Diamidine analogs for anti-inflammatory use

Abstract

The present invention provides a pharmaceutical composition for preventing or treating inflammation in a subject; the compounds have the formula 1: wherein R1 comprises one or more cycloalkyl groups; and R2 comprises one or more cycloalkyl groups, or a pharmaceutically acceptable salt thereof; the invention also provides a method of treating or preventing inflammation in a subject by administering to the subject in need thereof a therapeutically effective amount of the pharmaceutical composition.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 492,661, filed March 28, 2023, the contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure relates to the treatment and prevention of inflammatory diseases or disorders, and more specifically to the treatment and prevention of inflammatory diseases or disorders by using a class of compounds that are diamidine analogs, namely pharmaceutical compositions. Background Technology

[0003] Recent studies have investigated the binding of pentamidine to lipopolysaccharides (LPS), as described by Wu et al. (2022) in *Frontiers in Pharmacology*, doi:10.3389 / fphar.2022.835081. LPS is a large molecule comprising lipids and polysaccharides. It is a bacterial toxin found in the outer membrane of Gram-negative bacteria. Therefore, it induces an inflammatory response in the host. Prolonged exposure to LPS can lead to inflammatory diseases or conditions.

[0004] For example, when LPS is present in the gut (due to the significant presence of Gram-negative bacteria), it can lead to inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. In some cases, LPS may also be present in the liver, where the resulting inflammation can lead to fatty liver disease.

[0005] In Wu et al. (2022), *Frontiers in Pharmacology*, doi:10.3389 / fphar.2022.835081, it was determined that the ether backbone of pentamidine participates in binding with LPS, thus preventing LPS-induced inflammation in subjects. The anti-inflammatory properties of pentamidine are weakened when the ether backbone is modified by adding additional oxygen. However, despite its anti-inflammatory properties, pentamidine exhibits significant levels of toxicity in subjects. Therefore, due to its toxicological characteristics, it would be unsuitable for the treatment of inflammatory diseases or conditions, as the harm would outweigh the benefit, especially in cases where long-term use of pentamidine is necessary to treat or prevent inflammation caused by or associated with inflammatory diseases or conditions. Summary of the Invention

[0006] This disclosure relates to a class of diamidine analogs. At least some of these compounds are suitable for treating LPS-mediated anti-inflammatory conditions in subjects.

[0007] Furthermore, it has been determined that the toxicity of pentamidine in subjects is due to its affinity for endo-exonucleases. Endonucleases play a role in DNA repair and recombination. For example, in U.S. Patent No. 5,324,830, Resnick et al. described the isolation of a DNA fragment encoding the endo-exonuclease RhoNuc from Saccharomyces cerevisiae. Therefore, inhibiting endo-exonuclease function in healthy cells using pentamidine may lead to toxicity in subjects.

[0008] This disclosure relates to a class of diamidine analog compounds that have been modified to reduce or prevent binding of the diamidine analog to endonucleases, thereby reducing toxicity to subjects from ingestion of the diamidine analog compared to pentamidine.

[0009] Compared to pentamidine, the diamidine analog compounds of this disclosure exhibit reduced binding activity to endonuclease activity, thereby reducing their toxicity when administered to subjects, while maintaining their binding affinity for LPS. Therefore, these diamidine analog compounds, due to their reduced toxicity, can be used to treat inflammatory diseases or conditions, exhibiting significantly lower toxicity compared to pentamidine.

[0010] In a broad aspect of this disclosure, such diamidine analog compounds have the following structure 1:

[0011] R1 and R2 have been substituted to reduce or prevent the binding of diamidine analogs to endonucleases and exonucleases. Appropriate substituents need to be selected because certain substituents at the R1 and R2 positions may actually increase the binding affinity to endonucleases and exonucleases, as illustrated in Chow's U.S. Patent No. 11,103,468. Furthermore, the size and / or nature of R1 and R2 may not prevent these substituents from hindering the binding of the compound to LPS through the compound's backbone.

[0012] Therefore, R1 and R2, which contain at least one five-membered cycloalkyl group, appear to produce enough changes in the structure of pentamidine (perhaps at least in part due to the bulkiness of the substituents containing larger ring structures) to interfere with the binding of the diamidine analog to endonucleases, thereby reducing toxicity to subjects while maintaining its interaction with LPS to eliminate LPS and thus reduce inflammation.

[0013] Diamidine analogs can be ingested and therefore administered orally. By comparing their known properties and mechanisms of action with those of pentamidine, the structure of diamidine analog compounds is significantly preserved during passage through the subject's digestive system and is at least partially absorbed by the subject's liver. Therefore, diamidine analogs can react with LPS present in the digestive system and / or liver, thereby eliminating toxins from the digestive system and / or liver, reducing inflammation, and treating or preventing associated diseases or conditions.

[0014] The first broad aspect is the compounds of formula 1: in: R1 includes one or more cycloalkyl groups; and R2 includes one or more cycloalkyl groups.

[0015] In some implementations, R1 and R2 can be the same.

[0016] In some embodiments, one or more cycloalkyl groups of R1 may be composed of a single cycloalkyl group, and one or more cycloalkyl groups of R2 may be composed of a single cycloalkyl group.

[0017] In some embodiments, the ring of one or more cycloalkyl groups of R1 may include ring atoms selected from C and N, and the ring of one or more cycloalkyl groups of R2 may include ring atoms selected from C and N.

[0018] In some embodiments, one or more cycloalkyl rings of R1 may be partially substituted, and one or more cycloalkyl rings of R2 may be partially substituted.

[0019] In some embodiments, the ring of one or more cycloalkyl groups of R1 may be aromatic, and the ring of one or more cycloalkyl groups of R2 may be aromatic.

[0020] In some embodiments, one or more cycloalkyl groups of R1 may include imidazole, and one or more cycloalkyl groups of R2 may include imidazole.

[0021] In some implementations, the compound may be:

[0022] In some implementations, the compound may be:

[0023] In some implementations, the compound may be: Where A1 can be C 1-7 Alkyl or OH-C1-7 Alkyl; and A2 can be C 1-7 Alkyl or OH-C 1-7 alkyl.

[0024] In some implementations, A1 and A2 can be the same.

[0025] In some implementations, A1 is OH-C 1-7 Alkyl group, and A2 can be OH-C. 1-7 alkyl.

[0026] In some implementations, the compound may be: Where A1 can be C 1-7 Alkyl or OH-C 1-7 alkyl; A2 can be C 1-7 Alkyl or OH-C 1-7 alkyl; A3 can be C 1-7 Alkyl, OH-C 1-7 Alkyl or hydrogen; or A4 can be C 1-7 Alkyl, OH-C 1-7 Alkyl or hydrogen.

[0027] In some implementations, A1 and A2 can be the same.

[0028] In some implementations, A1 is OH-C 1-7 Alkyl group, and A2 can be OH-C. 1-7 alkyl.

[0029] In some implementations, each of A1 and A2 may be selected from the group consisting of ethanol, propanol, methanol and isopropanol.

[0030] In some implementations, each of A3 and A4 may be selected from the group consisting of ethanol, propanol, methanol and isopropanol.

[0031] In some implementations, A3 and A4 can be the same.

[0032] In some implementations, each of A3 and A4 can be hydrogen.

[0033] In some embodiments, R1 may include at least two N atoms in at least one cycloalkyl monocyclic or polycyclic ring of one or more cycloalkyl groups, and R2 may include at least two N atoms in at least one cycloalkyl monocyclic or polycyclic ring of one or more cycloalkyl groups.

[0034] Another broad aspect is a pharmaceutical composition for the prevention or treatment of inflammation in a subject, comprising a compound of formula 1: in: R1 includes one or more cycloalkyl groups; and R2 includes one or more cycloalkyl groups, Or its pharmaceutically acceptable salt.

[0035] In some implementations, R1 and R2 can be the same.

[0036] In some embodiments, one or more cycloalkyl groups of R1 may be composed of a single cycloalkyl group, and one or more cycloalkyl groups of R2 may be composed of a single cycloalkyl group.

[0037] In some embodiments, the ring of one or more cycloalkyl groups of R1 may include ring atoms selected from C and N, and the ring of one or more cycloalkyl groups of R2 may include ring atoms selected from C and N.

[0038] In some embodiments, one or more cycloalkyl rings of R1 may be partially substituted, and one or more cycloalkyl rings of R2 may be partially substituted.

[0039] In some embodiments, the ring of one or more cycloalkyl groups of R1 may be aromatic, and the ring of one or more cycloalkyl groups of R2 may be aromatic.

[0040] In some embodiments, one or more cycloalkyl groups of R1 may include imidazole, and one or more cycloalkyl groups of R2 may include imidazole.

[0041] In some implementations, the compound may be:

[0042] In some implementations, the compound may be:

[0043] In some implementations, inflammation may occur in the intestines, and the pharmaceutical composition may be suitable for oral administration.

[0044] In some implementations, the inflammation may be caused by inflammatory bowel disease.

[0045] In some implementations, inflammation may occur in the liver, and the pharmaceutical composition may be suitable for oral administration.

[0046] In some implementations, the subjects may have fatty liver disease associated with inflammation in the liver.

[0047] In some implementations, the compound may be: Among them, B1, B2, B3, B4, B5, B6, B7, B8, B9, B 10 Each optional free C in 1-7 Alkyl, OH-C 1-7 A group composed of alkyl groups and hydrogen.

[0048] In some embodiments, one of the groups B1-B5 may be C 1-7 Alkyl or OH-C 1-7 Alkyl groups, and other groups in groups B1-B5 can be H, and groups B6-B... 10 One of them could be C 1-7 Alkyl or OH-C 1-7 Alkyl group, and group B6-B 10 Other groups in it can be H.

[0049] Another broad aspect is a method of treating or preventing inflammation in at least one of the intestines and liver of a subject, comprising administering to the subject in need a therapeutically effective amount of the compound according to this disclosure or a pharmaceutically acceptable salt thereof.

[0050] In some implementations, administration can be done orally.

[0051] In some implementations, the compound may be found in a slow-dissolving capsule to delay the release of the compound after oral administration.

[0052] In some implementation schemes, administration may be parenteral.

[0053] In some implementations, the inflammation being treated or prevented can be in the gut.

[0054] In some implementations, the inflammation may be caused by or related to inflammatory bowel disease.

[0055] In some implementations, inflammatory bowel disease can be ulcerative colitis.

[0056] In some implementations, inflammatory bowel disease can be Crohn's disease.

[0057] In some implementations, the inflammation being treated or prevented can be located in the liver.

[0058] In some implementations, the inflammation may be caused by or related to fatty liver disease.

[0059] Another broad aspect is the use of the compounds disclosed herein for treating or preventing inflammation in subjects.

[0060] In some implementations, the inflammation being treated or prevented can be in the gut.

[0061] In some implementations, the inflammation may be caused by or related to inflammatory bowel disease.

[0062] In some implementations, inflammatory bowel disease can be ulcerative colitis.

[0063] In some implementations, inflammatory bowel disease can be Crohn's disease.

[0064] In some implementations, the inflammation being treated or prevented can be located in the liver.

[0065] In some implementations, the inflammation may be caused by or related to fatty liver disease.

[0066] Another broad aspect is a method for treating or preventing inflammatory bowel disease in a subject. This includes administering, when inflammatory bowel disease is detected in a subject, a therapeutically effective amount of a compound according to this disclosure or a pharmaceutically acceptable salt thereof to a subject in need.

[0067] In some implementations, the detection can be performed by measuring LPS from an intestinal sample received from the subject.

[0068] In some implementations, the method may include determining the efficacy of the treatment by obtaining indicators of inflammation in the subject's gut through receiving a subsequent gut sample obtained after administration.

[0069] In some implementations, indicators of inflammation can be determined by measuring the level of LPS in intestinal samples.

[0070] Another broad aspect is a method for treating or preventing fatty liver disease in a subject. This includes administering a therapeutically effective amount of the compound according to this disclosure or a pharmaceutically acceptable salt thereof to the subject in need, upon detection of inflammatory bowel disease in the subject.

[0071] In some implementations, the detection can be performed by measuring LPS from an intestinal sample received from the subject.

[0072] In some implementations, the method may include determining the efficacy of the treatment by obtaining indicators of inflammation in the liver of the subject.

[0073] In some implementations, indicators of inflammation can be determined by measuring the level of LPS in the liver. Detailed Implementation

[0074] This disclosure relates to a class of diamidine analog compounds suitable for treating or preventing inflammatory diseases or conditions in subjects by binding with LPS (an inflammatory agent), while exhibiting reduced toxicity to subjects compared to pentamidine. Therefore, due to the reduced toxicity of this class of diamidine analog compounds, they are suitable for treating or preventing inflammatory diseases or conditions.

[0075] Diamidine analog compounds retain the ether backbone of pentamidine, which has been shown to confer the ability of pentamidine to target and bind to LPS, thereby inactivating or eliminating LPS and reducing inflammation in subjects caused by it. However, diamidine analog compounds include cycloalkyl groups as terminal substitutions, which reduce or prevent the binding of diamidine analogs to endonucleases and exonucleases, thus reducing the associated toxicity.

[0076] The diamidine analog compounds according to this disclosure have the following structure (1):

[0077] R1 and R2 are each one or more cycloalkyl groups used to reduce the binding of the compound to endonucleases when compared with pentamidine.

[0078] Due to the ability of pentamidine and therefore the diamidine analog compounds of this disclosure to be administered orally and to maintain significant integrity in the digestive system of a subject, the diamidine analog compounds of this disclosure are suitable for the treatment or prevention of inflammatory diseases and / or conditions located in the intestine (such as inflammatory bowel disease – e.g., Crohn's disease; ulcerative colitis). Furthermore, since the diamidine analog compounds reach the liver in the same way as pentamidine, they can also treat and / or prevent inflammatory diseases and / or conditions located in the liver (such as fatty liver disease).

[0079] Unless the context otherwise requires, throughout the specification and the following claims, the word “comprising” and its variations, such as “including” and “having”, shall be interpreted in an open, inclusive sense, meaning “including but not limited to”.

[0080] Throughout this specification, the reference to "an embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Therefore, the phrases "in an embodiment" or "in one embodiment" appearing in different places throughout this specification do not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in one or more embodiments in any suitable manner.

[0081] As used in this specification and the appended claims, the singular forms “a” and “the” include plural references, unless otherwise expressly stated. It should also be noted that the term “or” is generally used in its sense as including “and / or”, unless otherwise expressly stated.

[0082] As can be understood from the foregoing, although specific embodiments have been described herein for illustrative purposes, various modifications may be made without departing from the spirit and scope of the teachings. Therefore, the claims are not limited to the disclosed embodiments.

[0083] definition:

[0084] As used in this article, the term "C" 1-7 "Alkyl" refers to a straight-chain or branched hydrocarbon chain radical composed of carbon and hydrogen atoms, without unsaturated bonds, having one to seven carbon atoms, and connected to the rest of the molecule by single bonds. 1-7 Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, etc.

[0085] As used in this article, the term "C" 2-7 "Alkene" refers to a straight-chain or branched hydrocarbon chain group composed of carbon and hydrogen atoms, containing one or more double bonds, having two to seven carbon atoms, and connected to the rest of the molecule by single bonds. 1-7 Examples of olefins include, but are not limited to: ethylene, propylene, butene, etc.

[0086] As used in this article, the term "C" 2-7 Alkynes are straight-chain or branched hydrocarbon groups composed of carbon and hydrogen atoms, containing one or more triple bonds, having two to seven carbon atoms, and connected to the rest of the molecule by single bonds. 1-7 Examples of alkynes include, but are not limited to, acetylene and propyne.

[0087] As used herein, the term "cycloalkyl" refers to a saturated, partially saturated, or unsaturated five- to ten-membered monocyclic or polycyclic ring, wherein the ring atoms are selected from N, O, S, and C. Examples of cycloalkyl groups include, but are not limited to, azetidinyl, tetrahydrofuran, dihydrofuran, dioxane, morpholine, etc. A cycloalkyl group may optionally be composed of one to five independently selected, for example, hydroxyl, thiol, cyano, nitro, straight-chain or branched OH-C groups. 1-7 Substituents in the group consisting of alkyl, sulfonyl, halogen or amino groups.

[0088] As used herein, the term "intestinal sample" refers to a sample derived from the gastrointestinal tract of a subject. In some examples, an intestinal sample may specifically refer to the colon. In other examples, an intestinal sample may specifically refer to the ascending or descending colon. An intestinal sample may be a fecal sample or any other non-invasive sample derived from the subject's intestine. In some examples, an intestinal sample may involve obtaining a mucosal lumen interface (MLI) sample from the subject. In some examples, an intestinal sample may be, for example, a biopsy sample taken from the subject during a colonoscopy.

[0089] As used in this article, the term "C" 1-7 "Alkoxy" refers to the formula -OR a The group, wherein R a It is C as defined above. 1-7 Alkyl group. C 1-7 Examples of alkoxy groups include, but are not limited to: methoxy, propoxy, butoxy, pentoxy, etc.

[0090] As used in this article, the term "OH-C" 1-7 "alkyl" refers to C 1-7 alkyl groups, wherein C 1-7 One of the alkyl groups or one of the hydrogen atoms is replaced by "OH". Example: OH-C 1-7 Alkyl groups include, but are not limited to, hydroxy-methyl, hydroxy-ethyl, 2-hydroxy-propyl, 2-hydroxy-butyl, etc.

[0091] As used in this article, the term "amino-C" 1-7 "alkyl" refers to C 1-7 alkyl groups, wherein C 1-7 One hydrogen atom of the alkyl group is replaced by an amino group. Example: amino-C 1-7 Alkyl groups include, but are not limited to, amino-methyl, amino-ethyl, amino-propyl, 2-amino-butyl, etc.

[0092] As used in this article, the term "halogen" refers to bromine, chlorine, fluorine, or iodine.

[0093] As used in this article, the term "halogen-C" 1-7 "alkyl" refers to C 1-7 Alkyl groups, wherein one or more hydrogen atoms are replaced by halogens.

[0094] As used in this article, the term “inflammatory bowel disease” refers to ulcerative colitis (UC), subjects with Crohn’s disease (CD), and / or subjects with unclassified IBD (IBD-U).

[0095] As used herein, the term “inhibition” refers to the reduction or suppression of a given condition, symptom, disease, or ailment, or a decrease in the baseline activity of a biological activity or process.

[0096] As used herein, the term “measurement” as used herein refers to the analysis of a sample using techniques such as immunoassays (e.g., ELISA), semi-quantitative immunoblotting, mass spectrometry, and other techniques known in the art for the quantitative and / or qualitative analysis of sample contents obtained from a subject.

[0097] As used herein, the term "pharmaceutical composition" refers to a compound or a pharmaceutically acceptable salt thereof according to the present disclosure in a form suitable for oral or parenteral administration.

[0098] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that can be used in the preparation of a pharmaceutical composition and includes, for example, one or more of the following: diluents, surfactants, preservatives, buffers, isotonic agents, salts, excipients, lubricants, wetting agents, flavoring agents, etc.

[0099] As used in this article, the term "prevent (preventing or prevention)" refers to preventive treatment of a disease or condition, or delaying the onset or progression of a disease or condition.

[0100] In this disclosure, "subject" means mammal, such as human. The term "subject" should not impose any limitations regarding sex or age.

[0101] As used herein, the term “treatment” refers to the reduction or improvement of a subject’s outcome for a given disease or condition, which can be quantified by improving at least one physical parameter, ailment, or biomarker of a subject suffering from that disease or condition.

[0102] Exemplary Synthesis-Reaction Scheme 1:

[0103] The diamidine analog compounds disclosed herein can be produced by organic synthesis methods known in the art, with reference to the following reaction schemes and examples.

[0104] The starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents, etc. used to synthesize this compound can be commercially available or produced using known organic synthesis techniques.

[0105] As used herein, the term "salt" or "salt" refers to the acidic or basic salt of the compounds of this disclosure. The term "pharmaceutically acceptable salt" refers to a salt that retains the biological functions and properties of the compounds of this disclosure. For example, the compounds of this disclosure are capable of forming acidic and / or basic salts due to amino and / or carboxyl groups (or similar groups).

[0106] Pharmaceutically acceptable acid salts can be formed using inorganic acids (e.g., hydrochloric acid, sulfuric acid, nitric acid, etc.) and / or organic acids (e.g., acetic acid; oxalic acid; succinic acid; etc.).

[0107] Pharmaceutically acceptable basic salts can be formed using inorganic bases (such as sodium hydroxide, potassium hydroxide, magnesium hydroxide, etc.) and / or organic bases (such as isopropylamine, choline salts, lysine, etc.).

[0108] Any formula provided in this article is intended to also represent unlabeled or isotopically labeled forms.

[0109] Intermediates and final products can be purified using known methods (e.g., chromatography; partitioning; recrystallization, etc.).

[0110] The method for synthesizing the compound of formula (1) is provided in Scheme 1 below: Scheme 1: Exemplary synthesis of a compound of formula (II), wherein R2 is hydrogen.

[0111] Synthetic strategies for preparing compounds of formula (1) include using p-bromophenol, with the nitrile moiety at the p-position being substituted to obtain compound 5. Dianitronic acid 6 can be obtained by treating at least two equivalents of compound 5 with a suitable alkyl dibromide or alkyl dihalide or an alkyl reagent having two leaving groups in the presence of a base to obtain compound 6. Further reaction of 6 to convert the dinitronic acid to substance 1 can be accomplished by treatment with ethanol in the presence of HCl, followed by treatment with an equivalent of a suitable amine (R₂NH₂), and then treatment with ammonia in ethanol.

[0112] Exemplary synthesis-reaction scheme 2:

[0113] The synthetic strategy for preparing the compound of formula (1) involves converting the dinitrile into substance 1 using compound 6, which can be accomplished by treatment with ethanol in the presence of HCl, followed by treatment with an equivalent of a suitable amine (R1NH2) and an equivalent of a suitable amine (R2NH2). Scheme 2: Exemplary synthesis of the compound of formula (1).

[0114] Exemplary preparation of pharmaceutical compositions:

[0115] The pharmaceutical compositions disclosed herein can be prepared into pharmaceutically acceptable salts by reacting the basic form of the compound with a pharmaceutically acceptable acid. In some examples, alternatively, the free acid form of the compound can be reacted with a base.

[0116] The basic or acidic form of a compound can be prepared from the corresponding basic or acidic salt form.

[0117] Any asymmetric atom (e.g., carbon) of the compounds disclosed herein can be prepared in a racemic or enantiomerically enriched form, such as (R), (S), or (RS) configurations. In some embodiments, each enantiomer atom has at least 50% enantiomer excess. In some embodiments, each enantiomer atom has at least 60% enantiomer excess. In some embodiments, each enantiomer atom has at least 70% enantiomer excess. In some embodiments, each enantiomer atom has at least 80% enantiomer excess. In some embodiments, each enantiomer atom has at least 90% enantiomer excess. Substituents for unsaturated bonds can be present in either a -cis or -trans configuration.

[0118] Any mixture of stereoisomers obtained can be separated based on the physicochemical differences between their components, for example, by using chromatography and / or fractional crystallization.

[0119] This disclosure further includes any variations of the process, wherein intermediates available at any stage of the process may be used as starting materials. It should be understood that the compounds and intermediates disclosed herein can be converted between each other using techniques known in the art. When the production of certain materials is not specifically described herein, it should be understood that their synthesis can be carried out in accordance with teachings known in the art.

[0120] It should be understood that the above-described method is merely representative of the preparation method of this compound, and other well-known methods can be used alternatively to prepare the compound disclosed herein.

[0121] The following exemplary studies are provided to enable those skilled in the art to better understand this disclosure. As they are merely illustrative and representative examples, they should not limit the scope of this disclosure and are added for illustrative and representative purposes only. It should be understood that other exemplary studies may be used to further illustrate and represent this disclosure without departing from these teachings.

[0122] Exemplary Study 1:

[0123] Inhibition of endonucleases was measured using pentamidine and pentamidine analogs in which the central site (carbon backbone) of pentamidine was altered. In vitro inhibition of endonucleases was also tested. The results are shown in Table 1 below: Table 1: Inhibition of endonuclease activity after modification of the central carbon backbone.

[0124] Therefore, as shown in Table 1, modification of the central carbon backbone of pentamidine did not eliminate endonuclease activity. Since endonuclease inhibition is associated with the compound's cytotoxicity in patients, this modification of the central backbone of pentamidine, when administered to subjects for the treatment of inflammatory conditions, does not significantly reduce cytotoxicity. Instead, the inhibition of endonucleases by pentamidine is related to the structure of the pentamidine terminals (substitution at either end of the pentamidine molecule via a single bond to a nitrogen atom bonded to an adjacent carbon atom – where the hydrogen bonded to the nitrogen is replaced by another group). It has been further determined that substitution at these R1 and R2 positions of pentamidine with cycloalkyl groups (preferably having one or more nitrogen atoms) results in reduced inhibition of endonuclease activity by pentamidine analogs while retaining the compound's binding affinity for LPS. For example, suitable substituents at the R1 and R2 positions can be 3a,4,5,6,7,7a-hexahydrobenzimidazole or N-hydroxyethylimidazole.

[0125] discuss:

[0126] It has been found that adding large groups at the R1 and R2 positions of the following diamidine analogs can reduce or prevent the binding of pentamidine to endonucleases:

[0127] For example, it has been found that both R1 and R2 are defined as: -3a,4,5,6,7,7a-hexahydrobenzimidazole; or -N-hydroxyethylimidazole, It can prevent the binding of diamidine analogs to endonucleases while retaining their binding to LPS. Therefore, these diamidine analogs exhibit significantly reduced toxicity due to their reduced or prevented binding to endonucleases (whose activity has been associated with human toxicity), while also exerting anti-inflammatory effects through binding to LPS.

[0128] However, it is speculated that including large groups only at the R1 and R2 positions may not be sufficient to reduce the binding affinity of diamidine analogs to endonucleases and thus reduce toxicity. This is illustrated in Chow’s U.S. Patent No. 11,103,468, where substituting phenyl groups for R1 and R2, compared to pentamidine, actually increases the binding affinity of diamidine analogs to endonucleases.

[0129] Therefore, it is reasonable to believe that the inclusion of cycloalkyl groups with one or more nitrogen atoms or atoms in the same column of the periodic table as nitrogen (such as phosphorus) at the R1 and R2 positions is the cause of the reduced binding affinity of diamidine analogs to endonucleases, thereby reducing the toxicity of diamidine analogs after administration to subjects.

[0130] In Zhao E et al. (2017) *Hepatology* 66:922-935, pentamidine was shown to reduce liver triglycerides, serum ALT, body weight, and food intake in mice. This study demonstrated that pentamidine has been shown to reduce non-alcoholic fatty liver disease in mice. However, due to the interaction between pentamidine and endonucleases, this could lead to cytotoxicity in patients when treating fatty liver disease; therefore, reducing the endonuclease inhibition of the administered compound would be beneficial in reducing cytotoxicity. This can be achieved by substitution at the R1 and R2 positions of pentamidine, where more specifically, these substitutions include one or more ring structures (in some examples, preferably, including at least one nitrogen atom in the ring structure). Cycloalkyl substitution at the R1 and R2 positions of pentamidine has been shown to result in a reduction in endonuclease activity while preserving the compound's binding affinity for LPS.

[0131] Furthermore, it has been found that substitution at the R1 and R2 positions does not affect the binding affinity of the diamidinium analog to LPS, thus retaining its anti-inflammatory properties despite the R1 and R2 substitutions. However, maintaining the carbon-ether backbone of the diamidinium analog has been shown to be important for preserving its LPS binding.

[0132] The aromaticity of the rings in R1 and R2 does not appear to be essential for reducing or eliminating the binding of diamidine analogs to endonucleases and exonucleases. However, the presence of one or more unsaturated bonds in the rings of R1 and R2 of diamidine analogs appears to at least contribute to reducing the binding activity of diamidine analogs to endonucleases and exonucleases.

[0133] Exemplary methods for treating inflammatory bowel disease:

[0134] Exemplary uses of the diamidine analog compounds disclosed herein are for the treatment and / or prevention of inflammation caused by or associated with inflammatory bowel disease. This document references exemplary methods of treating inflammatory bowel disease using the diamidine analog compounds described herein.

[0135] It can be determined whether a subject has inflammatory bowel disease (IBD). The presence of IBD can be detected by measuring one or more biomarkers (e.g., calprotectin; LPS; etc.) from one or more intestinal samples received from the subject, symptoms experienced by the subject, etc., for purposes known in the art for detecting IBD in a subject.

[0136] When detecting or determining the presence of inflammatory bowel disease in a subject, a therapeutically effective amount of one or more diamidine analog compounds according to these teachings may be administered. In some embodiments, the diamidine analogs may be administered orally. In some cases, capsules containing one or more diamidine analogs may be sustained-release capsules to, for example, delay the release of one or more diamidine analog compounds until the capsule reaches the colon.

[0137] In some cases, diamidine analogs can be administered parenterally.

[0138] After administration of a therapeutically effective amount of one or more diamidine analog compounds, intestinal samples may subsequently be obtained from the subject to determine whether the inflammatory bowel disease is being effectively treated (wherein biomarkers may be measured from these intestinal samples and compared with corresponding results obtained from the initial intestinal sample).

[0139] Exemplary methods for treating fatty liver disease:

[0140] Exemplary uses of the diamidine analog compounds disclosed herein are for the treatment and / or prevention of inflammation caused by or associated with fatty liver disease. This document references exemplary methods for treating fatty liver disease using the diamidine analog compounds described herein.

[0141] It can be determined whether a subject has fatty liver disease. The presence of fatty liver disease can be detected by measuring one or more biomarkers (e.g., apolipoprotein A1, apolipoprotein B, leptin, adiponectin, free fatty acids, LPS, ghrelin, tumor necrosis factor-α, etc.) from one or more samples obtained from the subject, determined by symptoms described by the subject, etc., as known in the art for the purpose of detecting fatty liver disease in a subject.

[0142] When a subject is detected or diagnosed with fatty liver disease, a therapeutically effective amount of one or more diamidine analog compounds according to these teachings may be administered. In some embodiments, the diamidine analogs may be administered orally. In some cases, capsules containing one or more diamidine analogs may be sustained-release capsules to, for example, delay the release of one or more diamidine analog compounds.

[0143] In some cases, diamidine analogs can be administered parenterally.

[0144] Following administration of a therapeutically effective amount of one or more diamidine analog compounds, subsequent samples may be obtained from the subject to determine whether fatty liver disease is being effectively treated (where biomarkers may be measured from these samples and compared with the results of the initial samples received from the subject).

[0145] Representative, non-limiting examples of the invention have been described in detail above with reference to the accompanying drawings. This detailed description is merely intended to teach those skilled in the art to practice preferred aspects of the teachings and is not intended to limit the scope of the invention. Furthermore, each additional feature and teaching disclosed above and below may be used alone or in combination with other features and teachings to provide useful amidine and diamidine compounds and methods of using them to treat cancer.

[0146] Furthermore, the combination of features and steps disclosed in the above detailed description and experimental examples may not be necessary for practicing the invention in the broadest sense, but is taught only to specifically describe representative examples of the invention. In addition, the various features of the representative examples described above, as well as the various independent and dependent claims below, can be combined in a manner that is not specifically and explicitly enumerated to provide additional useful embodiments of this teaching.

[0147] For the purposes of the original written disclosure and for the purpose of limiting the claimed subject matter, all features disclosed in the description and / or claims are intended to be disclosed separately and independently of each other, independent of the composition of the embodiments and / or features in the claims. Furthermore, indications of all value ranges or entity groups are intended to disclose every possible intermediate value or intermediate entity for the purposes of the original written disclosure and for the purpose of limiting the claimed subject matter.

Claims

1. A compound of formula 1: in: R1 includes one or more cycloalkyl groups; and R2 includes one or more cycloalkyl groups.

2. The compound according to claim 1, wherein R1 and R2 are the same.

3. The compound according to claim 1 or claim 2, wherein the one or more cycloalkyl groups of R1 are composed of a cycloalkyl group, and the one or more cycloalkyl groups of R2 are composed of a cycloalkyl group.

4. The compound according to any one of claims 1 to 3, wherein the ring of the one or more cycloalkyl groups of R1 comprises ring atoms selected from C and N, and the ring of the one or more cycloalkyl groups of R2 comprises ring atoms selected from C and N.

5. The compound of claim 4, wherein the ring of the one or more cycloalkyl groups of R1 is partially substituted, and the ring of the one or more cycloalkyl groups of R2 is partially substituted.

6. The compound of claim 5, wherein the ring of the one or more cycloalkyl groups of R1 is aromatic, and the ring of the one or more cycloalkyl groups of R2 is aromatic.

7. The compound of claim 6, wherein the one or more cycloalkyl groups of R1 comprise imidazole, and wherein the one or more cycloalkyl groups of R2 comprise imidazole.

8. The compound according to claim 7, wherein the compound is:

9. The compound according to any one of claims 1 to 5, wherein the compound is:

10. A pharmaceutical composition for preventing or treating inflammation in a subject, comprising a compound of formula 1: in: R1 includes one or more cycloalkyl groups; and R2 includes one or more cycloalkyl groups, Or its pharmaceutically acceptable salt.

11. The pharmaceutical composition of claim 10, wherein the one or more cycloalkyl groups of R1 are composed of a cycloalkyl group, and the one or more cycloalkyl groups of R2 are composed of a cycloalkyl group.

12. The pharmaceutical composition according to claim 10 or claim 11, wherein the ring of the one or more cycloalkyl groups of R1 comprises ring atoms selected from C and N, and the ring of the one or more cycloalkyl groups of R2 comprises ring atoms selected from C and N.

13. The pharmaceutical composition of claim 12, wherein the ring of the one or more cycloalkyl groups of R1 is partially substituted, and the ring of the one or more cycloalkyl groups of R2 is partially substituted.

14. The pharmaceutical composition of claim 13, wherein the ring of the one or more cycloalkyl groups of R1 is aromatic, and the ring of the one or more cycloalkyl groups of R2 is aromatic.

15. The pharmaceutical composition according to any one of claims 10 to 14, wherein the one or more cycloalkyl groups of R1 comprise imidazole, and wherein the one or more cycloalkyl groups of R2 comprise imidazole.

16. The pharmaceutical composition according to claim 15, wherein the compound is:

17. The pharmaceutical composition according to any one of claims 10 to 15, wherein the compound is:

18. The pharmaceutical composition according to any one of claims 10 to 17, wherein the inflammation occurs in the intestine, and the pharmaceutical composition is suitable for oral administration.

19. Use of the compound according to any one of claims 1 to 9 for the treatment or prevention of inflammation in a subject.

20. The method of claim 19, wherein the inflammation is caused by or related to fatty liver disease.

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