EA041412B1 - 5-[(2,4-DINITROPHENOXY)METHYL]-1-METHYL-2-NITRO-1H-IMIDAZOLE - Google Patents

Description

Technical field

This application proposes new phenyl derivatives. The novel compounds can be used to regulate mitochondrial activity, reduce obesity, treat diseases, including diabetes and complications associated with diabetes.

State of the art

Obesity is a well-known risk factor for many common diseases such as type 2 diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). Obesity should be considered any degree of excess fat deposition that poses a health risk. In the case where energy intake exceeds expenditure, excess calories accumulate predominantly in adipose tissue, and if this net positive balance is prolonged, obesity occurs, that is, there are two components of body weight stability, and abnormalities on either side (intake or expenditure) can lead to obesity. To prevent obesity, you can increase energy expenditure or reduce energy intake. Therefore, there is a need for pharmaceutical agents that are capable of controlling excess adipose tissue, for example by increasing energy expenditure or decreasing energy intake. The body obtains energy from the oxidation of food such as glucose and fatty acids. Mitochondria are known to control metabolism in individual cells by burning sugars and fats. One of the main functions of mitochondria is oxidative phosphorylation, which is the process by which energy obtained from the metabolism of energy sources such as glucose or fatty acids is converted into ATP (ATP). The formation of ATP in mitochondria is associated with the oxidation of NADH, which leads to the transport of protons in the electron transport chain. Chemical uncouplers (uncouplers) can interfere with effective energy production (ATP) in cells with mitochondria. They cleave oxidative phosphorylation by transporting protons across the mitochondrial membrane, resulting in rapid energy uptake (energy expenditure) without the formation of ATP. In other words, uncouplers flood the mitochondrial matrix with protons and NADH oxidation continues, but instead of producing energy in the form of ATP, the energy of the proton gradient is dissipated as heat. The use of chemical mitochondrial uncouplers to reduce fat deposits has been a scientific challenge for more than eighty years. See Simkins S Dinitrophenol and desiccated thyroid in the treatment of obesity: a comprehensive clinical and laboratory study. J Am Med Assoc 108: 2110-2117 (1937) and Fleury C et al, Nature Genetics 15, 269-272 (1997), Uncoupling Protein-2: A Novel Gene Linked to Obesity and Hyperinsulinemia. The best known chemical uncoupler is 2,4-dinitrophenol (DNP), which has been shown to increase energy expenditure in humans as well as animals. However, chemical uncouplers are often toxic. Concerns about dangerous side effects led to the removal of DNP from the market.

There is a need for safe mitochondrial uncouplers that can safely provide the desired medical effect without harm to humans. Disclosed in the present description of the new phenyl derivatives meet these needs.

The essence of the invention

A novel compound is proposed, 5-[(2,4-dinitrophenoxy)methyl]-1-methyl-2-nitro-1H-imidazole or a pharmaceutically acceptable salt thereof (Compound A). Also disclosed in the specification are compounds of formula I

Formula I or pharmaceutically acceptable salts of said compounds, wherein ring A is imidazole substituted with 1-3 substituents independently selected from -NO2 and methyl; each R ¹ is independently halogen, cyano, NO2, -C(O)H, -COOH, -C(O)O( _C1.4 alkyl), -C(O)( _C1.4 alkyl), _{C1- 4} alkyl, C _1-4 alkenyl, or C _1-4 alkynyl, wherein said C _1-4 alkyl, C _1-4 alkenyl, and C _1-4 alkynyl are each independently and optionally substituted with 1-3 substituents selected from the group consisting of halogen, NO2 and cyano; y is 1, 2, or 3; and x is an integer from 1 to 6.

These compounds can be used to regulate mitochondrial activity, reduce obesity, treat diseases including metabolic disorders, diabetes or diabetes-related complications such as heart disease and kidney failure, and reduce or control weight gain in a mammal.

Brief description of the drawings

In FIG. 1 shows the total exposure of DNP (gray) and Compound A (black) calculated from the area under the curve (AUC) during the first 24 hours after oral administration of the compound, and the corresponding concentration of the compound indicated under the x-axis. DNP did not survive the study, so the LD50 for DNP was 100 mpk (mgm/kg body weight). In FIG. 2 shows administration of Compound A to mice. Maximum concentration in

- 1 041412 plasma (Cmax) of the DNP residue is significantly reduced compared with the direct administration of DNP, the toxicity is also significantly less. In FIG. 3 shows that the total DNP exposure increases linearly up to at least 1500 mpk of Compound A. The total DNP exposure after administration of 100 mpk DNP is taken as 100% in this graph. Each data point is represented as a black dot. Straight linearity (Y = 0.1932-x+13.94) is represented by a solid black line, and the 95% confidence interval is represented by dotted lines. ^R2 = 0.9770. The exposure on the graph is expressed as a percentage of the total exposure to DNP, while the exposure after the introduction of 100 mpk DNP was taken as 100%.

In FIG. 4 shows that the maximum plasma concentration of DNP in mice that receive Compound A increases linearly with dose, but does not reach the levels observed with a dose of LD50 DNP. The maximum concentration of DNP after the introduction of 100 mpk DNP is taken in this graph as 100%. Each data point is represented as a black dot. Straight linearity (Y = 0/04178-x+11.34) is represented by a solid black line, and the 95% confidence interval is represented by dotted lines. ^R2 = 0.9770.

In FIG. 5 shows the plasma concentration of the liver enzymes ALT, AST and ALP in mice with induced fatty liver disease after 4 weeks of administration of Compound A.

In FIG. 6 shows the blood glucose levels in animals treated with Compound A compared with similar animals not receiving treatment, 120 min after glucose administration in all three treatment groups (p<0.05 for 25 and 100 mg/kg treatment, p <0.01 for 5 mg/kg treatment). All differences between vehicle and treatment recipients are statistically significant (p<0.05). An oral glucose tolerance test was performed after five weeks of treatment with Compound A.

In FIG. 7 shows lipid droplets in the liver of a control mouse in mice with MCD diet-induced NASH (NASH).

In FIG. 8 shows mouse lipid droplets in the liver of MCD diet-induced NASH mice treated with 5 mpk of Compound A.

In FIG. 9 shows the reduction in liver levels of TNFa and II.-1β in MCD diet-induced NASH mice treated with 5 mpk of Compound A.

In FIG. 10 shows the serum TG level for the study group from Example 5.

In FIG. 11 shows the serum FFA level for the study group from Example 5.

In FIG. 12 shows the level of TG in the liver for the study group from example 5.

In FIG. 13 shows the level of liver ceramides for the study group from example 5.

In FIG. 14 shows feed intake curves for the test groups from Example 6.

In FIG. 15 shows the level of free fatty acid (FFA) in the third group of example 6. It should be noted that p<0.05 compared to p<0.01 when compared with the vehicle treated group (mean ± SEM).

In FIG. 16 shows the blood levels of TG (triglycerides) from Example 6, which indicate that the results in the treatment groups were lower than in the vehicle control group in all study groups.

In FIG. 17 shows that TG in the liver were also lower in all study groups in example 6, and the decrease was statistically significant in the group receiving the highest levels of Compound A (5.0 mg/kg) with p<0.05 compared to the group treated with vehicle (mean ± SEM).

In FIG. 18 shows that blood insulin levels were lower in all treatment groups in Example 6.

In FIG. 19 shows the effect of sorafenib alone and in combination with Compound A in the treatment of the Hep3B-luc orthotopic model of human liver cancer as described in Example 7.

In FIG. 20 shows experimental design and disease progression in an animal model of diet-induced non-alcoholic fatty liver disease (see Example 8).

In FIG. 21 shows the change in body weight in an animal model of diet-induced non-alcoholic fatty liver disease after introduction of a Western diet (week 0) and during treatment (weeks 12-20). The data indicate a significant reduction in total body weight when treated with a high dose of Compound A (see Example 8).

In FIG. 22 shows a significant overall reduction in body weight in an animal model of diet-induced non-alcoholic fatty liver disease after eight weeks of high dose Compound A (see Example 8).

In FIG. 23 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on liver weight after eight weeks of administration (see Example 8).

In FIG. 24 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on liver weight after eight weeks of administration (see Example 8).

In FIG. 25 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on serum ALT (alanine aminotransaminase) levels (see Example 8).

In FIG. 26 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on serum Alk Phos (alkaline phosphatase) levels (see Example 8).

In FIG. 27 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on serum AST (aspartate transaminase) levels (see Example 8).

In FIG. 28 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on serum ALB (albumin) levels (see Example 8).

In FIG. 29 shows cell growth of NCI-60 cell lines treated with Compound A or DNP at 10 μM (see Example 10).

In FIG. 30 shows the formation of DNP from 2 μM Compound A in liver microsomes from different species and the need for NADPH for said formation (see Example 11).

In FIG. 31 shows a 7-day repeated dose toxicity study in mice to evaluate changes in behavioral and safety parameters.

Compound A, when administered orally at levels up to 500 mg/kg, did not cause renal dysfunction as measured by blood creatinine, while administration as low as 1 mg/kg or DNP resulted in an increase in blood creatinine. Changes in creatinine depend on Cmax.

In FIG. 32 shows the effect of a high dose of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on serum ALT, AST and ALP levels (see Example 8).

In FIG. 33 shows the effect of a high dose of Compound A on NAS and SAF activity indices. Both indexes were significantly lowered with Compound A.

Detailed description of the invention

Definitions

All terms used in this document have the meanings generally accepted by experts in the field of pharmaceuticals.

As used herein, an effective amount is defined as the amount necessary to produce a therapeutic effect on the patient being treated, and is generally determined based on the age, body surface area, weight, and condition of the patient.

As used herein, the term mammal, patient, or subject refers to any animal, including humans, livestock, and pets.

The phrase pet or pets refers to animals kept as pets. Examples of pets include cats, dogs and horses.

As used herein, the term control, treatment, includes: (1) inhibition of a disease, condition, or disorder, that is, stopping or reducing the development of a disease or its clinical symptoms/signs; or (2) alleviation of the disease, ie regression of the disease or its clinical symptoms/signs.

As used herein, the term pharmaceutically acceptable means suitable for use in mammals, domestic animals, or livestock.

As used herein, the term DNP refers to 2,4-dinitrophenol or a salt, solvate or adduct thereof.

As used herein, the term metabolic disorder refers to a condition characterized by an alteration or impairment of metabolic function.

As used herein, the phrase "pharmaceutically acceptable salt" refers to a salt that is a pharmaceutically acceptable non-toxic acid, including inorganic acids, organic acids, solvates, or hydrates.

As used herein, the term pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition, or carrier such as a liquid or solid excipient, stabilizer, dispersing agent, suspending agent, diluent, filler, thickener, solvent, or encapsulating material.

In one aspect, the present invention relates to compounds of Formula I

Formula I or a pharmaceutically acceptable salt of these compounds, wherein: Ring A is imidazole substituted with 1-3 substituents independently selected from -NO2 and methyl; each R ¹ is independently halogen, cyano, NO2, -C(O)H, -COOH, -C(O)O(C _1-4 alkyl), -C(O)(C _1-4 alkyl), C1 .4 alkyl, C1-4 alkenyl or C _1-4 alkynyl, where said C _1-4 alkyl, C _1-4 alkenyl and C _1-4 alkynyl are each

- 3 041412 are independently and optionally substituted with 1-3 substituents selected from the group consisting of halo, NO2 and cyano; y is 1, 2, or 3; and x is an integer from 1 to 6.

In some embodiments, ring A is imidazole substituted with 2 substituents independently selected from -NO ₂ and methyl; each R ¹ is independently halogen, NO ₂ , Ci-4 alkyl, Ci-4 alkenyl and C1.4 alkynyl, wherein said C1.4 alkyl and C1.4 alkenyl are each independently and optionally substituted with 1-3 substituents selected from the group , consisting of halogen, NO ₂ and cyano; y is 1, 2, or 3; and x is an integer from 1 to 3.

In some embodiments, ring A is imidazole substituted with 2 substituents independently selected from -NO ₂ and methyl; each R ¹ is independently halogen or NO ₂ ; y is 1, 2, or 3; and x is an integer from 1 to 2.

In some embodiments, ring A is imidazole substituted with 2 substituents independently selected from -NO ₂ and methyl; each R ¹ is independently halogen or NO ₂ ; y is 1, 2, or 3; and x is an integer from 1 to 2.

In some embodiments, ring A is imidazole substituted with 2 substituents independently selected from -NO ₂ and methyl; each R ¹ is NO ₂ ; y is 1 or 2 and x is an integer from 1 to 2.

In some embodiments, ring A is imidazole substituted with 2 substituents independently selected from -NO ₂ and methyl; each R ¹ is NO ₂ ; y is 2 and x is 1.

In some embodiments, Ring A is 1-imidazolyl, 5-imidazolyl, or 2-imidazolyl.

In some embodiments, Ring A is 1-imidazolyl or 5-imidazolyl.

In some embodiments, Ring A is 1-imidazolyl.

In some embodiments, Ring A is 5-imidazolyl.

In some embodiments, Ring A is 2-imidazolyl.

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, each R ¹ is independently halogen, cyano, NO ₂ , C 1.4 alkyl, or C 1.4 alkenyl, wherein said C 1.4 alkyl and C 1.4 alkenyl are each independently and optionally substituted with 1-3 cyano or fluoro substituents.

In some embodiments, the halogen substituent is selected from C1 and Br. In another embodiment, R ¹ is CH ₂ F, CHF ₂ or CF ₃ .

In some embodiments, each C1.4 alkyl is independently methyl, ethyl, propyl, or butyl. In some other embodiments, each C1.4 alkyl is independently propyl or butyl. In other embodiments, each C1.4 alkyl is independently butyl. In an additional embodiment, each C1.4 alkyl is t-butyl.

In some embodiments, each C1.4 alkenyl is independently ethenyl, allyl, but-3-en-1-yl, or but-2-en-1-yl optionally substituted with 1-3 cyano substituents. In some other embodiments, said C1.4 alkenyl is substituted with two cyano substituents. In another embodiment, said C _b4 alkenyl is

CN

CN

In some embodiments, R ¹ is NO ₂ .

In some embodiments, R ¹ is halo or NO ₂ .

In some embodiments, y is 2 and each R ¹ is NO ₂ or halogen.

In some implementations, the fragment

-4041412 selected from

In some embodiments, each R ¹ is independently halogen, cyano, NO2, C _1-4 alkyl, or C _1-4 alkenyl, wherein said C _1-4 alkyl and C _1-4 alkenyl are each independently and optionally substituted with 1-3 cyano substituents , and ring A is imidazole substituted with 2 substituents independently selected from -NO2 or methyl; or ring A is imidazole substituted with one -NO2 and one methyl; or ring A is selected from the group consisting of 1-imidazolyl, 5-imidazolyl and 2-imidazolyl; or ring A is selected from the group consisting of 1-imidazolyl and 5-imidazolyl; or ring A is 1-imidazolyl; or ring A is 5imidazolyl; or ring A is 2-imidazolyl; or ring A is or ring A is selected from

In some embodiments, each R ¹ is independently Cl, Br, cyano, NO2, methyl, ethyl, propyl, butylethenyl, allyl, but-3-en-1-yl, or but-2-en-1-yl, wherein said ethenyl, allyl, but-3-en-1-yl or but-2-en-1-yl is optionally substituted with 1-3 cyano substituents, and ring A is imidazole substituted with 2 substituents independently selected from -NO ₂ or methyl; or ring A is imidazole substituted with one —NO ₂ and one methyl; or ring A is selected from the group consisting of 1-imidazolyl, 5-imidazolyl and 2-imidazolyl; or ring A is selected from the group consisting of 1-imidazolyl and 5-imidazolyl; or ring A is 1imidazolyl; or ring A is 5-imidazolyl; or ring A is 2imidazolyl; or ring A is

or ring A is chosen from

In some embodiments, each R ¹ is independently Cl, Br, cyano, NO2, methyl, t-butyl, or ethenyl, wherein said ethenyl is optionally substituted with 1-3 cyano substituents and Ring A is imidazole substituted with 2 substituents independently selected from - NO ₂ or methyl; or ring A is imidazole substituted with one —NO ₂ and one methyl; or ring A is selected from the group consisting of 1-imidazolyl, 5-imidazolyl and 2-imidazolyl; or ring A is selected from the group consisting of 1-imidazolyl and 5-imidazolyl; or ring A is 1-imidazolyl; or ring A is 5-imidazolyl; or ring A is 2imidazolyl; or ring A is

- 5 041412 or ring A selected from selected from

In some embodiments, moiety and ring A is imidazole substituted with 2 substituents independently selected from -NO ₂ or methyl; or ring A is imidazole substituted with one —NO ₂ and one methyl; or ring A is selected from the group consisting of 1-imidazolyl, 5-imidazolyl and 2-imidazolyl; or ring A is selected from the group consisting of 1-imidazolyl and 5-imidazolyl; or ring A is 1-imidazolyl; or ring A is 5-imidazolyl; or ring A is 2-imidazolyl; or ring A is or ring A is selected from

In some implementations, y is 1. In some implementations, y is 2. In some implementations, y is 3.

In some implementations, x is an integer between 1 and 3. In another implementation, x is 1. In another implementation, x is 2.

In some embodiments, the compounds disclosed herein may be represented by Formula IIa:

or a pharmaceutically acceptable salt of a compound wherein R ¹ and y are as defined above. In some embodiments of Formula IIa, y is 1 and R ¹ is NO2. In another embodiment, y is 2 and each R ¹ is independently NO2 or halogen. In another embodiment, y is 2 and each R ¹ is independently NO2, Cl, or Br. In another embodiment, y is 3 and each R ¹ is independently NO2 or Cl. In another embodiment, y is 3 and each R ¹ is independently methyl or t-butyl. In another implementation,

CN is 3 and each R ¹ is independently t-butyl or .

- 6 041412

In some embodiments, the compounds disclosed herein may be represented by Formula IIb

Formula II or a pharmaceutically acceptable salt of a compound wherein R ¹ and y are as defined above. In some embodiments of Formula IIb, y is 1 and each R ¹ is independently NO2. In another embodiment, y is 2 and each R ¹ is independently NO2 or halogen. In another embodiment, y is 2 and each R ¹ is independently NO2 or Cl. In another embodiment, y is 3 and each R ¹ is independently NO2 or Cl.

In one embodiment, the novel compound of the present invention is selected from the compounds listed in Table 1. And lower.

Table A

Connection No. Name Structural formula 1 1-[2-(2,4-Dinitrophenoxy)ethyl]-2methyl-5-nitro-1H-imide azole ° ² N h o ₂ n ⁰ o ₂ N 2 (Compound A) 5 - [(2,4-Dinitrophenoxy)methyl] -1 methyl-2-nitro-1 H-imidazole '—Ν νό ₂ 3 1-Methyl-2-nitro-5-[(4nitrophenoxy)methyl]-1 H-imidazole N_/ ^N0 2 ο ₂ ν —* ^Ν 4 1-Methyl-2-nitro-5-[(3nitrophenoxy)methyl]-1 H-imidazole O0^'/r ^N ° ² ο ₂ ν ν 5 5 - [(3,5 - Dinitrophenoxy)methyl] -1 methyl-2-nitro-1 H-imidazole ο ₂ ν ο ₂ ν ^ν 6 5 - [(2,4-Dichl orphenoxy)methyl] -1 methyl-2-nitro-1 H-imidazole ¹ V°Xr° ² CI ^N 7 5 -[(2,4-Dibromophenoxy)methyl] -1 methyl-2-nitro-1 H-imidazole Br^^\ CH ₃ Br 8 5-[(2,6-Dichloro-4nitrophenoxy)methyl]-1-methyl-2nitro-1H-imidazole 1° ^! ^0%G ^g Cl 9 2-[3,5-Di-tert-butyl-4-((1-methyl-2nitro-1 H-imidazol-5 yl)methoxy)benzylidene]malononitri l and £ Az 47 _f ⁰ \ 7 O£ ^x Λ\----4-- ⁰ c /° 0 z 10 5-[(2,6-Di-tert-butyl-4methylphenoxy)methyl]-1-methyl-2nitro-1H-imidazole I _P I / \=/ I CO 4 ) ⁰ I J ΞΕ ω A=\ ^ω 1 zo A 3z AND 2-Methyl-5-nitro-1 -[(4nitrophenoxy)methyl] -1 H-imidazole O ₂ N-/A /-/4 O ₂ N 12 2-Methyl-5-nitro-1-[(3nitrophenoxy)methyl]-1 H-imidazole ABOUT 7z 13 1 - [(3,5 - Dinitrophenoxy)methyl] -2methyl- 5 -nitro-1 H-imide azole 0 z 0 p- Ύ/>- *—z

- 7 041412

- [(2,4-Dichl orphenoxy)methyl] -2methyl-5-nitro-1 H-imide azole

1-[(2,6-Dichloro-4nitrophenoxy)methyl]-2-methyl-5nitro-1H-imidazole

2-[(2,4-Dinitrophenoxy)methyl]-1 methyl-5-nitro-1 H-imide azole

2-[2-(2,4-Dinitrophenoxy)ethyl]-1 methyl-5-nitro-1 H-imide azole

1-Methyl-5-nitro-2-[(4nitrophenoxy)methyl]-1 H-imidazole

1-Methyl-5-nitro-2-[(3nitrophenoxy)methyl]-1 H-imidazole

2-[3,5 - Dinitrophenox and) methyl] -1 methyl- 5 -nitro-1 H-imide azole

2-[(2,4-Dichlorophenoxy)methyl]-1 methyl-5-nitro-1 H-imide azole

2-[(2,6-Dichloro-4nitrophenoxy)methyl]-1-methyl-5nitro-1H-imidazole

In one embodiment, the present invention provides a novel compound, 5-[(2,4Dinitrophenoxy)methyl]-1-methyl-2-nitro-1H-imidazole, or a pharmaceutically acceptable salt thereof.

In another embodiment, a novel compound of the present invention may be used to treat mitochondrial diseases including, but not limited to, obesity, diabetes, insulin resistance, and heart or kidney failure in a mammal in need thereof.

In another embodiment, a novel compound of the present invention may be used to treat diseases, disorders, and conditions associated with impaired mitochondrial function in a mammal in need thereof.

In another embodiment, a novel compound of the present invention can stimulate an oxygen consumption rate (OCR) in a mammal in need thereof.

In another embodiment, a novel compound of the present invention may be used to treat diabetic diseases including, but not limited to, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic steatosis, and type 2 diabetes (T2DM) in a mammal. in need of it.

In another embodiment, the novel compound of the present invention may be used to treat lipodystrophy (acquired or inherited) in a mammal in need thereof.

In another embodiment, a novel compound of the present invention may be used to treat hypertriglyceridemia in a mammal in need thereof.

In another embodiment, the implementation of a new compound in accordance with the present invention can be used to treat metabolic diseases or disorders in a mammal in need thereof.

In another embodiment, the implementation of a new compound in accordance with the present invention can be used to treat obesity or reduce obesity in a mammal in need thereof.

In another embodiment, a novel compound of the present invention may be used to treat or prevent weight gain or maintain weight in a mammal in need thereof.

In another embodiment, the novel compound of the present invention may be used to treat or prevent obesity or excess body fat in a mammal in need thereof.

In another embodiment, a novel compound of the present invention may be used to treat dyslipidemia in a mammal in need thereof.

- 8 041412

In another embodiment, the implementation of a new compound in accordance with the present invention can be used to treat cardiovascular diseases in mammals in need of it.

In another embodiment, the implementation of the new compound in accordance with the present invention can be used to treat heart disease in mammals in need of it.

In another embodiment, the implementation of a new compound in accordance with the present invention can be used to treat cardiovascular diseases in mammals in need of it.

In another embodiment, the novel compound of the present invention may be used to treat atherosclerosis in a mammal in need thereof.

In another embodiment, a novel compound of the present invention may be used to treat or prevent ischemic reperfusion injury in a mammal in need thereof.

In another embodiment, the implementation of a new compound in accordance with the present invention can be used to treat inflammation and fibrosis leading to NASH.

In another embodiment, the present invention provides pharmaceutical compositions containing a pharmaceutically acceptable carrier and a new compound in accordance with the present invention.

Routes of administration

In therapeutic use for the treatment or prevention of weight gain in a mammal, the compound of the present invention, or pharmaceutical compositions thereof, may be administered orally or parenterally.

In some embodiments, a compound of the present invention, or pharmaceutical compositions thereof, may be administered orally once a day.

Pharmaceutical salts

The compound of formula I may be used in native form or as a salt. In cases where a stable non-toxic acid or basic salt is desired, it may be appropriate to administer the compound as a pharmaceutically acceptable salt.

Suitable pharmaceutically acceptable salts include those derived from inorganic and organic acids, including sulfate, hydrosulfate, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, phosphoric, formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4hydroxybenzoic, phenylacetate, almond, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, sulfanilic, stearic,2 -hydroxyethanesulfonic, p-toluenesulfonic, cyclohexylaminosulfonic, salicylic, galactaric, β-hydroxybutyric and galacturonic acids; or derived from ammonium salts and metal salts, including calcium, magnesium, potassium, sodium and zinc salts.

Composition/Composition

Pharmaceutical compositions in accordance with the present invention can be prepared by methods well known in the art, for example, using known mixing, dissolving, granulating, panning, levitating, emulsifying, encapsulating, trapping, lyophilizing or spray drying processes.

Pharmaceutical compositions for use in accordance with the present invention can be prepared in a known manner using one or more pharmaceutically acceptable carriers containing excipients and excipients to facilitate the incorporation of the active compounds into preparations that can be used for pharmaceutical use. The appropriate preparation depends on the mode of administration chosen. Pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the present invention. These excipients and carriers are described, for example, in Remington's Pharmaceutical Sciences Mack Pub. Co., New Jersey (1991). Dosage

Pharmaceutical compositions suitable for use in the present invention include compositions in which the active ingredients are contained in an amount sufficient to achieve the intended purpose, that is, to treat or prevent weight gain or maintain body weight.

The amount of the active ingredient, which is a new compound according to the present invention, in the pharmaceutical composition and its single dose can be changed or adjusted depending on the activity of the particular compound and the desired concentration. Determining a therapeutically effective amount is within the skill of the art. As a rule, the amount of the active component is from 0.01 to 99.9 wt.% of the composition.

In general, a therapeutically effective dose amount of the active ingredient may be from about 0.001 mg/kg body weight/day to about 1000 mg/kg body weight/day. The required dose for ease of administration may be presented as a single dose or as multiple doses administered through

- 9 041412 appropriate intervals, for example, in the form of two, three, four or more parts of the dose per day.

In other embodiments, an effective amount of a novel compound of the present invention is from about 0.01 to about 1000 mg/kg, and any and all whole or partial increments in between, including about 0.1 mg/kg, about 1 mg /kg, about 0.01 mg/kg, about 0.1 mg/kg, about 1 mg/kg, about 10 mg/kg and about 100 mg/kg.

In some embodiments, the effective amount of the new compound is about 100-50 mg/kg. In some embodiments, the effective amount of the novel compound is about 50-10 mg/kg. In other embodiments, the effective amount of the novel compound is about 10-5 mg/kg. In other embodiments, the effective amount of the new compound is about 5-2.5 mg/kg.

Examples

Definitions:

ALT = alanine aminotransaminase.

AST = aspartate transaminase.

ALP = alkaline phosphatase.

ALB = albumin.

General synthesis scheme

Path B

Compounds of formula I can be obtained by synthetic methods known to those skilled in the art. The two methods are shown in Scheme 1, where the ring variables A, R ¹ , x and y are defined above and are not limited in any way. Many other methods for synthesizing compounds according to the invention are possible. As indicated in Scheme A, the Mitsunobu reaction can be used to activate the hydroxyl oxygen in the imidazole compound using a combination of reagents such as diisopropyl azodicarboxylate (DIAD) or diethyl azodicarboxylate (DEAD) with triphenylphosphine, resulting in nucleophilic displacement of the activated hydroxyl by phenol.

Compounds of formula I can also be prepared by nucleophilic aromatic substitution according to scheme B. In this case, the fluorophenyl compound is reacted with an imidazole compound under moderately basic conditions, for example, potassium carbonate in dimethylformamide. Replacement of the fluoride with the hydroxyl group of the imidazole compound provides an ether linkage of the compound of formula I.

Example 1 Plasma Concentration of DNP and Compound A Following Administration of DNP or Compound A

Materials and methods:

Male C57BL/6 mice aged 5-7 weeks weighing 18-20 g were obtained from Beijing Vital River Co., LTD. Animals were quarantined in polycarbonate cages in a well-ventilated, environmentally controlled facility at temperature (22 ± 3°C), relative humidity 40-80%, laminar flow, 3 animals in each cage for 7 days before and during research. Illumination with fluorescent lamps was provided for about 12 hours per day. Corn stalks bedding was changed once a week. Each animal was assigned an identification number. Mice had unlimited access to irradiation-sterilized dry kibble (Beijing Keaoxieli Feed Co., Ltd., Beijing, China) and sterile drinking water throughout the study period.

Animals were randomly distributed (n = 4) into appropriate groups depending on body weight using computer randomization. The following doses were given by gavage in 7.1% DMSO in saline: vehicle only (7.1% DMSO in saline), 100 mg/kg DNP and 1, 5, 10, 50, 100, 200, 300, 400 , 500, 600, 1000, 1250, 1500 mg/kg Compound A. DNP was obtained from Sinopharm Chemical Reagent Beijing Co., Ltd. DMSO was obtained from Sigma Aldrich.

Plasma was collected by ophthalmic puncture into 0.5 ml heparin-coated centrifuge tubes at 0, 15, 30, 45 min, 1, 2, 3, 4, 6, 8, 12, 20, and 24 h.

Samples were centrifuged for 5 min at 4000 rcf on a benchtop centrifuge. The clear supernatant was transferred to a new tube and stored at -80°C for PK (pharmacokinetic) analysis.

A full statistical analysis was performed, the significance level was set at P<0.05. Mean values for groups and standard errors were calculated for all measurement parameters in accordance with the study plan. Comparative One way ANOVA analyzes between groups were performed using SPSS 17.0 software.

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Results:

Two (50%) animals treated with DNP at 100 mg/kg died within the first 2 hours after administration, and one of the animals treated with 1500 mg/kg Compound A was found dead 12 hours later. no pathological clinical symptoms were observed during the entire experiment. PK analysis showed that Compound A was hydrolyzed to the remainder of DNP and Compound A was found in plasma only in small amounts in animals at the maximum dose. In FIG. 1 and 2 show that in animals treated with Compound A, the maximum plasma concentration (Cmax) of the DNP residue was significantly lower than with direct administration of DNP. None of the Compound A groups achieved the same Cmax as the DNP group. Tmax was delayed in Compound A treated animals. Significantly higher total DNP residue exposure was found in Compound A treated animals compared to DNP treated animals. Thus, Compound A is a safer drug than DNP due to the reduction in Cmax. Both the total exposure and the Cmax of the DNP residue increase linearly with increasing dose of Compound A.

Example 2 Plasma Concentration of Liver Enzymes ALT, AST and ALP Following Administration of Compound A to Mice with Induced Fatty Liver Disease

Male C57BL/6 mice were obtained from Beijing Vital River Co., LTD. Animals were quarantined in polycarbonate cages in a well-ventilated, environmentally controlled facility at temperature (22 ± 3°C), relative humidity 40-80%, laminar flow, 3 animals in each cage for 7 days before and during research. Illumination with fluorescent lamps was provided for about 12 hours per day. The mice had ad libitum access to irradiation sterilized dry kibble (Beijing Keaoxieli Feed Co., Ltd., Beijing, China) and sterile drinking water for the first week. After acclimatization and throughout the study period, the diet was changed to a methionine/choline-deficient diet (MCD) to induce non-alcoholic fatty liver disease (NAFLD) in the animals. After four weeks on MCD, animals were divided into four groups (n=8) and Compound A was administered at 0 mpk, 5 mpk, 25 mpk, or 100 mpk orally via gavage in 7.1% DMSO in saline.

Blood was collected in a tube without anticoagulant, serum samples were immediately processed by centrifugation at 4°C, 6000 g for 15 min, and then placed in a new tube. Samples were analyzed for three liver enzymes - ALT, ACT and ALP - using an automatic biochemistry analyzer TOSHIBA TBA-40FR.

Results:

As shown in FIG. 5, ALT and AST levels increased dramatically in mice treated with MCD. These levels decreased with the dose of Compound A. Statistically significant reductions were observed at 5 and 100 mpk, while statistical significance was only achieved in the 25 mpk dose level group with two outliers removed from the analysis.

Example 3 Oral glucose tolerance test following administration of Compound A to mice with induced fatty liver disease

Mice were prepared as described in Example 2. An oral glucose tolerance test (OGTT) was performed on all test animals after five weeks of treatment with Compound A. Baseline (time 0) glucose levels were measured after 16 hours of fasting. Following oral administration of 2 g/kg glucose, blood glucose levels were measured at 30, 60 and 120 minutes using the Accu-Chek Performa system.

Results:

Blood glucose levels were significantly lower 120 min after the glucose test in all three treatment groups (p < 0.05 for 25 and 100 mg/kg treatment, p 0.01 for 5 mg/kg treatment). See fig. 6.

Example 4 Evaluation of the effect of Compound A in a mouse model of NASH (NASH) induced by the MCD diet.

The present inventors have shown that Compound A reduces steatohepatitis and inflammatory cytokines in the liver of MCD diet-induced NASH mice. Lipid drops decreased after 6 weeks of treatment. See fig. 7 and 8. These images show a dramatic reduction in liver fat accumulation after treatment with 5 mg/kg Compound A. FIG. 7 shows lipid droplets in the liver of a control mouse in MCD diet-induced NASH mice. In FIG. 8 shows lipid droplets in the liver of MCD diet-induced NASH mice treated with 5 mpk of Compound A. Lipid droplets decreased dramatically after 6 weeks of treatment.

TNFa and IL-1e in the liver also decreased in treated mice (see Fig. 9).

Example 5 Evaluation of the effect of Compound A on the NAFLD model in HFD-induced rats.

To evaluate the effect of Compound A in rats fed a high fat diet (HFD), Compound A was administered orally via gavage once a day for 14 days. 50 SD rats aged 6 to 8 weeks were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were placed in quarantine at least 7 days before the start of the study. Animals

- 11 041412 were kept in laminar flow rooms at constant temperature and humidity, sterile drinking water was available without restriction, there was one animal in each cage. After a 7 day acclimatization period, rats were kept on a high fat diet (D12492, Research Diets) for a two week induction period. After the induction period, the animals were randomly assigned to appropriate groups based on body weight. The studied groups and detailed information on treatment are given in table. 1.

Table 1. Group and treatment

Group Treatment Dose (mg/kg) HFD Route of administration Scheme Qty 1 Carrier - Yes RO QD H14d 10 2 Compound A 0.1 Yes RO QD H14d 10 5 Compound A 0.5 Yes RO QD H14d 10 4 Compound A 5 Yes RO QD H14d 10 5 DNP 1 Yes RO QD H14d 10

Animals were treated with 5 ml/kg PO of Compound A, DNP, or vehicle alone (7.5% DMSO in water) orally via gavage daily for 14 days (day 15 to day 28). The body weight of all animals was measured twice a week throughout the study. Food intake was recorded for animals in all groups twice a week throughout the study. Blood samples were collected by ophthalmic puncture in a tube without anticoagulant on day 15 (before treatment), day 22 and day 29. Blood samples were centrifuged at 6000 g for 15 min at 4°C, then serum samples were collected and placed in another tube. If the analysis was not carried out immediately, serum samples were stored at -80°C. Lipid levels, including triglycerides (TG), total cholesterol (TCHO), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and free fatty acid (FFA), were measured at the end of the study using automated biochemical analyzer TOSHIBA TBA-40FR. After euthanasia of animals in all study groups on the 29th day, an autopsy was performed.

Liver tissue samples were taken from all animals, each liver sample was cut into 3 fragments: one for analysis of lipid levels in the liver, one for histology, and the last fragment was quickly frozen as a backup. At the end of the study, TG, TSHO, HDL-C, LDL-C and FFA levels in the liver were analyzed using a chemical analyzer, and ceramide levels in the liver were analyzed using the LC-MS/MS method.

The results of the study showed no significant changes or trends in glucose tolerance (measured using an oral glucose tolerance test), body weight, or food intake between study groups.

Serum FFA levels showed significant differences after seven days of dosing compared to vehicle, however serum FFA and TG levels tended to decrease in a dose dependent manner compared to vehicle control (see FIGS. 8 and 9). The data in FIG. 9 are statistically significant with p < 0.05 (T-test) when compared with the vehicle treated group (mean ± SEM). The results show that Compound A can be used to reduce the risk of cardiovascular disease, NASH and NAFLD. In the diagram in Fig. 10 shows serum TG levels. In FIG. 10 p<0.05 (ANOVA) (mean ± SEM). In the diagram in Fig. 11 shows serum FFA levels.

All liver lipid levels, including TG, TCHO, HDL-C, LDL-C, FFA, and ceramides, tend to decrease at all treatment doses. A significant decrease in FFA was observed at the highest two doses of Compound A. The decrease in hepatic ceramide levels also reached significance in the highest dose group. In FIG. 12 shows TG levels in the liver. In FIG. 13 shows the levels of ceramides in the liver.

Example 6 Evaluation of the effect of Compound A in diabetic obese rats (ZDF).

To determine the effect of Compound A administered orally by gavage once a day for 28 days to diabetic Zucker (ZDF) rats, 50 male rats from Beijing Vital River Laboratory Animal Technology Co. were obtained. studies were quarantined for 7 days. The rats were housed in laminar flow rooms at constant temperature and humidity, one animal per cage, water provided ad libitum during the quarantine and study periods. After a 7 day acclimatization period, 50 ZDF rats were kept on a special diet (Purina 5008 diet) for 4 weeks to induce type 2 diabetes. After 4 weeks of induction, animals were randomized into appropriate groups based on body weight and fasting glucose levels. The studied groups and the number of animals in the group are presented in table. 2.

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Table 2. Groups and treatment

Group Treatment Dose (mg/kg) Route of administration Scheme Quantity 1 Carrier - RO QD X28 d 10 2 DNP 1 RO QD X28 d 10 3 Compound A OD RO QD X28 d 10 4 Compound A 0.5 RO QD X28 d 10 5 Compound A 5.0 RO QD X28 d 10

The test preparations were dissolved in 7% aqueous DMSO (Sigma) (v/v) and administered orally in a volume of 5 ml/kg once a day for 30 days from day 29 to day 58. The compositions were prepared twice a week.

Body weight was measured twice a week throughout the study, food intake (feeding/withdrawal) was recorded for animals in all groups weekly throughout the study.

Fasting blood glucose levels of study animals were measured weekly after the induction period by tail vein blood sampling using the Accu-Chek Performa system. All trials were performed on the 29th (baseline), 36th, 43rd, 50th and 57th days. The animals were fasted overnight (16 hours from 17:00 to 09:00 the next day) before measurement.

Serum lipid profile and liver enzyme levels were measured weekly after the induction period, specific blood chemistry parameters are shown in Table 1. 3. All tests were performed on the 29th (baseline), 36th, 43rd, 50th and 57th days. Blood was collected from the ophthalmic veins in a tube without anticoagulant, serum samples were immediately processed by centrifugation at 4°C, 6000 g for 15 min, and then placed in a new tube. Lipid levels and total blood chemistry were measured using a TOSHIBA TBA40FR automatic chemistry analyzer.

Table 3. Biochemical analysis of blood

Category Reduction Definitions liver enzyme ALT Alanine aminotransferase AST Aspartate aminotransferase ALP Alkaline phosphatase blood lipid TG Triglycerides TCHO total cholesterol HDL-C high density lipoprotein cholesterol LDL-C LDL cholesterol FFA free fatty acid

Insulin levels in all test animals were measured on day 57 by ELISA. Serum was used for this analysis.

On the last day (day 59), a full autopsy was performed and the liver was removed from all animals. Liver samples were divided into 3 fragments: 1/3 was fixed in 10% formalin and processed in histological paraffin block, 1/3 was processed for lipid measurements (TG, TCHO, HDL-C, LDL-C and FFA), and the remaining 1 /3 was quickly frozen and stored at -80°C for further analysis.

Statistical tests were performed for all data, the significance level was set to 5% or P<0.05. Mean values for groups and standard deviations for all measurement parameters were calculated in accordance with the study plan. One-way analysis of variance (ANOVA) was used for groups with GraphPad Prism 6.0 software.

No significant changes in body weight and food intake were observed between the study groups, however, a trend towards higher food intake in the highest Compound A dosage group was observed in combination with a trend towards weight loss in all different dosage groups after day 36 in comparison with the carrier. In FIG. 14 shows feed intake curves for the test groups from Example 6.

In FIG. 15 shows the levels of NEFA (non-esterified fatty acid, i.e. free fatty acid (FFA)), which were significantly lower in the two groups that received the highest levels of Compound A. The third group, 0.1 mg/kg, also tended to decrease . See fig. 15. In FIG. 16 *p<0.05, **p<0.01 compared to vehicle treated group (mean ± SEM). In FIG. 16 shows blood levels of TG (triglycerides) that were similar and lower than vehicle control in all study groups (mean ± SEM). See fig. 16. On

- 13 041412 fig. 17 shows that liver TG levels were also lower in all study groups, and the reduction did indeed reach statistical significance in the group receiving the highest levels of Compound A (5.0 mg/kg) *p<0.05 compared to the group treated with vehicle (mean ± SEM). See fig. 17. In FIG. 18 shows that blood insulin levels were lower in all treatment groups. *p<0.05 compared to the vehicle treated group (mean ± SEM) (See Fig. 18). The study showed that Compound A may be effective in reducing the risk of heart and cardiovascular disease and may be used to treat NAFLD, NASH and type 2 diabetes.

Example 7 Evaluation of the effect of Compound A in an orthotopic mouse model of human liver cancer Hep3B-luc.

Sixty (60) female BALB/c nude mice were quarantined 7 days prior to the study. During the entire study period, animals were kept under standard laboratory conditions and provided free access to radiation-sterilized dry granular food and sterile drinking water. After a quarantine period, mice were inoculated in situ with 1 x 10E6 luciferase-expressing Hep3B-luc cells suspended in 10 μl of MEM/Matrigel (7:3). The skin and peritoneum were cut to expose the left lobe of the liver in the anesthetized mice, the cells were slowly injected into the left lobe of the liver so that a clear bubble of cells was visible through the liver capsule. Tumor growth was monitored by image analysis. On day 14, mice were randomized using computer randomization into 6 groups based on body weight and bioluminescent imaging (BLI) values (10 mice per group) to ensure a similar mean BLI among groups. The test animals were monitored not only for tumor growth, but also for behaviors such as mobility, food and water intake (only by checking the cage), body weight (BW), eye/hair dullness, and any other abnormal effect. To measure BLI, mice were intraperitoneally injected with 15 mg/ml (at 5 μl/g body weight) of D-luciferin (Pharmaron) and anesthetized by inhalation of 1-2% isoflurane. Ten minutes after luciferin injection, mice were imaged using IVIS Lumina II (Caliper) twice a week.

Living Image software (Caliper) was used to calculate regions of interest (ROI) and determine the total bioluminescent signal in each ROI. Bioluminescent signals (photons/s) from the ROI were quantified and used as an indicator of tumor growth and antitumor activity. Treatment was started when the average bioluminescent tumor signals reached about 52 x 10E6 photons/s on day 14 after tumor cell inoculation. Animals were divided into the following treatment groups (n = 10/group):

1. Carrier control

2. Sorafeniba tosylate 80 mg/kg

3. Sorafeniba tosylate 80 mg/kg + 25 mg/kg Compound A

4. Sorafeniba tosylate 80 mg/kg +100 mg/kg Compound A

5. Sorafeniba tosylate 80 mg/kg + 200 mg/kg Compound A

6. Sorafeniba tosylate 80 mg/kg + 300 mg/kg Compound A

All drugs were dissolved in 7% DMSO + 20% hydroxypropyl beta-cyclodextrin (HPBCD).

No changes in body weight were observed either during the trials or between the study groups. Sorafenib, as a single agent, was effective in reducing the BLI of human liver tumor Hep3B-luc by in vivo bioluminescence after 28 days of sequential treatment (see FIG. 19). However, this effect was not enhanced by Compound A at any of the dosage levels tested. Compound A does not appear to improve the effect of sorafenib and does not increase the sensitivity of cells to apoptosis. In fact, Sorafenib alone had the lowest BLI at the end of the study. All test preparations were well tolerated under the indicated test conditions in mice with orthotopic tumors.

No other major clinical abnormalities were observed in all animals during the treatment period. In FIG. 19 shows the effect of Sorafenib alone and in combination with Compound A in the treatment of Hep3B-luc, an orthotopic model of human liver cancer.

Example 8 Description of the effect of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease.

This study evaluated the effect of Compound A on steatohepatitis and fibrosis progression in a diet-induced non-alcoholic liver disease (DIAMOND) model in C57BL/6J(B6)-129s1/SvImJ male mice (S129). The Sanyal DIAMOND mouse model represents key physiological, metabolic, histological, transcriptomic, and cell signaling-related changes observed in humans with progressive NASH. See also Journal of Hepatology Volume 65, Issue 3, September 2016, Pages 579-588 A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer by A. Asgharpour et al.

Two dose levels of Compound A (daily dose of 1 or 5 mg/kg in vehicle for 8 weeks) were compared with vehicle control and data from previous studies in negative control mice in a strain dependent manner under normal diet (Harlan Normal Rodent Chow, TD 7012 Teklad

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LM-485) and reverse osmosis (RO) water.

At baseline (week 0), 30 male mice aged 8-12 weeks were fed an unrestricted Western diet, Harlan 42% calories from fat (Harlan TD. 88137), and sweetened water (SW 23.1 g/L d-fructose + 18 .9 g/l d-glucose). After disease progression in mice to steatohepatitis for 12 weeks, mice were randomly divided into three treatment groups (n = 10):

1. Vehicle control - 0.5% aqueous sodium carboxymethylcellulose with 0.1% Tween-80 (VC)

2. Compound A 1.0 mg/kg/day in vehicle (low dose of Compound A)

3. Compound A 5.0 mg/kg/day in vehicle (high dose of Compound A)

Two additional groups (data from previous studies) were also used for comparison.

4. Negative control - mice fed a normal diet (negative control 20 weeks)

5. Positive Control - Mice Fed on a Western Sweetened Water Diet, No Treatment, No Forced Feeding (Positive Control 20 Weeks) After eight weeks of treatment, all mice were autopsied after week 20. The authors of the present invention found that animals receiving a dose of 5 mg/kg had a significant decrease in body weight at week 20. Animals in this group also had a statistically significant reduction in liver weight, total cholesterol, ALT, ALP and ASP. Lobular inflammation was significantly reduced with Compound A. NAS and SAF activity indices were significantly reduced with Compound A. There was a significant reduction in progression to NASH with Compound A (only one mouse progressed to NASH when treated with Compound A, whereas in vehicle control, all mice progressed to NASH). According to the results of the study, Compound A is effective in the treatment of NAFLD and NASH, and may also be effective in reducing BMI in the treatment of obesity and in reducing the risk of heart disease. These results are shown in the following figures. In FIG. 20 shows experimental design and disease progression in an animal model of diet-induced non-alcoholic fatty liver disease. In FIG. 21 shows the change in body weight in an animal model of diet-induced non-alcoholic fatty liver disease after introduction of a Western diet (week 0) and during treatment (weeks 12-20). The data show a significant reduction in total body weight when treated with a high dose of Compound A. FIG. 22 shows a significant overall weight loss in an animal model of diet-induced non-alcoholic fatty liver disease after eight weeks of a high dose of Compound A. FIG. 23 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on liver weight after eight weeks of administration. In FIG. 24 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on liver weight after eight weeks of administration. In FIG. 25 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on serum ALT (alanine aminotransaminase) levels. In FIG. 26 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on serum ALP (alkaline phosphatase) levels. In FIG. 27 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on serum AST (aspartate transaminase) levels. In FIG. 28 shows the effect of low and high doses of Compound A in an animal model of diet-induced non-alcoholic fatty liver disease on serum ALB (albumin) levels.

Example 9 Preparation of 5-[(2,4-dinitrophenoxy)methyl]-1-methyl-2-nitro-1H-imidazole (Compound No. 2 in Table A or Compound A).

2,4-dinitrophenol (wetted with about 20% water, from TCI America, catalog number DO109) (wet wt 269 mg, dry wt 215 mg, 1.17 mmol) was dissolved in methylene chloride (2 mL) and stirred with anhydrous sodium sulfate at room temperature for 3 hours. The methylene chloride solution was decanted from the reaction flask and the sodium sulfate was washed with additional methylene chloride (2 ml). To the solution were added (1-methyl-2-nitro-1H-imidazol-5yl)methanol (115 mg, 0.732 mmol, prepared by the method described in US Pat. No. 8,003,625 B2) and triphenylphosphine (211 mg, 0.805 mmol). The mixture was stirred at room temperature until a solution was obtained. The solution was then cooled in an ice bath and treated with diisopropyl azodicarboxylate, DIAD (158 μl, 0.805 mmol). After 1 hour, it was removed from the ice bath and the mixture was stirred overnight at room temperature. The crude product was purified on a silica gel column to isolate the product mixed with triphenylphosphine oxide. The solids were triturated with tert-butyl methyl ether

- 15 041412 to remove triphenylphosphine oxide to give 5-[(2,4-dinitrophenoxy)methyl]-1-methyl-2-nitro1H-imidazole (70 mg, 0.236 mmol, 30% yield).

¹ H NMR (DMSO-d6) δ 8.80 (d, J=2.4 Hz, 1H), 8.58 (dd, J=9.6, 2.4 Hz, 1H); δ 7.82 (D, J=9.6 Hz, 1H), 7.40 (s, 1H), 5.66 (s, 2H), 3.95 (s, 3H). MS (ESI+) for C11H9N5O7 m/z 324.1 (M + H) ⁺ .

Example 10 Evaluation of the inhibitory effect of Compound A on a panel of NCI-60 tumor cell lines.

Human tumor cell lines of the Cancer Screening Panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening test, cells were inoculated into 96-well microtiter plates at 100 μl at seeding densities of 5,000 to 40,000 cells/well, depending on the doubling time of the individual cell lines. After cell inoculation, microtiter plates were incubated at 37° C., 5% CO ₂ , 95% air, and 100% relative humidity for 24 h before addition of experimental preparations.

After 24 hours, two plates of each cell line were fixed in situ with TCA to measure the cell population for each cell line at the time of drug addition (Tz). Experimental preparations were solubilized in dimethyl sulfoxide to 400 times the required final maximum test concentration and stored frozen prior to use. During drug addition, an aliquot of the frozen concentrate was thawed and diluted to twice the required final maximum test concentration with complete medium containing 50 μg/ml gentamicin. Aliquots of 100 µl of diluted drug were added to appropriate microtiter wells already containing 100 µl of medium to provide the desired final drug concentration.

After drug addition, the plates were incubated for an additional 48 hours at 37°C, 5% CO ₂ , 95% air and 100% relative humidity. For adherent cells, the assay was terminated by adding cold TCA. Cells were fixed in situ by carefully adding 50 μl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4°C. The supernatant was removed and the plates were washed five times with tap water and air dried. A solution of sulforodamine B (SRB) (100 μl) at a concentration of 0.4% (w/v) in 1% acetic acid was added to each well and the plates were incubated for 10 minutes at room temperature. After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were dried in air. The bound dye was then solubilized with 10 mM Trizma base and absorbance read on an automated plate reader at 515 nm. For suspension cells, the same procedure was used, except that the assay was terminated by fixing settled cells at the bottom of the wells with the careful addition of 50 μl of 80% TCA (final concentration, 16% TCA). Using seven absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of drug at five concentration levels (Ti)], the percent growth for each drug concentration level was calculated. The percentage of growth inhibition was calculated as:

[(Ti-Tz)/(C-Tz)] x 100 for concentrations where Ti >/= Tz >

[(Ti-Tz)/Tz] x 100 for concentrations where Ti<Tz.

Single dose data are presented as an average graph of percent growth of treated cells. The indicated number represents the growth relative to the control without drug and relative to the number of cells at the beginning of the countdown. This allows detection of growth inhibition (values from 0 to 100) as well as lethality (values less than 0).

In NCI-60 for Compound A, the mean percent growth over control was 98.63 with a delta of 26.47% for all cell lines tested and a range of 40.14%. Similarly, DNP had a mean percent control growth of 98.4% with a delta of 28.44 for all tested cell lines with a range of 44.43%.

The results for each cell line are presented below. Compound A and DNP did not cause strong growth inhibition (at least 50%) at 10 μM in any of the NCI-60 cell lines. In FIG. 29 shows growth of NCI-60 cell lines treated with Compound A or DNP at 10 μM.

Example 11 Formation of DNP from Compound A in liver microsomes.

Stage 1: The solution was prepared in accordance with the table. 4.

Table 4. Stock solution preparation

Reagent Initial concentration Volume Final concentration Phosphate buffer 200 mm 200 µl 100 mM High purity H2O - 106 µl - MgCh solution 50 mM 40 µl 5 mM Microsomes (20 mg/ml) 10 µl 0.5 mg/ml

- 16 041412

Step 2: 40 µl of 10 mM NADPH solution was added to each well. Final NADPH concentrations were 1 mM. The mixture was preheated at 37° C. for 5 minutes. Negative controls were prepared by replacing the NADPH solutions with 40 µl of ultra pure H ₂ O. The negative control was used to rule out an incorrect reading caused by chemical instability. Samples with NADPH were prepared in duplicate. Negative controls were prepared in a single copy (see Fig. 30, Formation of DNP from 2 μm Compound A in different types of liver microsomes). The source of obtaining the tested species is given in table. 5.

Table 5. Information on liver microsomes

View Catalog number Batch number Strain and gender Provider Human 452117 38291 United, males and females Corning Rat 452501 62547 United, male Sprague-Dawley Corning Mouse 452701 4133003 Consolidated, male CD-1 Corning Monkey (cyno) - ZDD United, male Cynomolgus RILD (Shanghai) Dog D1000 1310086 Consolidated, male beagle Xenotech

Step 3: The reaction was started by adding 4 µl of 200 µM solutions of control compound or test compound. Verapamil was used as a positive control in this study. The final concentration of test compound or control compound was 2 μM.

Step 4: 50 µl aliquots were taken from the reaction solution at 0, 15, 30, 45 and 60 minutes. The reaction was stopped by adding 4 volumes of cold methanol with IS (200 nM imipramine, 200 nM labetalol and 2 μM ketoprofen). Samples were centrifuged at 3220 g for 40 min. An aliquot of 90 μl of the supernatant was mixed with 90 μl of ultrapure H ₂ O and then used for LCMS/MS analysis.

Stage 5: Data analysis

All calculations were performed using Microsoft Excel.

Peak areas were determined on the extracted ion chromatograms.

The value of the slope to was determined using linear regression of the natural logarithm of the remaining percentage of the original drug on the curve of incubation time.

The results show that DNP formation is feasible between species and easily occurs with the help of shared liver enzymes.

Example 12 Proposed Criteria for Inclusion in a Phase 1/2 Clinical Trial

Inclusion criteria 2-3 markers of metabolic syndrome 10% liver fat ALT 40 or higher FIB4 greater than 1.1

Exclusion criteria: BMI <25, alcohol consumption

History of sinus tachycardia, ischemic disease, or renal dysfunction

Limit values of the studied performance indicators obtained at the research stage 1/2

Liver fat quantification using MRI-PDFF and MRE

Circulating CK18

Non-invasive breath test to assess liver mitochondrial activity (BreathID® System, Exalenz Biosciences)

Verification of target exposure and respondent PD ID and patient stratification

Quick validation of set parameters

The BreathID® system has been used in several NASH Phase II studies (NCT02314026, NCT01244503, NCT01281059).

Example 13 Synthesis of 1-[2-(2,4-dinitrophenoxy)ethyl]-2-methyl-5-nitro-1H-imidazole (Compound No. 1 in Table A).

2,4-dinitrophenol (wet with about 20% water) (wet weight 269 mg, dry weight 215 mg, 1.17 mmol) was dissolved in methylene chloride (2 ml) and stirred with anhydrous

- 17 041412 sodium sulfate at room temperature for 3 hours. The methylene chloride solution was decanted from the reaction flask and the sodium sulfate was washed with additional methylene chloride (2 ml). 2-(2-Methyl-5-nitroimidazol-1-yl)ethanol (125 mg, 0.732 mmol) and triphenylphosphine (211 mg, 0.805 mmol) were added to the solution. The mixture was stirred at room temperature until a solution was obtained. The solution was then cooled in an ice bath and treated with diisopropyl azodicarboxylate, DIAD (158 μl, 0.805 mmol). After 1 hour, it was removed from the ice bath and the mixture was stirred overnight at room temperature. The crude product was purified on a silica gel column to isolate the product mixed with triphenylphosphine oxide. The solids were triturated with tert-butyl methyl ether to remove triphenylphosphine oxide to give 1-[2-(2,4-dinitrophenoxy)ethyl]-2-methyl-5-nitro-1H-imidazole. MS (ESI+) for C ₁₂ HnN ₅ O ₇ m/z 338.1 (M + H) ⁺ .

Example 14 Synthesis of 1-methyl-2-nitro-5-[(4-nitrophenoxy)methyl]-1H-imidazole (Compound No. 3 in Table A).

4-Nitrophenol (162 mg, 1.17 mmol) was dissolved in methylene chloride (2 ml). (3methyl-2-nitro-3H-imidazol-4-yl)methanol (115 mg, 0.732 mmol) and triphenylphosphine (211 mg, 0.805 mmol) were added to the solution. The mixture was stirred at room temperature until a solution was obtained. The solution was then cooled in an ice bath and treated with diisopropyl azodicarboxylate, DIAD (158 μl, 0.805 mmol). After 1 hour, it was removed from the ice bath and the mixture was stirred overnight at room temperature. The crude product was purified on a silica gel column to isolate the product mixed with triphenylphosphine oxide. The solids were triturated with tert-butyl methyl ether to remove triphenylphosphine oxide to give 1-methyl-2-nitro-5-[(4-nitrophenoxy)methyl]-1H-imidazole. MS (ESI+) for C11H10N4O5 m/z 279.1 (M + H) ⁺ .

Example 15 Synthesis of 1-Methyl-2-nitro-5-[(3-nitrophenoxy)methyl]-1H-imidazole (Compound No. 4 in Table A).

3-Nitrophenol (162 mg, 1.17 mmol) was dissolved in methylene chloride (2 ml). (3methyl-2-nitro-3H-imidazol-4-yl)methanol (115 mg, 0.732 mmol) and triphenylphosphine (211 mg, 0.805 mmol) were added to the solution. The mixture was stirred at room temperature until a solution was obtained. The solution was then cooled in an ice bath and treated with diisopropyl azodicarboxylate, DIAD (158 μl, 0.805 mmol). After 1 hour, it was removed from the ice bath and the mixture was stirred overnight at room temperature. The crude product was purified on a silica gel column to isolate the product mixed with triphenylphosphine oxide. The solids were triturated with tert-butyl methyl ether to remove triphenylphosphine oxide to give 1-methyl-2-nitro-5-[(3-nitrophenoxy)methyl]-1H-imidazole. MS (ESI+) for C11H10N4O5 m/z 279.1 (M + H) ⁺ .

Example 16 Synthesis of 5-[(3,5-dinitrophenoxy)methyl]-1-methyl-2-nitro-1H-imidazole (Compound No. 5 in Table A).

3,5-Dinitrophenol (215 mg, 1.17 mmol) was dissolved in methylene chloride (2 ml). (3-Methyl-2-nitro-3H-imidazol-4-yl)methanol (115 mg, 0.732 mmol) and triphenylphosphine (211 mg, 0.805 mmol) were added to the solution. The mixture was stirred at room temperature until a solution was obtained. The solution was then cooled in an ice bath and treated with diisopropyl azodicarboxylate, DIAD (158 μl, 0.805 mmol). After 1 hour, it was removed from the ice bath and the mixture was stirred overnight at room temperature. The crude product was purified on a silica gel column to isolate the product mixed with triphenylphosphine oxide. The solids were triturated with tert-butyl methyl ether to remove triphenylphosphine oxide to give 5-[(3,5-dinitrophenoxy)methyl]-1-methyl-2-nitro-1H-imidazole.

MS (ESI+) for C11H ₉ N ₅ O ₇ m/z 324.1 (M+H)+.

Example 17 Synthesis of 5-[(2,4-dichlorophenoxy)methyl]-1-methyl-2-nitro-1H-imidazole (Compound No. 6 in Table A).

2,4-Dichlorophenol (190 mg, 1.17 mmol) was dissolved in methylene chloride (2 ml). To the solution was added

- 18 041412 (3-methyl-2-nitro-3H-imidazol-4-yl) methanol (115 mg, 0.732 mmol) and triphenylphosphine (211 mg, 0.805 mmol). The mixture was stirred at room temperature until a solution was obtained. The solution was then cooled in an ice bath and treated with diisopropyl azodicarboxylate, DIAD (158 μl, 0.805 mmol). After 1 hour, it was removed from the ice bath and the mixture was stirred overnight at room temperature. The crude product was purified on a silica gel column to isolate the product mixed with triphenylphosphine oxide. The solids were triturated with tert-butyl methyl ether to remove triphenylphosphine oxide to give 5-[(2,4-dichlorophenoxy)methyl]-1-methyl-2-nitro-1H-imidazoleα.

MS (ESI+) for Sc^C^O3 m/z 301.1 (M+H) ⁺ .

Example 18 Synthesis of 5-[(2,6-dichloro-4-nitrophenoxy)methyl]-1-methyl-2-nitro-1H-imidazole (Compound No. 8 in Table A).

CI

2,6-Dichloro-4-nitrophenol (243 mg, 1.17 mmol) was dissolved in methylene chloride (2 ml). (3-Methyl-2-nitro-3H-imidazol-4-yl)methanol (115 mg, 0.732 mmol) and triphenylphosphine (211 mg, 0.805 mmol) were added to the solution. The mixture was stirred at room temperature until a solution was obtained. The solution was then cooled in an ice bath and treated with diisopropyl azodicarboxylate, DIAD (158 μl, 0.805 mmol). After 1 hour, it was removed from the ice bath and the mixture was stirred overnight at room temperature. The crude product was purified on a silica gel column to isolate the product mixed with triphenylphosphine oxide. The solids were triturated with tert-butyl methyl ether to remove triphenylphosphine oxide to give 5-[(2,6-dichloro-4-nitrophenoxy)methyl]-1-methyl-2-nitro-1-nimidazole. MS (ESI+) for C11H ₈ Cl ₂ N ₄ O ₅ m/z 347.0 (M+H) ⁺ .

Example 19 Synthesis of 2-[(2,4-dinitrophenoxy)methyl]-1-methyl-5-nitro-1H-imidazole (Compound No. 16 in Table A).

F

Mixture of (1-methyl-5-nitro-1H-imidazol-2-yl)methanol (176 mg, 1.12 mmol) and 1-fluoro-2,4-dinitrobenzene (250 mg, 1.34 mmol) and K ₂ CO ₃ (465 mg, 3.36 mmol) in DMF (5 ml) was stirred for 2 h at ambient temperature. The reaction product was isolated by extraction. The residue was purified by preparative HPLC under the following conditions. Column: XBridge C18 OBD preparative column 19 x 150 mm, 5 µm; mobile phase A: water (10 mmol/l NH4HCO3), mobile phase B: ACN; flow rate: 20 ml/min; gradient elution. Fractions containing the product were collected and then lyophilized to give 2-[(2,4-Dinitrophenoxy)methyl]-1-methyl-5-nitro-1H-imidazole as a yellow solid. LC-MS: (ES, m/z) 324. (M+H) ⁺ .

¹ H-NMR: (400 MHz, DMSO-d ₆ ) δ 8.79 (d, J=2.8 Hz, 1H), 8.57 (dd, J1=2.8 Hz, J ₂ =9.2 Hz, 1H), 8.10 (s, 1H), 7.79 (d, J=9.6 Hz, 1H), 5.72 (s, 2H), 3.95 (s, 3H); analysis: C, 42.79; H, 3.43; N, 20.68; Oh, 33.25.

Formation of DNP from Compound B by liver microsomes

In accordance with the experimental procedure described in example 11, without changing the experimental conditions, but with the replacement of Compound A with the title compound in the human enzyme, the following results were obtained:

View Structure analysis DNP formation (µM) Percentage of education (%) 0 min. 15 minutes. 30 min. 45 min. 60 min. 0 min. 15 minutes. thirty min. 45 min. 60 min. Human With NADPH 0.031 0.038 0.025 0.023 0.018 1.56 1.90 1.26 1.13 0.91 Without NADPH 0.017 0.015 0.014 0.014 0.013 0.83 0.75 0.72 0.69 0.63

Example 20 Synthesis of 2-[2-(2,4-dinitrophenoxy)ethyl]-1-methyl-5-nitro-1H-imidazole (Compound No. 17 in Table A).

A mixture of 2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethanol (1.32 mmol) and 1-fluoro-2,4-dinitrobenzene (1.65 mmol) and K ₂ CO ₃ (465 mg, 3.36 mmol) in DMF (DMF) (5 ml) was stirred for 2 h at ambient temperature. The reaction product was isolated by extraction. The residue was purified by preparative HPLC under the following conditions. Column: XBridge C18 OBD preparative column 19 x 150 mm, 5 µm; mobile phase A: water (10 mmol/l NH4HCO3), mobile phase B: ACN; flow rate: 20 ml/min; gradient elution. Product-containing fractions were collected and then lyophilized with

- 19 041412 obtaining 2-[2-(2,4-dinitrophenoxy)ethyl]-1-methyl-5-nitro-1H-imidazole. LC-MS: (ES, m/z) 338.07 (M+H) ⁺ ; analysis: C, 42.62; H, 3.20; N, 20.83; Oh, 33.32.

Example 21 Synthesis of 1-methyl-5-nitro-2-[(4-nitrophenoxy)methyl]-1H-imidazole (Compound No. 18 in Table A).

A mixture of 2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethanol (1.24 mmol) and 1-fluoro-4-nitrobenzene (1.45 mmol) and K ₂ CO ₃ (465 mg, 3.36 mmol) in DMF (5 ml) was stirred for 2 hours at ambient temperature, after which it was heated. The reaction product was isolated by extraction. The residue was purified by preparative HPLC under the following conditions. Column: XBridge C18 OBD preparative column 19 x 150 mm, 5 µm; mobile phase A: water (10 mmol/l NH4HCO3), mobile phase B: ACN (acetonitrile); flow rate: 20 ml/min; gradient elution. Fractions containing the product were collected and then lyophilized to give 1-methyl-5-nitro-2-[(4-nitrophenoxy)methyl]-1H-imidazole. LC-MS: (ES, m/z) 279.07 (M+H) ⁺ ; analysis: C, 47.35; H, 3.71; N,20.24; Oh, 28.82.

Example 22 Synthesis of 1-methyl-5-nitro-2-[(3-nitrophenoxy)methyl]-1H-imidazole (Compound No. 19 in Table A).

A mixture of 2-(1-methyl-5-nitro-III-imidazol-2-yl)ethanol (1.32 mmol) and 1-fluoro-3-nitrobenzene (1.67 mmol) and K ₂ CO ₃ (465 mg, 3.36 mmol) in DMF (5 ml) was stirred for 2 h at ambient temperature, after which it was heated. The reaction product was isolated by extraction. The residue was purified by preparative HPLC under the following conditions. Column: XBridge C18 OBD preparative column 19 x 150 mm, 5 µm; mobile phase A: water (10 mmol/l NH ₄ HCO ₃ ), mobile phase B: ACN; flow rate: 20 ml/min; gradient elution. Fractions containing the product were collected and then lyophilized to give 1-methyl-5-nitro-2-[(3-nitrophenoxy)methyl]-1H-imidazole. LC-MS: (ES, m/z) 279.07 (M+H)+; analysis: C, 47.53; H, 3.69; N, 20.24; Oh, 28.85.

Example 23 Synthesis of 2-[(3,5-dinitrophenoxy)methyl]-1-methyl-5-nitro-1H-imidazole (Compound No. 20 in Table A).

A mixture of 2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethanol (1.22 mmol) and 3,5-dinitro-1-fluorobenzene (1.54 mmol) and K ₂ CO ₃ (465 mg, 3.36 mmol) in DMF (5 ml) was stirred for 2 h at ambient temperature, after which it was heated. The reaction product was isolated by extraction. The residue was purified by preparative HPLC under the following conditions. Column: XBridge C18 OBD preparative column 19 x 150 mm, 5 µm; mobile phase A: water (10 mmol/l NH4HCO3), mobile phase B: ACN; flow rate: 20 ml/min; gradient elution. Fractions containing the product were collected and then lyophilized to give 2-[(3,5-dinitrophenoxy)methyl]-1-methyl-5-nitro-1H-imidazole. LC-MS: (ES, m/z) 324.05 (M+H) ⁺ ; analysis: C, 40.90; H, 2.89; N, 21.72; Oh, 34.60.

Example 24 Synthesis of 2-[(2,4-dichlorophenoxy)methyl]-1-methyl-5-nitro-1H-imidazole (Compound No. 21 in Table A).

A mixture of 2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethanol (1.25 mmol) and 2,4-dichloro-1-fluorobenzene (1.45 mmol) and K ₂ CO ₃ (465 mg, 3.36 mmol) in DMF (5 ml) was stirred for 2 h at ambient temperature, after which it was heated. The reaction product was isolated by extraction. The residue was purified by preparative HPLC under the following conditions. Column: XBridge C18 OBD preparative column 19 x 150 mm, 5 µm; mobile phase A: water (10 mmol/l NH4HCO3), mobile phase B: ACN; flow rate: 20 ml/min; gradient elution. Fractions containing the product were collected and then lyophilized to give 2-[(2,4-dichlorophenoxy)methyl]-1-methyl-5-nitro-1H-imidazole. LC-MS: (ES, m/z) 302.00 (M+H) ⁺ ; analysis: C, 43.78; H, 3.08; C1, 23.52; N, 13.81; Oh, 15.99.

Example 25 Synthesis of 2-[(2,6-dichloro-4-nitrophenoxy)methyl]-1-methyl-5-nitro-1H-imidazole (Compound No. 22 in Table A).

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Claims (12)

A mixture of 2-(1-methyl-5-nitro-1H-imidazol-2-yl)ethanol (1.15 mmol) and 1,3-dichloro-2-fluoro-5nitrobenzene (1.24 mmol) and K ₂ CO ₃ (465 mg, 3.36 mmol) in DMF (5 ml) was stirred for 2 h at ambient temperature, after which it was heated. The reaction product was isolated by extraction. The residue was purified by preparative HPLC under the following conditions. Column: XBridge C18 OBD preparative column 19 x 150 mm, 5 µm; mobile phase A: water (10 mmol/l NH ₄ HCO ₃ ), mobile phase B: ACN; flow rate: 20 ml/min; gradient elution. Fractions containing the product were collected and then lyophilized to give 2-[(2,6-dichloro-4-nitrophenoxy)methyl]-1-methyl-5-nitro-1Himidazole. LC-MS: (ES, m/z) 346. (M+H)+; analysis: C, 38.01; H, 2.22; C1, 20.13; N, 16.19; 0.23.15. Example 26. Synthesis of Compounds 11, 12, 13, 14 and 15 in the table. A. In a reaction vessel, a mixture of an imidazole compound (1 molar equivalent), methylene iodide (1 molar equivalent), a potassium salt of a phenolic compound (1 molar equivalent), dry triethylamine (1 molar equivalent), and a catalytic amount of TBAB was dissolved in dry acetonitrile. The solution was refluxed for 2 hours followed by evaporation of the solvent. Then the residue was dissolved in CHCl ₃ and washed with water (2 x 200 ml). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated to give the crude product. Further purification was performed using silica gel column chromatography. CLAIM 1. 5-[(2,4-Dinitrophenoxy)methyl]-1-methyl-2-nitro-1H-imidazole or a pharmaceutically acceptable salt thereof. 2. A method of treating mitochondrial disorders or conditions in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the disorder or condition is selected from obesity, diabetes, insulin resistance or intolerance, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic steatosis, dyslipidemia, cardiovascular disease, heart disease, or atherosclerosis. 3. The method of claim 2, wherein the disorder is obesity, diabetes, or insulin resistance or insulin intolerance. 4. The method of claim 2, wherein the disorder is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic steatosis, or type 2 diabetes (T2DM). 5. The method of claim 2 wherein the disorder is obesity or excess body fat. 6. The method of claim 2 wherein the disorder is dyslipidemia. 7. The method of claim 2 wherein the disorder is a cardiovascular disease. 8. The method of claim 2 wherein the disorder is heart disease. 9. The method of claim 2 wherein the disorder is atherosclerosis. 10. A method of reducing obesity, controlling or preventing weight gain in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof. 11. A method of treating inflammation and fibrosis leading to non-alcoholic steatohepatitis (NASH) in a mammal in need thereof, comprising administering to the mammal an effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof. 12. A pharmaceutical composition for the treatment of mitochondrial disorders or conditions in a mammal in need thereof, containing a pharmaceutically acceptable carrier and a compound according to claim 1 or a pharmaceutically acceptable salt thereof, where the disorder or condition is selected from obesity, diabetes, insulin resistance or insulin intolerance, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic steatosis, dyslipidemia, cardiovascular disease, heart disease, or atherosclerosis. -