cells are obtained from peripheral blood or bone marrow. They may be obtained after mobilizing the cells in vivo by growth factor treatment.

[0161] Mobilization may be carried out using, for example, G-CSF, plerixaphor, or a combination thereof. Other agents such as NSAIDs and dipeptidyl peptidase inhibitors may also be useful as mobilizing agents.

[0162] Since the stem cell growth factors GM-CSF and G-CSF have become available, most current hematopoietic stem cell transplantation procedures are now performed using stem cells collected from peripheral blood rather than bone marrow. Collection of peripheral blood stem cells provides a larger graft, eliminates the need to anesthetize the donor for graft collection, shortens the time to engraftment, and may provide a reduction in the long-term relapse rate. Bone marrow can be collected using standard aspiration methods (steady state or after mobilization) or next-generation collection tools (e.g., Marrow Miner). Additionally, hematopoietic stem cells and / or progenitor cells may 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 generated 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 further differentiate to provide induced hematopoietic stem cells and / or progenitor cells. Induced hematopoietic stem cells and / or progenitor cells can further differentiate into other cells of the hematopoietic lineage.

[0163] HSCs typically have low forward and side scatter profiles by flow cytometry. Some are metabolically quiescent, as shown by rhodamine labeling that can determine mitochondrial activity. HSCs may include certain cell surface markers such as CD34, CD45, CD133, CD90, and CD49f. They may also be defined as cells lacking the expression of CD38 and CD45RA cell surface markers. However, the expression of some of these markers depends on the developmental stage of HSCs and tissue-specific circumstances. Some HSCs called "side population cells" exclude the Hoechst 33342 dye detected by flow cytometry. Thus, HSCs have descriptive characteristics that allow for identification and separation.

[0164] CD38 is the most established and useful single-negative marker for human HSCs. Human HSCs can 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 used in combination for HSC enrichment. A "negative marker" is understood as human HSCs lacking the expression of these markers.

[0165] CD34 and CD133 are the most useful positive markers for HSCs. Some HSCs are also positive for lineage markers such as CD90, CD49f, and CD93. However, these markers may need to be used in combination for HSC enrichment. A "positive marker" is understood as human HSCs expressing these markers.

[0166] Differentiated cells are more specialized cells compared to stem cells and progenitor cells. Differentiation occurs during the development of multicellular organisms, and the organism changes from a single zygote into a complex system of tissues and cell types. Differentiation is also a common process in adults: adult stem cells divide during tissue repair and normal cell turnover, producing fully differentiated daughter cells. Differentiation causes dramatic changes in cell size, shape, membrane potential, metabolic activity, and responsiveness to signals. These changes are mainly due to highly regulated modifications of gene expression. In other words, differentiated cells are cells that have a specific structure and perform a specific function through a developmental process involving the activation and inactivation of specific genes. In this specification, examples of differentiated cells include differentiated hematopoietic cells such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells, T cells, B cells, and NK cells. For example, differentiated hematopoietic cells can be distinguished from stem cells and progenitor cells by detecting cell surface molecules that are not expressed or are expressed at low levels in 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).

[0167] A "separated cell population" is understood to mean that the cell population has been previously removed from the body. The separated cell population can be cultured and manipulated ex vivo or in vitro using standard techniques known in the art. The separated cell population can be reintroduced into a subject at a later time. The subject can be the same subject from which the cells were originally separated or a different subject. The methods and uses performed on the separated cell population are ex vivo or in vitro methods and uses.

[0168] A cell population can be selectively purified from cells that exhibit a specific phenotype or characteristic, and other cells that do not exhibit or exhibit to a lesser extent that phenotype or characteristic. For example, a cell population that expresses a specific marker (such as CD34) can be purified from the starting cell population. Alternatively, or in addition thereto, a cell population that does not express another marker (such as CD38) can also be purified.

[0169] "Enriching" a cell population for a particular type of cell is understood to mean an increase in the concentration of that type of cell within the population. At the same time, the concentration of other types of cells may decrease.

[0170] By purification or enrichment, the cell population may become substantially pure of other types of cells. Purification or enrichment of a cell population that expresses a specific marker (e.g., CD34 or CD38) can be achieved by using an agent that binds to that marker, preferably an agent that binds substantially specifically to that marker.

[0171] The agent that binds to the cell marker may be an antibody, such as an anti-CD34 antibody or an 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, modified antibodies such as chimeric, CDR-grafted, and humanized antibodies, and artificially selected antibodies generated using phage display or alternative techniques. Furthermore, in the present invention, alternatives to classical antibodies, such as "avidibodies", "avimers", "anticalins", "nanobodies", and "DARPins" can also be used.

[0172] Agents that bind to specific markers can be labeled so that they can be identified using any of a number of techniques known in the art. The agent may be inherently labeled or may be modified by attaching a label. "Binding" means that the agent and the label are operably linked. This means that the agent and the label are linked together in such a way that both can perform their functions (e.g., bind to a marker, enable fluorescence identification, or enable separation when placed in a magnetic field) without substantial interference. Suitable binding methods are well known in the art and will be readily identifiable by those skilled in the art.

[0173] The label enables, for example, purification of the labeled agent and the cells to which it is bound from their environment (e.g., the agent can be labeled with magnetic beads or an affinity tag such as avidin), detection, or both. Detectable markers suitable for use as labels include fluorescent dyes (e.g., green, cherry, cyan, orange fluorescent proteins) and peptide tags (e.g., His tag, Myc tag, FLAG tag, and HA tag).

[0174] Many techniques for separating cell populations that express a specific marker are known in the art. These include magnetic bead-based separation techniques (e.g., closed-circuit magnetic bead-based separation), flow cytometry, fluorescence-activated cell sorting (FACS), affinity tag purification (e.g., using an affinity column or beads such as a biotin column to separate an avidin-labeled agent), and microscopy-based techniques. It may also be possible to perform the separation using a combination of different techniques, such as sorting the cell population obtained by flow cytometry for one or more additional (positive or negative) markers after a magnetic bead-based separation step. Clinical-grade separation can be performed, for example, using the CliniMACS® system (Miltenyi). This is an example of a closed-circuit magnetic bead-based separation technique.

[0175] It is also contemplated to enrich HSCs using dye exclusion properties (e.g., side population or rhodamine labeling) or enzyme activity (e.g., ALDH activity).

[0176] The cells of the present invention can be formulated for administration to a subject together with a pharmaceutically acceptable carrier, diluent or excipient. Suitable carriers and diluents include isotonic saline solutions, such as phosphate buffered saline, which may contain human serum albumin. The handling of Cell Therapy Product B is preferably carried out in accordance with the FACT-JACIE international standards for cell therapy.

[0177] Vector A vector is a tool that enables or facilitates the transfer of an entity from one environment to another. The vectors used in the present invention for transducing one or more cells selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 may be viral vectors. The viral vectors may be derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus. All of these viruses are lentiviruses. By "vector derived from a particular type of virus" is understood that the vector contains at least one component part derived from that type of virus.

[0178] A retroviral vector may be from or derived from any suitable retrovirus. A number of different retroviruses have been identified. Examples include murine leukemia virus (MLV), human T-cell leukemia virus (HTLV), murine 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), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (AEV). A detailed list of retroviruses can be found in Coffin, J.M. et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63.

[0179] Retroviruses can be broadly classified into two categories: "simple" and "complex". Further, retroviruses are divided into seven groups. Five of these groups represent retroviruses with oncogenic properties. The remaining two groups are lentiviruses and spumaviruses. A review of these retroviruses is presented in Coffin, J.M. et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63.

[0180] The basic structures of retrovirus and lentivirus genomes share many common characteristics, such as the 5’ LTR and 3’ LTR. Between or within these, there are packaging signals that enable the packaging of the genome, primer binding sites, integration sites that enable integration into the host cell genome, and the gag, pol, and env genes that encode packaging components. These are polypeptides necessary for the assembly of virus particles. Lentiviruses have additional features such as the HIV rev sequence and RRE sequence, which enable the efficient export of the RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of the infected target cells.

[0181] In the provirus, these genes are adjacent at both ends to regions called long terminal repeats (LTRs). The LTRs are responsible for provirus integration and transcription. The LTRs also function as enhancer-promoter sequences and can control the expression of viral genes.

[0182] The LTR itself is the same sequence divided into three elements: U3, R, and U5. U3 is derived from the sequence unique to the 3’ end of the RNA. R is derived from the sequence repeated at both ends of the RNA. U5 is derived from the sequence unique to the 5’ end of the RNA. The sizes of the three elements can vary greatly depending on the retrovirus.

[0183] In defective retroviral vector genomes, gag, pol, and env may be absent or non-functional.

[0184] In a typical retroviral vector, at least a portion of one or more protein coding regions essential for replication can be removed from the virus. This renders the viral vector replication-incompetent. Also, a portion of the viral genome can be replaced with a library encoding candidate regulatory portions operably linked to regulatory control regions and reporter portions within the vector genome to generate a vector capable of transducing a target host cell and / or integrating its genome into the host genome.

[0185] Lentiviral vectors are part of a large group of retroviral vectors. A detailed list of lentiviruses can be found in Coffin, J.M. et al. (1997) Retroviruses, Cold Spring Harbour Laboratory Press, 758-63. Briefly, lentiviruses are 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 "slow virus" 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).

[0186] 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, P.F. 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 that make up tissues such as muscle, brain, lung, and liver.

[0187] As used in the specification, a lentiviral vector is a vector that contains at least one component part that can be derived from a lentivirus. Preferably, the component part is involved in the biological mechanism by which the vector infects cells and expresses or replicates genes.

[0188] The lentiviral vector may be a "primate" vector. The lentiviral vector may be a "non-primate" vector (i.e., derived from a virus that does not naturally infect primates, particularly humans). Examples of non-primate lentiviruses may be any member of the lentivirus members that do not naturally infect primates.

[0189] Examples of lentivirus-based vectors include HIV-1 and HIV-2-based vectors, which are described below.

[0190] The HIV-1 vector contains cis-acting elements also found in simple retroviruses. Sequences extending to the gag open reading frame have been shown to be important for HIV-1 packaging. Therefore, HIV-1 vectors often contain a relevant part of gag with a mutated translation initiation codon. Furthermore, most HIV-1 vectors also contain a part of the env gene that includes the RRE. Rev binds to the RRE, thereby enabling the transport of full-length or single-spliced mRNA from the nucleus to the cytoplasm. In the absence of Rev and / or the RRE, full-length HIV-1 RNA accumulates in the nucleus. Alternatively, the need for Rev and the RRE can be reduced by using the constitutive transport elements of certain simple retroviruses such as Mason-Pfizer monkey virus. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0191] Most of the HIV-2-based vectors are structurally very similar to the HIV-1 vectors. Similar to the HIV-1-based vectors, the HIV-2 vectors also require the RRE for efficient transport of full-length or single-spliced viral RNA.

[0192] In one system, the vector and helper construct are derived from two different viruses, and the reduced nucleotide homology may reduce the likelihood of recombination. In addition to vectors based on primate lentiviruses, FIV-based vectors have also been developed as an alternative to vectors derived from the pathogenic HIV-1 genome. The structure of these vectors is also similar to that of HIV-1-based vectors.

[0193] The viral vectors used in the present invention preferably have a minimal viral genome. By "minimal viral genome" it is understood that the viral vector is engineered to remove non-essential elements and retain essential elements in order to provide the functions necessary to infect, transduce and deliver the nucleotide sequence of interest to the target host cell. Details of this strategy can be found in WO1998 / 017815.

[0194] Preferably, the plasmid vector used to generate the viral genome in the host cell / packaging cell has sufficient lentiviral genetic information to package the RNA genome into viral particles that can infect the target cell in the presence of packaging components but cannot replicate independently to generate infectious viral particles in the final target cell. Preferably, the vector lacks a functional gag-pol gene and / or env gene and / or other genes essential for replication.

[0195] However, the plasmid vector used to generate the viral genome in the host cell / packaging cell also includes transcriptional regulatory control sequences operably linked to the lentiviral genome to direct genomic transcription in the host cell / packaging cell. These regulatory sequences can be native sequences associated with the transcribed viral sequences (i.e., the 5'U3 region), or heterologous promoters such as another viral promoter (e.g., the CMV promoter).

[0196] The vector can be a self-inactivating (SIN) vector from which the enhancer and promoter sequences of the virus have been deleted. The SIN vector can be generated in vivo and transduced into non-dividing cells with an efficiency similar to that of the wild-type vector. Transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilization by replication-competent viruses. This should also enable regulated expression of the gene from an internal promoter by eliminating the cis-acting effects of the LTR.

[0197] The vector may be integration-deficient. Integration-deficient lentiviral vectors (IDLVs) can be generated, for example, by packaging the vector with a catalytically inactive integrase (such as 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, A.D. et al. (1996) J. Virol. 70: 721-8), by modifying or deleting the essential at sequences from the vector LTR (Nightingale, S.J. et al. (2006) Mol. Ther. 13: 1121-32), or by any combination of the above.

[0198] The HIV-derived vector used in the present invention is not particularly limited with respect to the HIV strain. A number of examples of HIV strain sequences can be found in the HIV sequence database (http: / / www.hiv.lanl.gov / content index). For example, the HIV-1-derived vector may be derived from any of the HIV-1 strains NL4-3, IIIB_LAI or HXB2_LAI (X4 tropic), or BAL (R5 tropic), or a chimera thereof. The HIV-2-derived vector may be derived, for example, from the HIV-2 strain ROD.

[0199] As described elsewhere in this specification, the cyclosporin analogs described in the present invention have little, minimal, or no ability to bind to, inhibit, and / or degrade CypA. Many viral vectors utilized in gene therapy applications recruit CypA to help with efficient infection (e.g., efficient infection of HSCs) in order to protect the capsid from restriction by another antiviral protein called TRIM5. Inhibition of CypA recruitment by cyclosporin analogs can, as a result, risk reducing the efficiency of transduction to sub-optimal levels.

[0200] In one embodiment, a viral vector that is insensitive or has limited sensitivity to CypA (e.g., a viral vector that does not bind to CypA) is utilized. Non-limiting examples of such vectors include HIV capsid mutants that are insensitive to CypA, such as A92E and G94D, and are resistant to TRIM5 restriction (see, e.g., Ylinen et al., Journal of Virology 83(4), 2009, p. 2044-2047).

[0201] However, in a more general embodiment of the present invention, a viral vector that is sensitive to CypA (e.g., a viral vector that binds to CypA) is utilized. For example, the viral vector may be a vector that is not selected from HIV capsid mutants that are insensitive to CypA, such as A92E and G94D, and are resistant to TRIM5 restriction.

[0202] Transduction of cells In one aspect, the present invention provides for the use of a cyclosporine analog according to the present invention to enhance the efficiency of transduction of an isolated population of one or more cells selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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.

[0203] Enhancing the efficiency of transduction means that in the presence of a drug (e.g., a cyclosporine analog according to the present invention), transduction of one or more cells selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 compared to transduction achieved under substantially the same conditions but in the absence of the drug. Thus, an increase in the efficiency of transduction can potentially allow for a reduction in the multiplicity of infection (MOI) and / or the transduction time required to achieve effective transduction.

[0204] In one embodiment, the proportion of one or more cells selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 are transfected by a vector, increases. In another embodiment, the copy number of the vector per cell increases. Preferably, both are achieved simultaneously.

[0205] Methods for determining the proportion of cells transfected by a vector are known in the art. Suitable methods include flow cytometry, fluorescence-activated cell sorting (FACS), and fluorescence microscopy. The technique employed is preferably one that allows for automation and / or high-throughput screening.

[0206] For example, a cell population can be transfected with a vector carrying a reporter gene. The vector can be constructed such that the reporter gene is expressed when the vector transfects the cell. Suitable reporter genes include, for example, genes encoding green, yellow, cherry, cyan, or orange fluorescent proteins. When the cell population is transfected by the vector, the numbers of both cells expressing the reporter gene and cells not expressing it can be quantified using a suitable technique such as FACS. Next, the proportion of cells transfected by the vector can be calculated.

[0207] Alternatively, quantitative PCR (qPCR) can also be used to determine the proportion of cells transfected with a vector that does not carry a reporter gene. For example, single colonies of CD34+ cells can be picked from semi-solid cultures and qPCR can be performed separately on each colony to determine the proportion of vector-positive colonies among the analyzed colonies.

[0208] Methods for determining the copy number of a vector are also known in the art. The techniques employed are preferably those that are acceptable for automation and / or high-throughput screening. Suitable techniques include quantitative PCR (qPCR) and Southern blot-based approaches.

[0209] The concentration at which the cyclosporine analogs described in the present invention can be applied to one or more cell populations selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 adjusted to optimize the transfection efficiency for different vector systems. The method for determining the transfection efficiency is described above.

[0210] When applied at high concentrations, the cyclosporine analogs described in the present invention may be toxic to one or more cells selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 cyclosporine analogs described in the present invention to one or more cells selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 determined by quantifying the number of viable cells remaining after exposure to the cyclosporine analog for a certain period of time. Methods for quantifying the number of viable cells are known in the art. Thus, one skilled in the art can select an appropriate concentration of the cyclosporine analogs described in the present invention using the approaches described herein to maximize the transduction efficiency while minimizing the effects of toxicity.

[0211] For example, the concentration applicable to one or more cell populations selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 may be about 0.1-50 μM, about 1-50 μM, about 5-50 μM, about 10-50 μM, about 5-40 μM, about 10-40 μM, about 10-25 μM or about 10-15 μM.

[0212] The present invention encompasses the use of the cyclosporine analogs described in the present invention. The cyclosporine analogs of the present invention may increase the efficiency of transduction of an isolated population of one or more cells selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, CAEV or visna lentivirus.

[0213] The cyclosporine analogs described in the present invention are preferably of low toxicity to mammals, particularly humans, and are preferably of low toxicity to one or more cells selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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.

[0214] Therapeutic uses: Gene therapy and transplantation The vectors used in the present invention preferably contain the nucleotide of interest (NOI). Preferably, the NOI produces a therapeutic effect and is thus useful for gene therapy. Suitable NOIs include, but are not limited to, sequences encoding enzymes, cytokines, chemokines, hormones, antibodies, antioxidant molecules, modified immunoglobulin-like molecules, single-chain antibodies, fusion proteins, immunostimulatory molecules, immunomodulatory molecules, antisense RNAs, microRNAs, shRNAs, siRNAs, ribozymes, miRNA target sequences, transdominant negative mutants of target proteins, toxins, conditional toxins, antigens, tumor suppressor proteins, growth factors, transcription factors, membrane proteins, surface receptors, anti-cancer molecules, vasoactive proteins and peptides, anti-viral proteins and ribozymes, and derivatives thereof (such as derivatives having a relevant reporter group). The NOI can also encode a prodrug activating enzyme. An example of NOI is the beta-globin chain that can be used for gene therapy of thalassemia / sickle cell anemia.

[0215] Examples of NOI include treatment of chronic granulomatous disease (CGD, e.g., gp91 phox transgenic), leukocyte adhesion deficiency, other phagocyte disorders in patients with non - progressive severe infections, and hereditary bone marrow failure syndromes (e.g., Fanconi anemia), and other diseases that require non - urgent / selective gene correction in the myeloid system such as primary immunodeficiency (SCID). NOI may also include those useful for the treatment of lysosomal storage disorders and immunodeficiency diseases.

[0216] The present invention also provides one or more cell populations selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, for use in treatment, for example gene therapy, according to the method of the present invention. The use may be as part of a transplantation procedure for mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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.

[0217] Hematopoietic stem cell transplantation (HSCT) is the transplantation of hematopoietic stem cells from bone marrow (in this case known as bone marrow transplantation) or blood. Stem cell transplantation is a medical procedure in the fields of hematology and oncology and is most frequently performed on humans with blood or bone marrow diseases, or certain types of cancer. Many of the recipients of HSCT are patients with multiple myeloma or leukemia who do not benefit from, or are already resistant to, long-term chemotherapy. Candidates for HSCT include pediatric cases where the patient has a congenital deficiency such as severe combined immunodeficiency or congenital neutropenia with defective stem cells, as well as children and adults with aplastic anemia who have lost their stem cells after birth. Other conditions treated with stem cell transplantation include sickle cell disease, myelodysplastic syndromes, neuroblastoma, lymphoma, Ewing sarcoma, fibrosarcoma, small round cell tumor, and Hodgkin's disease. Recently, a procedure has been developed that requires less preparative chemotherapy and radiation, so-called "mini-transplantation", which is non-myeloablative of the bone marrow. This has made it possible to perform HSCT on elderly and other patients who were previously considered too weak to withstand conventional treatment regimens.

[0218] In one embodiment, a population of one or more cells selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common progenitor cells, natural killer cells, α / β T cells, γδ T cells or regulatory T cells, B cells, and plasma cells, prepared according to the method of the present invention, is administered as part of an autologous stem cell transplantation procedure.

[0219] In another embodiment, one or more cell populations selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, are administered as part of an allogeneic stem cell transplantation procedure.

[0220] The "autologous stem cell transplantation procedure" is understood to mean that the starting population of cells (transduced according to the method of the invention) is obtained from the same subject as the subject to whom the transduced cell population is administered. Autologous transplantation procedures are advantageous as they avoid problems associated with immunological incompatibility and are available to the subject regardless of the availability of a genetically compatible donor.

[0221] The "allogeneic stem cell transplantation procedure" is understood to mean that the starting population of cells (transduced according to the method of the invention) is obtained from a subject different from the subject to whom the transduced cell population is administered. Preferably, the donor is genetically identical to the subject to whom the cells are administered in order to minimize the risk of immunological incompatibility.

[0222] An appropriate dose of the transduced cell population is an amount that is therapeutically and / or prophylactically effective. The dose administered may depend on the subject and condition being treated and can be readily determined by one of ordinary skill in the art.

[0223] The products, methods and uses of the present invention may be useful for the treatment of disorders listed in WO1998 / 005635. For ease of reference, a partial list thereof is provided below: cancer, inflammation or inflammatory diseases, skin disorders, fever, cardiovascular effects, bleeding, aggregation and acute phase response, cachexia, anorexia, acute infections, HIV infection, shock states, graft-versus-host reaction, autoimmune diseases, reperfusion injury, meningitis, migraine and aspirin-dependent antithrombosis; tumor growth, wetting and spreading, angiogenesis, metastasis, malignant ascites and malignant pleural effusion; 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 scar healing; rhinitis, allergic conjunctivitis, eczema, anaphylaxis; restenosis, congestive heart failure, endometriosis, atherosclerosis or intimal sclerosis.

[0224] Furthermore, or alternatively, the products, methods and uses of the present invention may be useful for the treatment of disorders listed in WO1998 / 007859. For ease of reference, a partial list thereof is provided below: cytokine and cell growth / differentiation activity; immunosuppressive or immunostimulatory activity (e.g., treatment of immunodeficiencies such as infection by the human immunodeficiency virus; regulation of lymphocyte proliferation; treatment of cancer and many autoimmune diseases, and prevention of transplant rejection or induction of tumor immunity); hematopoietic regulation, e.g., treatment of myeloid or lymphoid diseases; promotion of growth of bone, cartilage, tendon, ligament and nerve tissue, e.g., treatment of wound healing, burns, ulcers, periodontal diseases and neurodegeneration; inhibition or activation of follicle-stimulating hormone (regulation of fertility); chemotactic / chemokinetic activity (e.g., for mobilizing specific cell types to sites of injury or infection); hemostatic and thrombolytic activity (e.g., for the treatment of hemophilia and stroke); anti-inflammatory activity (e.g., for the treatment of septic shock or Crohn's disease); as an antibacterial agent; e.g., as a regulator of metabolism or behavior; as an analgesic; treatment of specific deficiencies; e.g., treatment of psoriasis, in human or veterinary medicine.

[0225] Furthermore, or alternatively, the products, methods and uses of the invention may be useful for the treatment of the disorders listed in WO1998 / 009985. For ease of reference, a partial list thereof is provided below: macrophage inhibition and / or T cell inhibition activity and thus anti-inflammatory activity; anti-immune activity, i.e., an inhibitory effect on 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 such as arthritis such as rheumatoid arthritis, inflammation associated with allergies, allergic reactions, asthma, systemic lupus erythematosus, collagen diseases and other autoimmune diseases, atherosclerosis, arteriosclerosis, atherosclerotic heart disease, reperfusion injury, cardiac arrest, myocardial infarction, vascular inflammatory disorders, inflammation associated with respiratory distress syndrome or other cardiopulmonary diseases, peptic ulcer, ulcerative colitis and other inflammatory disorders associated with gastrointestinal diseases, liver fibrosis, cirrhosis or other liver diseases, thyroiditis or other glandular diseases, glomerulonephritis or other kidney and urinary tract diseases, otitis media or other otolaryngological diseases, dermatitis or other skin diseases, periodontal disease or other dental diseases, orchitis or epididymitis, infertility, testicular trauma or other immune-related testicular diseases, placental insufficiency, placental failure, habitual abortion, eclampsia, preeclampsia and other immune and / or inflammation-related gynecological diseases, posterior uveitis, intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveoretinitis, optic neuritis, intraocular inflammation, e.g., retinitis or cystoid macular edema, sympathetic ophthalmia, scleritis, retinitis pigmentosa, immune and inflammatory elements of degenerative fundus diseases, inflammatory elements of ocular trauma, ocular inflammation due to infection, proliferative vitreoretinopathy, acute ischemic optic neuropathy, excessive scarring, e.g., after glaucoma filtration surgery, immune and / or inflammatory responses to intraocular implants and other immune and inflammation-related ocular diseases, inflammation associated with autoimmune diseases or conditions or disorders where suppression of immune and / or inflammation is beneficial in both the central nervous system (CNS) and other organs, Parkinson's disease, complications and / or side effects of the treatment of Parkinson's disease, AIDS-related dementia complex, HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other degenerative diseases,The state or disorder of the CNS, the inflammatory elements of stroke, post-polio syndrome, the immune and inflammatory elements of mental disorders, 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, the inflammatory elements of compression of the CNS or trauma to the CNS or infection of the CNS, the inflammatory elements of muscular atrophy and muscular dystrophy, and immune and inflammation-related diseases, states or disorders of the central and peripheral nervous systems, post-traumatic inflammation, septic shock, infections, 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., infection by viral carriers, or inflammation associated with AIDS, suppression or inhibition of humoral and / or cellular immune responses, treatment or alleviation (by reducing the amount of monocytes or leukocytes) of monocytic or leukocytic proliferative diseases, e.g., leukemia, prevention and / or treatment of graft rejection in the case of transplantation of natural or artificial cells, tissues, organs such as cornea, bone marrow, organs, lens, pacemaker, natural or artificial skin tissue.

[0226] Furthermore, or alternatively, the products, methods and uses of the present invention may be useful for the treatment of β-thalassemia, chronic granulomatous disease, metachromatic leukodystrophy, mucopolysaccharidosis disorders, and other lysosomal storage disorders.

[0227] Gene therapy can occur by the product encoded by the NOI, for example, by the sustained or transient release of the encoded product described above. For example, hematopoietic progenitor cells typically provide short-term engraftment. Thus, gene therapy by administering transduced hematopoietic progenitor cells may provide a non-permanent effect in a subject. For example, the effect may be limited to 1 to 6 months after administration of the transduced hematopoietic progenitor cells. The advantage of this approach is that safety and tolerability are improved due to the self-limiting nature of the therapeutic intervention. Such hematopoietic progenitor cell gene therapy may be suitable for the treatment of acquired disorders such as cancer, where limited expression of the desired (potentially toxic) anti-cancer nucleotide may be sufficient to eradicate the disease. In contrast, HSCs are likely to provide long-term engraftment and thus may be suitable for providing longer-term effects in a subject or effects that persist throughout the subject's lifetime. For example, the effect may be limited from 3 months to 30 years after administration of the transduced HSCs. Longer-term or sustained effects may be suitable for the treatment of genetic gene disorders such as SCID where long-term expression of the desired nucleotide may be desirable.

[0228] After gene therapy, the encoded product may be released systemically in the subject, for example, into the circulatory system. As a result of systemic release, the activity of the encoded product may increase 1.1, 1.2, 1.5, 2, 5, 10, 25, 50, 100, 250, 500, or 1000-fold compared to its activity prior to gene therapy. Assays for measuring the activity of the encoded product will be apparent to those of skill in the art.

[0229] Alternatively, after gene therapy, the encoded product may be released in a targeted manner to target a specific group of tissues and / or organs. For example, the encoded product may target the central nervous system (CNS), heart, face, mouth, eyes, bones, liver, spleen, and / or lungs. Targeted release may result in a 1.1, 1.2, 1.5, 2, 5, 10, 25, 50, 100, 250, 500, or 1000-fold increase in the activity of the encoded product in the targeted tissues and / or organs as compared to the activity in the same tissues and / or organs prior to gene therapy. Additionally, or alternatively, targeted release may result in a 1.1, 1.2, 1.5, 2, 5, 10, 25, 50, 100, 250, 500, or 1000-fold increase in the activity of the encoded product in the targeted tissues and / or organs after gene therapy as compared to the activity in non-targeted tissues and / or organs. Assays for measuring the activity of the encoded product will be apparent to those of ordinary skill in the art.

[0230] Accordingly, the present invention provides means for modifying, treating, preventing, alleviating, and / or preventing the pathological phenotypes associated with the above-mentioned indications. Modification can refer to both partial, complete, and overcorrection. Modification can be achieved after about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300 days, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, or 5 years. The effects of modifying, treating, preventing, alleviating, and / or preventing the phenotype can be transient. Alternatively, the effects of modifying, treating, preventing, alleviating, and / or preventing the phenotype can be long-term or persistent.

[0231] Treatment of mammals, particularly humans, is preferred. However, treatment of both humans and animals can be within the scope of the present invention.

[0232] Kit In other aspects, the present invention provides a kit comprising a cyclospolin analog of the present invention and / or a cell population. The cyclospolin analog of the present invention and / or the cell population can be provided in a suitable container. The kit can also include instructions for use.

[0233] Treatment of viral infections and other pathologies related to IFITM3 expression Cyclosporin A (CsA) has previously been disclosed to have antiviral activity against, for example, coronaviruses (see, e.g., de Wilde 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). More recently, it has been proposed that the IFITM protein can actively promote SARS-Cov-2 infection, for example, by hijacking normally antiviral proteins for efficient viral infection. This raises the prospect that such viral infections can be treated by inhibiting IFITM and disrupting this hijacking mechanism to reduce the efficiency of viral infection.

[0234] Accordingly, the compounds of the present invention are useful for the treatment or prevention of viral infections in a patient. Typically, the patient is a mammal such as a human or a cat, preferably a human.

[0235] 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 SARS). 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.

[0236] 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 a significant morbidity rate among people worldwide, such as the alpha, delta, and omicron variants. As shown in the Examples section of the present disclosure, the compounds of the present invention may have evolved to utilize the IFITM protein for efficient survival and / or replication in an infected host, and may be particularly useful for the treatment of certain variants of COVID-19 (e.g., the delta and / or omicron variants, particularly the omicron variant).

[0237] The compounds of the present invention can be administered to humans in various ways, such as oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, bronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual administration routes. The administration method and dosing schedule are selected by the attending physician considering various factors such as the patient's age, weight, condition, etc.

[0238] Compounds are typically administered as pharmaceutical compositions that usually include a derivative of the present invention and a pharmaceutically acceptable excipient, diluent, or carrier. Thus, pharmaceutical compositions containing the compounds of the present invention are usually formulated with appropriate pharmaceutically acceptable excipients, carriers, or diluents depending on the particular method of administration employed. For example, parenteral formulations are usually injectable liquids that use pharmaceutically and physiologically acceptable liquids such as physiological saline, balanced salt solutions, etc. as the medium. On the other hand, oral formulations may be solids such as tablets or capsules, or liquid solutions or suspensions.

[0239] The composition can be formulated in unit dosage form, i.e., individual parts containing a unit dose, or in multiples or subunits of a unit dose.

[0240] The dosage of the compounds of the present invention administered to a patient depends on the activity of the particular compound in question. Further factors include the condition being treated, the nature of the patient being treated, and the severity of the condition during treatment. The timing of compound administration should be determined by medical personnel. As understood by a skilled physician, and as with other drugs, the compound can be toxic at very high doses. For example, the compound can be administered at a dosage of from 0.01 to 30 mg per kg of body weight, such as from 0.1 to 10 mg, more preferably from 0.1 to 5 mg.

[0241] The compounds of the present invention can be administered alone or in combination with one or more additional antiviral agents, preferably one or more agents useful for the treatment of 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.

[0242] One such antiviral agent is remdesivir.

[0243] Antiviral agents useful for the treatment of 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 apricitabine. Preferred NNRTIs include efavirenz, nevirapine, delavirdine, etravirine, and rilpivirine. Preferred NtRTIs include tenofovir and adefovir.

[0244] Antiviral agents useful for the treatment of influenza virus include (a) neuraminidase inhibitors such as oseltamivir and zanamivir, and (b) M2 protein inhibitors such as amantadine and rimantadine.

[0245] Antiviral agents useful for the treatment of human cytomegalovirus (hCMV) include human cytomegalovirus antibodies and antiviral agents such as ganciclovir, valganciclovir, foscarnet, and cidofovir.

[0246] Antiviral agents useful for the treatment of hepatitis C virus (HCV) include pegylated interferon alpha and ribavirin.

[0247] An antiviral agent useful for the treatment of vaccinia virus is cidofovir.

[0248] Antiviral agents useful for the treatment of feline immunodeficiency virus (FIV) include lymphocyte T cell immunomodulators.

[0249] Antiviral agents useful for the treatment of coronaviruses such as COVID-19 include dexamethasone, remdesivir, paxlovid, sotrovimab, bebtelovimab, REGEN-COV, bamlanivimab / etesevimab, and molnupiravir.

[0250] The active ingredient is typically administered as a combination formulation.

[0251] Accordingly, the present invention also provides a combination comprising the compound of the present invention and one or more of the said additional antiviral agents. The combination is typically used for the treatment or prevention of the viral infection in a patient.

[0252] The present invention further provides a compound of the present invention for use in the treatment or prevention of viral infection in a patient by co-administration with one or more of the said additional antiviral agents. The co-administration can be simultaneous, parallel, separate, or sequential.

[0253] The present invention further provides one or more of the said additional antiviral agents for use in the treatment or prevention of the viral infection in a patient by co-administration with the compound of the present invention. The co-administration can be simultaneous, parallel, separate, or sequential.

[0254] The present invention further provides a product comprising the compound of the present invention and one or more of the said additional antiviral agents as a combination formulation for simultaneous, parallel, separate, or sequential use in the treatment or prevention of the viral infection in a patient.

[0255] The compound of the present invention, which is an IFITM3 inhibitor, is also more generally useful in the treatment of pathological conditions associated with IFITM3 expression. The term "associated with IFITM3 expression" can be used interchangeably herein with "sensitive to treatment by IFITM3 inhibition". Non-limiting examples of such pathological conditions include viral infections and Alzheimer's disease as described elsewhere herein. However, since IFITM3 is known to play a role in regulating innate immune signaling, the compound of the present invention can be useful as a medicament in any clinical situation where manipulating IFITM3 has a favorable effect on the immune system.

Examples

[0256] Example 1: Representative Synthesis of a Specific Exemplary Compound 2-(1H-Imidazol-1-yl)ethyl 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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)benzoate (JW3-158).

[0257]

Chemical Structure

[0258] To a solution of cyclosporin A (500 mg, 0.42 mmol) in DCM (5 mL) were added 4-vinylbenzoic acid (185 mg, 1.25 mmol) and Hoveyda-Grubbs second-generation catalyst (17 mol%). The reaction mixture was stirred in an MW reactor (90 °C, 40 min) and then cooled. The solvent was removed under reduced pressure, the crude product was redissolved in MeOH, passed through a Stratospheres PL Thiol MP SPE cartridge (polymer Lab, Varian Inc) to remove the catalyst, and concentrated under vacuum. 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 r.t. for 15 h, the solvent was removed under vacuum, 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. HRMS (m / z): found 1402.91;calc. for C 73 H119 N 13 O 14 [MH] + : 1402.83.

[0259] 2-(1H-Imidazol-1-yl)-ethyl 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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (BG147).

[0260]

Chem.

[0261] Methyl 4-bromo-1-naphthoate (530 mg, 1.99 mmol) was dissolved in a THF:water = 9:1 (28 mL) solution, followed by the addition of potassium vinyltrifluoroborate (1.34 g, 9.99 mmol), cesium carbonate (1.95 g, 5.99 mmol), and the catalyst Pd(dppf)Cl2·DCM (5 mol%). The reaction mixture was stirred at 70 °C for 15 h, warmed to r.t., and diluted with H2O. The organic layer was separated, and the aqueous layer 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 column (EtOAc in cyclohexane 0% → 100%) to give 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 the addition of 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 min), then cooled to r.t. The solvent was removed under reduced pressure, the residue was redissolved in MeOH, passed through a Stratospheres PL Thiol MP SPE cartridge (polymer Lab, Varian Inc) to remove the catalyst, and concentrated under vacuum 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, flushed with N2, and anhydrous THF (18 mL) was added thereto with stirring. The mixture was cooled to 0 °C, and 2-(1H-imidazol-1-yl)ethan-1-ol (118 mg, 1.06 mmol) was added. After stirring for 15 min, a solution of the aforementioned crude compound in anhydrous THF was added, and the mixture was stirred at r.t. overnight. Thereafter, the reaction mixture was quenched with saturated ammonium chloride solution (1 mL) and concentrated under vacuum. The crude residue was dissolved in DCM (20 mL), washed with H2O (3 x 20 mL), and concentrated under vacuum. 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. LCMS (m / z): found 1452.9; calc. for C 77 H 121 N 13 O 14 1452.9 [M+H + . 1 H-NMR (600 MHz, MeOD, δ ppm, J Hz) δ 8.90 (d, Napthyl 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). (1H-NMR signals characteristic of BG147)

[0262] ((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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)trifluoroborate (BG158).

[0263]

Chem.

[0264] To a solution of cyclosporin A (1 g, 0.83 mmol) in DCM (10 mL) were added vinylboronic acid pinacol ester (58 mg, 0.38 mmol) and Hoveyda-Grubbs second-generation catalyst (6 mol%). The reaction mixture was stirred in an MW reactor under N2 atmosphere (70 °C, 30 min) and then cooled. The solvent was removed under reduced pressure, the raw material was dissolved in MeOH, passed through a Stratospheres PL Thiol MP SPE cartridge (polymer Lab, Varian Inc) to remove the catalyst, and concentrated under vacuum. The crude product (1.1 g, 0.83 mmol) was dissolved in MeOH (10 mL), and potassium hydrogen fluoride solution (4.5 M, 1 mL) was added. The reaction mixture was stirred overnight at r.t., concentrated under reduced pressure, dissolved in acetone (10 mL x 3) and filtered. The filtrate was precipitated in Et2O (50 mL) to obtain an orange solid, which was collected by filtration, washed with Et2O (50 mL), and concentrated under reduced pressure to obtain the final product (50 mg, 0.04 mmol, 5%).

[0265] 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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-3,5-dimethoxybenzoate (BG186).

[0266]

Chemical formula

[0267] BG158 (6 mg, 0.005 mmol) was dissolved in a THF:water = 9:1 solution (1.4 mL), followed by the addition of 2-(1H-imidazol-1-yl)ethyl 4-bromo-3,5-dimethoxybenzoate (3 g, 0.01 mmol), cesium carbonate (9 mg, 0.03 mmol), and the catalyst Pd(dppf)Cl2·DCM (10 mol%). The reaction mixture was stirred at 90 °C for 30 min under a N2 atmosphere, warmed to r.t., and diluted with H2O. The organic layer was separated and the aqueous phase was extracted with DCM (x3). The combined organic layers were washed with brine (x3), 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 afford the final product BG186 (0.2 mg, 3%).

[0268] In the Example 2-model system, JW3-158 is a more potent transduction enhancer than the control compounds CsA, CsH, and JW115 HP-1 cells (human monocyte cell line) were treated overnight with 10 ng / ml of type I interferon (IFNβ) to induce IFITM3 expression. The next day, the cells were infected with a VSV-G pseudotyped HIV-1 vector (HIV-GFP) encoding GFP at a multiplicity of infection (MOI) of 0.2 IU / cell. The cells were infected in the presence of 5 μM of the compound or an equivalent amount of DMSO vehicle. After 48 h, the infection level was determined by counting GFP-positive green cells by flow cytometry.

[0269] Infection of cells without IFN treatment is high and is decreased by IFN (baseline). The inhibitor rescues IFN-mediated infection by inhibiting the antiviral action 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. The rescue fold of the test compounds compared to the inhibited baseline was 24.6 for CsH, 9.9 for CsA, 29.2 for JW3-158, and 14.2 for JW-115.

[0270] Note that in this experiment, it is shown that the molecule improves infection when the cells are in an antiviral state due to IFN exposure. Since the variability is likely to be affected by the level of the antiviral state of the patient cells, the differences in transduction efficiency between patients are expected to be equalized.

[0271] JW-115 is disclosed in WO2021 / 229237 and has the following structure:

[0272] [Chemical formula]

[0273] Example 3 - Representative compounds show reduced binding to CypA compared to CsA SPR binding experiment - Cyp inhibitor CypA binding was performed using surface plasmon resonance as described in (Warne J Biol Chem. 2016 Feb 26;291(9):4356-73). Cyclosporin A was used as a positive control.

[0274] Protocol To study the binding interactions between CypA and various inhibitors, surface plasmon resonance (SPR) using Biacore was performed at 25°C. In the SPR binding experiment, a dual flow cell with a blank reference was used. The sensor chip surface was first activated with a mixture of N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N'-ethyl-carbodiimide hydrochloride (EDC) at a flow rate of 10 μl / min for 420 s in both flow cell 2 and the blank flow cell 1.

[0275] The CypA protein was diluted to a concentration of 50 μg / mL by adding a 10 mM sodium acetate solution (pH 5), and then immobilized and covalently bound to the carboxymethylated dextran matrix of a Biacore CM5 sensor chip. CypA was flowed over the surface for 420 s, and then the unreacted surface was quenched with ethanolamine for 420 s.

[0276] Immobilization was performed in the presence of JW47 (Warne J Biol Chem. 2016 Feb 26;291(9):4356-73). The Cyp inhibitor compound stock in DMSO (10 mM concentration) was diluted with HBS-EP+ buffer to a final DMSO concentration of 2% (e.g., 5 μl was dissolved in 245 μl of buffer).

[0277] Kinetics / Affinity Experiments The contact time of the inhibitor with the surface was 120 s, followed by a dissociation time of 600 s. In the case of very strong binders such as CsA, the dissociation time was extended up to 1200 s. If the dissociation time is sufficient, the regeneration step is not required.

[0278] In each experiment, eight different DMSO dilutions were used as reference: 2.8%, 2.6%, 2.4%, 2.2%, 2%, 1.8%, 1.6% and 1.5% to perform solvent correction.

[0279] To include a wash cycle in the experiment, a 50 / 50 mixture of buffer and DMSO is required.

[0280] Examples 50 μl of CypA (0.1 mg / ml) + 40 μL of 10 mM sodium acetate pH5 + 10 μl of 600 μM JW47 DMSO solution. This mixture was set in an injection rack after being left at room temperature for 10 min.

[0281] The Cyp inhibitor compound stock in DMSO (10 mM concentration) was diluted with HBS-EP+ buffer to a final DMSO concentration of 2% (e.g., 5 μl was added to 245 μl of buffer).

[0282] Results The results are summarized in the following table.

[0283]

Table 1

[0284] 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.

[0285]

Chemical formula

[0286] Example 4 - Complementary cell line assay for identifying selective IFITM3 inhibitors An experiment similar to Example 2 was performed on IFN-treated THP-1 cells.

[0287] THP-1 cells (human monocyte cell line) were treated overnight with 10 ng / ml of type 1 interferon (IFNβ) to induce IFITM3 expression. The next day, the cells were infected with a VSV-G pseudotyped HIV-1 vector (HIV-GFP) encoding GFP at a multiplicity of infection (MOI) of 0.35. The cells were infected in the presence of three doses of the compound at 1.25, 2.5, and 5 μM or 0.1% v / v DMSO vehicle. After 48 hours, the infection level was determined by counting GFP-positive green cells by flow cytometry.

[0288] The results are shown in Figure 1. The bars without virus represent uninfected (green) cells. The infection of cells without IFN treatment is high (dark gray bars), and the infection is reduced by IFN (the third bar). The inhibitor rescues the IFN-induced infection by inhibiting the antiviral action of IFITM3. The higher the bar, the higher the rescue of infection and the better the inhibition of IFITM3. In this experiment, BG147 completely rescued the IFN-induced infection and was superior to other molecules. For each compound, three concentrations were tested (the bars from left to right are 1.25 μM, 2.5 μM, and 5 μM, respectively).

[0289] Next, experiments were conducted on U87 cells. These cells differ from THP-1 in that they express IFITM3 and TRIM5. Therefore, only the inhibitor with reduced CypA inhibition rescues infection. This functionally tests whether the inhibitor has lost CypA inhibition but retains IFITM3 inhibition. The results are shown in Figure 2.

[0290] 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 inhibitor (left bar to right bar for each of the compounds described). Inhibition of IFITM3 enhances infection. Unlike the other inhibitors, BG147 produces the greatest enhancement of infection at the lowest concentration of 2.5 μM. The other molecules achieve the greatest rescue of infection at the highest concentration.

[0291] Explanation of differences in drug potency in different cell lines Cyclosporin A is a complex molecule that targets multiple pathways within cells. Specifically, CsA targets cyclophilin A (an enzyme inhibited by CsA) and IFITM3 (an antiviral membrane protein that is rerouted to the lysosomal degradation pathway upon addition of CsA). Cyclophilin A (CypA) functions as a cofactor for HIV infection. This serves to protect HIV from the antiviral protein TRIM5. Thus, inhibiting CypA with CsA reduces infection via TRIM5 activity, but this only occurs in cells that produce TRIM5. On the other hand, since CsA also inhibits IFITM3, the HIV infectivity in cells that produce IFITM3 is increased. Therefore, the effect of CsA depends on the levels of TRIM5 and IFITM3. When TRIM5 is high and IFITM3 is low, CsA inhibits infection. When TRIM5 is low and IFITM3 is high, CsA rescues infection (IFN-treated THP-1 has high IFITM3 and low TRIM5, so the effect of drugs on IFITM3 can be isolated). Most cells produce both proteins to some extent. This is why CsA is not an effective transduction enhancer and also why the results vary between cell lines depending on the production levels of CypA and IFITM3. Since stem cells produce TRIM5, CsA cannot be used. Therefore, in this study, we have aimed to develop molecules such as BG147 that inhibit IFITM3 but not CypA.

[0292] Further research THP-1 cells (human monocyte cell line) were treated overnight with 10 ng / ml of type 1 interferon (IFNβ) to induce IFITM3 expression.

[0293] The next day, IFN-treated (closed symbols) and untreated cells (open symbols) were infected with a VSV-G pseudotyped HIV-1 vector (HIV-GFP) encoding GFP at a multiplicity of infection (MOI) of 0.4. Cells were infected in the presence of titrations of either BG147 or cyclosporin H (CsH). After 48 hours, the infection level was determined by counting GFP-positive green cells by flow cytometry.

[0294] The results are shown in Figure 3. In each of panels A and B, the results for the IFN-treated cells are shown by closed symbols, and the results for the untreated cells are shown by open symbols. Panel A shows the results with BG147, and panel B shows the results with CsH.

[0295] Both BG147 and CsH rescue infection from the antiviral effect. BG147 is more potent than CsH.

[0296] Example 5 - Stem Cell Research In this specification, inhibitors were tested for their ability to improve the transduction of HIV vectors 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 in gene therapy.

[0297] Human stem cells (HSCs) were infected with amounts of a VSV-G pseudotyped HIV gene therapy vector encoding GFP that infected 40% of the cells. The cells were infected in the presence and absence of various transduction enhancers as follows: Lentiboost (LB), protamine sulfate (PS), JW3-158, BG147 or CsH (concentrations 2.5, 1.25 and 0.5 μM). Forty-eight hours after transduction, the infected (green) cells were counted by flow cytometry. The results are shown in Figure 4.

[0298] BG147 is superior to other inhibitors and is as effective as the state-of-the-art transduction enhancer (the combination of LB + PS). The competing molecule CsH has been reported to achieve good enhancement but to be toxic at a high concentration of 12.5 μM.

[0299] In this first experiment, an effective concentration range was established.

[0300] When cell viability was measured from cells in the above experiment, it was shown 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, which is reflected in the decrease in transduction.

[0301] This experiment was repeated as follows with cells from the second HSC donor.

[0302] HSCs were infected with a dose of a VSV-G pseudotyped HIV gene therapy vector encoding GFP that infects 40% of the cells. Cells were infected in the presence and absence of various transduction enhancers as follows: Lentiboost (LB), protamine sulfate (PS), JW3-158, BG147 or CsH (concentrations 2.5, 1.25 and 0.5 μM). Forty-eight hours after transduction, the infected (green) cells were counted by flow cytometry. The results are shown in Figure 6.

[0303] In this experiment, BG147 was superior to other inhibitors including the combination of the state-of-the-art transduction enhancer LB+PS.

[0304] Importantly, 2.5 μM BG147 is additive with PS and LB, and the number of transduced (infected) cells is the highest. This is presumably because PS and LB improve virus attachment to cells, while BG147 improves the virus entry process, so both act well together.

[0305] Protocol Primary CD34+ HSCs were cultured in Stemspan II supplemented with 1% Pen / Strep, 100 ng / mL TPO, SCF and FLT3R, and 60 ng / mL IL-3. After thawing, the cells were resuspended at a concentration of 10^6 / mL and allowed to recover for 24 hours. On day 2, the cells were resuspended and incubated with GFP lentivirus (MOI 10), Lentiboost+ protamine sulfate, JW3-158, BG-147 and CsH, either in combination at various concentrations or without. The final volume was 125 uL in all cases and 250,000 cells per condition. The cells were incubated for an additional 48 hours to allow GFP expression. Next, the cells were transferred to FACS tubes and centrifuged at 300 xg for 10 minutes. The supernatant was aspirated, the cells were resuspended in 1 mL of FACS buffer and centrifuged again for washing. Thereafter, the cells were resuspended in 400 uL of FACS buffer containing DAPI. Viability and GFP expression were measured by flow cytometry.

[0306] Example 6 - Further Stem Cell Research In this specification, different measurements of transgene expression were performed using cells obtained from the same experiment as above (Example 5). Instead of measuring the number of transduced cells (infection %), the mean fluorescence intensity (greenness of transgene-expressing cells) was measured. The results are shown in Figure 7.

[0307] This indicates that the most green cells are those treated with 2.5 μM BG147 + PS + LB. This suggests that the infection after this treatment is the highest among all, and the cells have turned green because they are infected with one or more HIVGFP viruses. This also suggests that the maximum number of infected cells has been achieved, i.e., the remaining 20% cannot be infected with HIVGFP regardless of the dose.

[0308] Figure 8 summarizes the viability staining data from the previous experiment, showing that the cells did not significantly die with any of the treatments.

[0309] BG147 functions best. Without being limited by theory, it is proposed that JW3-158 does not function as well because the molecule should optimally be removed or at least further reduced to maintain anti-CypA activity for optimal function in stem cells.

[0310] Example 7 - Antiviral Activity Against COVID-19 Western blot THP-1 cells (human monocyte cell line) were treated overnight with 10 ng / ul of type I interferon (IFNβ) to induce IFITM3 expression. The next day, the cells were treated with 5 μM of CsA, CsH, or JW3 (JW3-158) or an equivalent amount of DMSO vehicle. Protein samples were harvested after 6 hours, and IFITM3 levels were measured by immunoblotting with actin detected as a loading control. IFN induces IFITM3. The results are shown in panel A of Figure 9. The inhibitors resulted in a decrease in IFITM3 protein levels compared to DMSO-treated cells (second lane), and JW3-158 had the most potent effect.

[0311] Calu-3 lung epithelial cells were pretreated with 2.5 uM JW3 (JW3-158) or an equivalent amount of DMSO for 4 hours and then infected with either the alpha, delta, or omicron isolate of SARS-CoV-2 at an MOI of 1000 viral E RNA copies / cell. The compound was present during infection. RNA was harvested 48 hours after infection, and viral replication levels were quantified by RT-qPCR measuring E copies. The results are shown in panel B of Figure 9. The fold change between the conditions treated with DMSO and JW3-158 is shown for each variant, and JW3-158 inhibits the replication of delta and omicron more than alpha.

[0312] Example 8 - Further Synthetic Studies Additional work was directed towards: (a) the synthesis of a larger batch of BG147; and (b) the synthesis of further compounds of the present disclosure. Procedure Method

[0313]

Chemical Structure

[0314] Synthesis and Characterization of BG147 and Its Analogs Methyl 4-vinyl-1-naphthoate (VP43)

[0315] [Chemical formula]

[0316] Methyl 4-bromonaphthalene-1-carboxylate (530 mg, 2 mmol) was dissolved in THF:water = 9:1 (28 mL), followed by the addition of potassium vinyltrifluoroborate (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 at 70 °C overnight, the reaction mixture was cooled and concentrated in vacuo. The residue was diluted with CH2Cl2 (10 mL), washed with water (3x10 mL) and brine, dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by chromatography column on silica gel (EtOAc 0% → 100% in Cyclohexane) to give VP43 (378 mg, 1.779 mmol, 89%) as a yellow oil.

[0317] 4-Vinyl-1-naphthoic acid (VP57)

[0318] [Chemical formula]

[0319] VP43 (2.2 g, 10 mmol) was dissolved in THF (12 mL) and treated with 5 M LiOH solution (25 mL). After stirring at 50 °C overnight, THF was removed under vacuum, the residue was acidified to pH 2 with 1 M HCl, extracted with EtOAc (30x3), dried over MgSO4, filtered and concentrated under reduced pressure to give VP57 (2 g, 10 mmol, 97%) as a yellow solid.

[0320] 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 - undecaoxo - 1,4,7,10,13,16,19,22,25,28,31 - undecaazacyclotritriacontan - 2 - yl)-5 - hydroxy - 4 - methylpent - 1 - en - 1 - yl)-1 - naphthoic acid (VP47)

[0321]

Chem.

[0322] VP57 (270 mg, 1.36 mmol) was dissolved in anhydrous DCE (4.5 mL), and then cyclosporin A (360 mg, 0.299 mmol) and Hoveyda - Grubbs second - generation catalyst (15 mol%) were added. The reaction mixture was stirred in an MW reactor under N2 atmosphere (70 °C, 30 min), and then cooled to r.t. The solvent was removed under reduced pressure, the raw materials were dissolved in MeOH, and the catalyst was removed by passing through a Stratospheres PL Thiol MP SPE cartridge (polymer Lab, Varian Inc). The untreated 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). The excess VP57 was removed by reverse - phase chromatography (C18, MeCN 10% → 100% in H2O + 10 mM NH3), and VP47 (115 mg, 0.085 mmol, 28%, trans / cis = 3 / 1) was obtained as an off - white solid. 1H-NMR (500 MHz, CDl3, δ ppm) δ 3.56 (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). 13 C-NMR (500 MHz, CDCl3, δ ppm) δ 173.92 (C=O), 173.89 (C=O), 173.66 (C=O), 171.71 (C=O), 171.50 (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). LCMS (m / z): [MH] + calcd. for C 72 H 115 N 11 O 14, 1358.9;found 1358.4

[0323] General procedure for the synthesis of BG147 and its ester and amide analogs To a solution of VP47 (1 eq) in DMF (1 mL), DIPEA (7 eq) and HATU (6 eq) were added. After stirring at r.t. 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. Then the solvent was removed under reduced pressure and the crude product was purified by chromatography columns on silica gel (MeOH 0% → 10% in DCM) and reverse phase C4 (MeCN 30% → 100% in H2O 10 mM NH3) to obtain the final product.

[0324] 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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (BG147)

[0325]

Chem.

[0326] BG147 was obtained as an off-white solid (17 mg, 0.01 mmol, 20%, trans / cis = 3 / 1) using 2-(1H-imidazol-1-yl)ethan-1-ol as the appropriate alcohol according to the above general procedure. 1 H-NMR (600 MHz, CDl3, δ ppm) δ 3.55 (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). 13 C-NMR (600 MHz, CDCl3, δ ppm) δ 173.97 (C=O), 173.88 (C=O), 173.63 (C=O), 173.13 (C=O), 172.80 (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). HRMS (m / z): [MH] + calcd. for C 77 H121 N 13 O 14, 1452.91560; found 1452.92287

[0327] 2-Morpholinoethyl 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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (VP50)

[0328]

Chem.

[0329] VP50 was obtained as an off-white solid (3.0 mg, 0.002 mmol, 17%, trans / cis = 3 / 1) using 2-morpholinoethanol as the appropriate alcohol according to the general procedure above. 1 H-NMR (600 MHz, CDCl3, δ ppm) δ 3.56 (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). LCMS (m / z): [MH] + calcd. for C 78 H 126 N 12 O 15, 1471.9; found 1471.6

[0330] (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)naphthalen-1-yl)-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-undecaazacyclotritriacontane-2,5,8,11,14,17,20,23,26,29,32-undecanone (undecaone) (VP51)

[0331]

Chem.

[0332] VP51 was obtained as an off-white solid (3.0 mg, 0.002 mmol, 18%, trans / cis = 3 / 1) using 2-(piperazin-1-yl)ethan-1-ol as the appropriate amine according to the above general procedure. 1 H-NMR (600 MHz, CDCl3, δ ppm) δ 3.55 (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). LCMS (m / z): [MH] + calcd. for C 78 H 127 N 13 O 14, 1,471.0; found 1471.2

[0333] 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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthamide (VP53)

[0334]

Chem.

[0335] VP53 was obtained as a yellow solid (8.0 mg, 0.006 mmol, 47%, trans / cis = 3 / 1) using 2-(1H-imidazol-1-yl)ethan-1-amine as the appropriate amine according to the general procedure above. 1 H-NMR (600 MHz, CDCl3, δ ppm) δ 3.55 (s, NMe, 3H), 3.37 (s, NMe, 3H), 3.29 (s, NMe, 3H), 3.08 (s, NMe, 3H), 3.03 (s, NMe, 3H), 2.71 (s, NMe, 3H), 2.67 (s, NMe, 3H). LCMS (m / z): [MH] + calcd. for C 77 H 122 N 14 O 13, 1451.9; found 1452.2

[0336] 2-(1H-Pyrazol-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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (VP72)

[0337]

Chem.

[0338] VP72 was obtained as a yellow solid (3.0 mg, 0.002 mmol, 14%, trans / cis = 3 / 1) using 2-(1H-pyrazol-1-yl)ethan-1-ol as the appropriate alcohol according to the above general procedure. 1 H-NMR (500 MHz, CDCl3, δ ppm) δ 3.56 (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). LCMS (m / z): [MH] + calcd. for C 77 H 121 N 13 O 14, 1451.9; found 1453.0

[0339] 3-(1H-Pyrazol-1-yl)propyl 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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (VP73)

[0340]

Chem.

[0341] VP73 was obtained as a yellow solid (3.0 mg, 0.002 mmol, 14%, trans / cis = 3 / 1) using 3-(1H-pyrazol-1-yl)propan-1-ol as the appropriate alcohol according to the general procedure above. 1 H-NMR (500 MHz, CDCl3, δ ppm) δ 3.56 (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). LCMS (m / z): [MH] + calcd. for C 78 H 123 N 13 O 14, 1465.9; found 1467.1

[0342] 4-(1H-Imidazol-1-yl)butyl 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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (VP74)

[0343]

Chem.

[0344] VP74 was obtained as a yellow solid (4.0 mg, 0.003 mmol, 18%, trans / cis = 1 / 1) using 4-(1H-imidazol-1-yl)butan-1-ol as the appropriate alcohol according to the above general procedure. 1 H-NMR (500 MHz, CDCl3, δ ppm) δ 3.55 (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). LCMS (m / z): [MH] + calcd. for C 79 H 125 N 13 O 14, 1479.9;found 1481.1

[0345] Methyl 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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-1-naphthoate (VP75)

[0346]

Chem.

[0347] VP75 was obtained as a yellow solid (11.0 mg, 0.008 mmol, 54%, trans / cis = 3 / 1) using methanol as the appropriate alcohol according to the above general procedure. 1 H-NMR (500 MHz, CDCl3, δ ppm) δ 3.56 (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). LCMS (m / z): [MH] + calcd. for C 73 H 117 N 11 O 14, 1371.9; found 1373.0

[0348] 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-undecaoxo-1,4,7,10,13,16,19,22,25,28,31-undecaazacyclotritriacontan-2-yl)-5-hydroxy-4-methylpent-1-en-1-yl)-N-(2-morpholinoethyl)-1-naphthamide (VP76)

[0349]

Chem.

[0350] VP76 was obtained as a white solid (10.0 mg, 0.007 mmol, 46%, trans / cis = 3 / 1) using 2-morpholinoethan-1-amine as the appropriate amine according to the general procedure above. 1 1H-NMR (500 MHz, CDCl3, δ ppm) δ 3.55 (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). LCMS (m / z): [MH] + calcd. for C 78 H 127 N 13 O 14, 1470.0;found 1471.3

[0351] Materials and Methods ·Commercially available solvents and reagents were used without further treatment unless otherwise specified. ·Evaporation of solvents was performed under reduced pressure (40 - 60 °C) using a Buchi Rotavapor R-210. · The microwave-assisted reactions were carried out using a Biotage Initiator for synthesis + apparatus. · The reactions under anhydrous conditions were carried out using commercially available N2. · Thin layer chromatography (TLC) was performed for qualitative purposes on aluminum silica gel plates (Alugram Sil G / UV 254) by detection with UV light (λ254 nm) and staining with [(NH4)6MoO4, Ce(SO4)2, H2SO4, H2O]. · Purification was carried out using a Biotage Isolera Four Flash Chromatography System. The chromatography columns used were a) normal phase Biotage Sfar Silica (5 g, 10 g, 25 g), b) reverse phase Biotage Snap Bio C4 300 Å 25 g or Biotage Sfar C18D-Duo 100 Å (12 g, 30 g, 60 g). PE-AX 10 g, a strong base ion exchange resin, was used when indicated. · 1 H- and 13 C-NMR spectra were recorded at the UCL Chemistry NMR Facility using a Bruker DRX 500, 600, or 700 MHz spectrometer. Chemical shifts (δ) are reported in ppm relative to TMS as an internal standard, and coupling constants (J) are reported in Hz. J values were assigned and not repeated. Unless otherwise indicated, CDCl3 and CD3OD were used as solvents at room temperature. All assignments were confirmed by 2D spectra (COSY and HSCQ). · Accurate mass measurements were performed at the UCL Chemistry Mass Spectrometry Facility using ASAP-HESI ionisation connected to a Q Exactive Plus mass spectrometer. · The LC-MS spectra were obtained using an analytical C4 column (Symmetry300, 50x4.6 mm, 3.5 μm) and a C18 column (Kinetex 5μm 100Å, 50x4.6 mm), and a single quadrupole LC / MSD XT mass spectrometer equipped with electrospray ionisation (ESI). The gradients used were as follows: a) 10% → 95% of MeCN + 0.1% FA (FA = formic acid) in H2O + 0.1% FA (6.5 min) b) 30% → 95% of MeCN + 0.1% FA in H2O + 0.1% FA (9 min) c) 30% → 95% of MeCN in H2O 10 mM NH3(9 min)

[0352] Example 9 - Study on the Activity of Further Compounds of the Present Disclosure Further studies were carried out to test the properties of additional compounds of the present disclosure.

[0353] Study 1: THP-1 cells (human monocyte cell line) were treated overnight with 10 ng / ml of interferon type I (IFNβ) to induce IFITM3 expression. The next day, the IFN-treated cells were infected with a VSV-G pseudotyped HIV-1 vector (HIV-GFP) encoding GFP at a multiplicity of infection (MOI) of 0.25. As shown in Figure 10, the cells were infected in the presence of a titration (1.25 - 5 μM) of one of various control compounds and comparative compounds. After 48 hours, the cells were fixed and the GFP-positive cells were counted by flow cytometry to determine the level of infection. For comparison, DMSO-treated THP-1 cells are shown with black bars in the presence or absence of IFNβ. The results are shown in Figure 10 (N = 2). A high level of % GFP-positive cell level at a specific test compound concentration indicates a high level of rescue of infection due to the antiviral effect of IFN.

[0354] Study 2 (Western blot): THP-1 cells (human monocyte cell line) were treated overnight with 10 ng / ml of type I interferon (IFNβ) to induce IFITM3 expression. Next, the cells were treated with 5 μM of the designated test compound. After 24 hours, the cells were lysed and electrophoresed on an SDS PAGE gel. Western blot analysis was performed using anti-actin (control) and anti-IFITM3 antibodies to visualize IFITM3 degradation. The results are shown in Figure 11.

[0355] Study 3 (Cell viability assay (MTT)): In parallel with the infection experiment, an MTT colorimetric viability assay of THP1 cells in the presence of the designated test compound was performed. THP-1 cells (human monocyte cell line) were treated overnight with 10 ng / ml of type I interferon (IFNβ) to induce IFITM3 expression. The next day, as shown in Figure 12, the cells were titrated (1.25 - 5 μM) and treated with one of various control compounds and comparative compounds. Triton X was a control for indicating 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 solubilized and the absorbance at 570 nM was measured using a spectrophotometer. The results are shown in Figure 12. Error bars represent the standard deviation of three technical replicates from one experiment. N = 1. Three bars are shown for each test compound, and each bar corresponds to the result obtained when using 1.25, 2.5, and 5 μM of the test compound, respectively, from left to right.

[0356] Example 10 - Synthesis of Intermediates Useful for the Preparation of Target Compounds VP_130

[0357]

Chemical formula

[0358] CsA (500.0 mg, 0.4161 eq) was dissolved in DCE (10 mL) in a microwave vial, and Grubbs’-Hoyveda G2 (18.3 mg, 0.029 mmol) was added, followed by sparging with ethylene gas. The vial was irradiated at 70 °C for 30 minutes (MW). The reaction mixture was passed through an SH resin column to remove the catalyst, rinsed with MeOH, and concentrated. The crude product weighed 490 mg (0.412 mmol, 92%). The product was used without further purification (purity by lcms, C4 acetonitrile: water: 0.1% formic acid). [MH]+ 1189.

[0359] VP_136

[0360]

Chemical Structure

[0361] Bromide (80.3 mg, 0.303 mmol, 5.000 eq) was dissolved in THF * :water = 9:1 (2 mL), and VP130 ((72.0 mg, 0.061 mmol, 1.000 eq)), cesium carbonate (59.2 mg, 0.182 mmol, 3.000 eq), and Pd(dppf)Cl·CH2Cl2 (2.5 mg, 0.003 mmol, 0.050 eq) (5 mol%) were added. The mixture was stirred in a microwave at 70 °C for 4 days. The residue was partitioned between DCM (10 mL) and water (10 mL), the organic phase was washed with brine, dried over MgSO4, and concentrated under reduced pressure. Purification was performed using an acetonitrile / water 0.1% formic acid, C4 column to afford the product (25 mg, 0.022 mmol, 30%). [MH]+ 1373.

[0362] The resulting methyl ester compound VP_136 can be readily converted to the target compound (but not limited to) by hydrolysis (e.g., using LiOH) to the corresponding carboxylic acid, i.e., VP47, followed by esterification with an appropriate alcohol (see Representative Example 8 showing a similar reaction using VP47).

Claims

1. A compound of formula (I): 【Chemical 1】 (wherein: R １ is hydrogen, C １ -C ４ -alkyl or C ２ -C ４ -alkenyl; R ２ is [Chemical Formula 2] represents; R ３ represents ethyl or isopropyl; R ４ represents methyl or ethyl; R ５ is -CH ２ CH(CH ３ ) ２ , -CH ２ CH(CH ３ )CH ２ CH ３ , -CH(CH ３ )CH ３ or -CH(CH ３ )CH ２ CH ３ represents; R ７ is a hydrogen atom or a C １－２０ alkyl group, a C ２－２０ alkenyl group or a C ２－２０ alkynyl group, and this part is unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups, where (a) 0, 1, 2 or 3 carbon atoms are C ６－１０ arylene, 5- to 10-membered heteroarylene, C ３－７ replaced by a group selected from carbocyclylene and 5- to 10-membered heterocyclylene groups, and (b) -CH ２ - group, the largest half of which is replaced by a group selected from -O-, -S-, -C(O)- and -N(C １－６ alkyl)-groups, where: (i) the arylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted with one or more substituents 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) (where n = 0 or 1), -(C １－６ alkyl) ｎ OC(O)(C １－６ alkyl) (where n = 0 or 1), C １－６ alkylthiol, -N(R Ｎ ) ２ (wherein each R Ｎ independently represents a hydrogen atom or a C １－６ alkyl group), -CN, -S(O) ２ NH ２、 nitro and sulfonic acid groups; and (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and heterocyclylene groups are replaced by a -C(O)- group; and Ring A represents a monocyclic ring or a bicyclic ring system, and C ６－１０ is an arylene group or a 5- to 10-membered heteroarylene group, unsubstituted or substituted with one or more substituents selected from a halogen atom 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) (where n = 0 or 1), -(C １－６ alkyl) ｎ OC(O)(C １－６ alkyl) (where n = 0 or 1), C １－６ alkylthiol, -N(R Ｎ ) ２（ (wherein each R Ｎ independently represents a hydrogen atom or a C １－６ alkyl group), -CN, -S(O) ２ NH ２ , nitro and sulfonic acid groups; and moiety-R Ｃ -R ６ -N(R ８Ａ )(R ８Ｂ is defined according to either (X) or (Y): (X): R Ｃ is -C(O)O-, -OC(O)-, -C(O)N(R Ｎ ), -N(R Ｎ ), -S(O) ２ N(R Ｎ ), -N(R Ｎ ), -N(R ２ ), -N(R Ｎ ), -C(O)-N(R Ｎ ), -N(R Ｎ ), -C(S)-N(R Ｎ ), -C(O)CH ２ -, -CH ２ C(O)-, -C(CF ３ ), -N(R Ｎ ), -N(R Ｎ ), -C(CF ３ ), -C(O)NF-, -NFC(O)-, -C(CN)=N-O-, -O-N=C(CN)-, -N(R Ｎ ), -C(O)O-, -OC(O)N(R Ｎ ), -phenylene, 5- to 6-membered heteroarylene, C ５－６ carbocyclylene and 5- to 6-membered heterocyclylene selected from the group consisting of, wherein each R Ｎ is independently a hydrogen atom or a C １－６ alkyl group, and the phenylene, the heteroarylene, the carbocyclylene and the heterocyclylene are each unsubstituted, or a halogen atom 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)(where n = 0 or 1), -(C １－６ alkyl) ｎ OC(O)(C １－６ alkyl)(where n = 0 or 1), C １－６ alkylthiol, -N(R Ｎ )([[]]wherein each R ２ is independently a hydrogen atom or a C Ｎ alkyl group １－６ ), -CN, -S(O) ２ NH ２ substituted with one or more substituents selected from nitro and sulfonic acid groups; R ６ is C １－６ an alkylene group, C ２－６ an alkenylene group or C ２－６ an alkynylene group, represents unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups; and R ８Ａ and R ８Ｂ are: (a) together with the nitrogen atom to which they are attached, form a 5- to 10-membered heteroaryl group or a 5- to 10-membered heterocyclyl group, said heteroaryl group and said heterocyclyl group being unsubstituted or substituted with one or more substituents 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) (where n = 0 or 1), -(C １－６ alkyl) ｎ OC(O)(C １－６ alkyl) (where n = 0 or 1), C １－６ alkylthiol, -N(R Ｎ )([[]ID=23]] ２ wherein each R Ｎ independently represents a hydrogen atom or a C １－６ alkyl group), -CN, -S(O) ２ NH ２ , nitro and sulfonic acid groups; or​ (b) independently C １－６ an alkyl group, C ２－６ an alkenyl group 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 hydroxy group; and is any of (Y): -R Ｃ -R ６ -N(R ８Ａ )(R ８Ｂ ), together form a group of formula (VI) 【Chemical Formula 3】 Here, R Ｃ２ is -C(O)-, -S(O) ２ -, -N(R Ｎ ), -C(O)-, -N(R Ｎ ), -C(S)-, -C(CF ３ ), or -OC(O)-, and Ring B is R Ｃ２ a 5- to 10-membered heterocyclylene ring containing both a nitrogen atom bonded to R ８Ｂ２ and a nitrogen atom bonded to R Ｎ２ wherein R ６２ and R are each an alkylene group, and R ８Ｂ２ is C １－６ an alkyl group, C ２－６ an alkenyl group or C ２－６ an alkynyl group, and is unsubstituted or substituted with one or more substituents selected from halogen atoms, sulfonic acid groups, and hydroxy groups) A ciclosporin analog or a pharmaceutically acceptable salt thereof, wherein is.

2. Part - R Ｃ -R ６ -N(R ８Ａ )(R ８Ｂ ) is defined according to (X), where R ８Ａ and R ８Ｂ are: (a) together with the nitrogen atom to which they are attached, form a 5- to 10-membered heteroaryl group or a 5- to 10-membered heterocyclyl group, said heteroaryl group and said heterocyclyl group being unsubstituted or substituted with one or more substituents 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) (where n = 0 or 1), -(C １－６ alkyl) ｎ OC(O)(C １－６ alkyl) (where n = 0 or 1), C １－６ alkylthiol, -N(R Ｎ )([[]ID=23] ２ wherein each R Ｎ independently represents a hydrogen atom or a C １－６ alkyl group), -CN, -S(O) ２ NH ２ , nitro and sulfonic acid groups; or​ (b) independently C １－６ an alkyl group, C ２－６ an alkenyl group or C ２－６ an alkynyl group, which is unsubstituted or substituted with one or more substituents selected from halogen atoms and sulfonic acid groups, and is any of the above, the cyclosporin analog according to claim 1.

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

4. Said C ６－１０ The cyclosporin analog according to claim 3, wherein the arylene group or 5- to 10-membered heteroarylene group is a naphthylene group.

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

6. The ciclosporin analog according to claim 3 or 5, wherein the 9- to 10-membered heteroarylene group is a quinolinylene, isoquinolinylene or indolinylene group.

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

8. In ring A, the substituent is C １－４ alkyl, C １－４ , haloalkyl and C １－４ alkoxy groups, preferably C １－４ alkyl group, the cyclosporin analog according to any one of the preceding claims.

9. The ciclosporin analog according to any one of the preceding claims, wherein ring A is unsubstituted or substituted with one or two of said substituents.

10. The ciclosporin analog according to claim 8, wherein ring A is unsubstituted.

11. Part-R Ｃ -R ６ -N(R ８Ａ )(R ８Ｂ ) is defined according to (X), and R Ｃ is -C(O)O-, -C(O)NH-, -S(O) ２ NH-, -NH-C(O)-NH-, -NH-C(S)-NH-, -NH-C(O)-O- and 【Chemical 4】 The ciclosporin analog according to any one of the preceding claims, which represents a group selected from the group consisting of.

12. R Ｃ The cyclosporine analog according to claim 1, wherein R is -C(O)O-.

13. Part-R Ｃ -R ６ -N(R ８Ａ )(R ８Ｂ ) is defined according to (X), and R ６ represents an alkylene group or a C ２－４ alkenylene group, preferably: (a) R ２－４ is ethylene, -(CH ６ ) ２ -; or (b) R ２ is -(CH ６ ) ２ ) ４ -, the cyclosporine analog according to any one of the preceding claims.

14. Part - R Ｃ -R ６ -N(R ８Ａ )(R ８Ｂ ) is defined according to (X), and R ８Ａ and R ８Ｂ are: (a) Together with the nitrogen atom to which they are attached, form a 5- to 6-membered heteroaryl group or a 5- to 6-membered heterocyclyl group; or (b) independently C １－４ represents an alkyl group or a C ２－４ alkenyl group, The ciclosporin analog according to any one of the preceding claims.

15. R ８Ａ and R ８Ｂ together with the nitrogen atom to which they are attached form an imidazolyl group, preferably of the general formula 【Chemical Formula 5】 The ciclosporin analog according to claim 14, which forms.

16. R １ represents hydrogen; R ２ is [Chemical Formula 6] represents; R ３ represents ethyl; R ４ represents methyl; R ５ is - CH ２ CH(CH ３ ) ２ represents; and R ７ The cyclosporin analog according to any one of the preceding claims, wherein R represents hydrogen.

17. The ciclosporin analog according to any one of the preceding claims, having at least one, preferably both, of (a) and (b): (a) The ciclosporin analog has a lower binding affinity for cyclophilin A (CypA) than for ciclosporin A (CsA); (b) The ciclosporin analog is an IFITM3 inhibitor.

18. The ciclosporin analog according to claim 1, which is a compound selected from the following compounds or a pharmaceutically acceptable salt thereof: 【Chemical Formula 7】 【Chemical Formula 8】 【Chemical Formula 9】 【Chemical 10】 【Chemical 11】 。

19. Use of a cyclosporin analog according to any one of claims 1 to 18 for enhancing the efficiency of transduction of an isolated mammalian cell population by a vector derived from HIV-1, HIV-2, SIV, FIV, BIV, EIAV, CAEV or visna lentivirus, preferably wherein the mammalian cells are human cells, more preferably wherein the mammalian 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, myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, and optionally, one or more cells selected from mammalian cells, human cells, 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, myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 are transduced by the vector, wherein the proportion of the one or more cells increases and / or the copy number of the vector per cell increases.

20. A method of transducing a mammalian cell population, preferably the mammalian cell is a human cell, more preferably the mammalian 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, such as 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 method comprising the following steps: a) contacting the cell population with a cyclosporine analog according to any one of claims 1 to 18; and 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 carried out ex vivo or in vitro; (ii) the proportion of one or more cells selected from 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 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, transduced by the vector increases and / or the copy number of the vector per cell increases; (iii) one or more cells selected from a mammalian cell population, 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, are obtained from or are obtained from mobilized peripheral blood, bone marrow or umbilical cord blood; and (iv) the method comprises a further step of enriching the population for one or more cells selected from 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 method of gene therapy comprising the following steps: a) transducing the mammalian cell population according to claim 20, preferably the mammalian cells are human cells, more preferably the mammalian 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, such as myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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; and b) administering the transduced cells to a subject; and optionally, the transduced cells are administered to the subject as part of an autologous stem cell transplantation procedure or an allogeneic stem cell transplantation procedure.

22. A mammalian cell population, 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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, prepared by the method according to claim 18, one or more cells selected therefrom.

23. A pharmaceutical composition comprising one or more cells selected from the mammalian cell population, 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 described in claim 22.

24. One or more cells selected from the mammalian cell population, 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 myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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 described in claim 22, for use in therapy, 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 analog according to any one of claims 1 to 18 for use in gene therapy of mammalian cells, human cells, hematopoietic stem cells and / or progenitor cells, induced hematopoietic stem cells and / or progenitor cells, and / or myeloid common progenitor cells, megakaryocytes, erythroblasts, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, lymphoid common 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.

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

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

28. Formula 【Chemical Formula 12】 Compound.

29. Formula 【Chemical 13】 Compound.

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