RU2776845C2 - Celastrol analogues - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a compound having the structure

The invention also relates to a pharmaceutical composition for the treatment of obesity, based on the specified compound.

EFFECT: new compounds having improved oral bioavailability, and a pharmaceutical composition based on them are obtained that can be used in medicine for the treatment of obesity.

11 cl, 17 dwg, 17 tbl, 109 ex

Description

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority based on U.S. Provisional Application No. 62/245,356, filed Oct. 23, 2015, the entire contents of which are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

In 2008, the World Health Organization (WHO) estimated that 1.4 billion adults worldwide were overweight; of these, 200 million men and 300 million women were obese. More than one billion people in the world are predicted to be obese by 2030. Obesity is the leading cause of debilitating conditions such as type 2 diabetes, cardiovascular disease, osteoarthritis (a health problem that causes pain, swelling and stiffness of one or more joints), stroke, hypertension, cancer (of the breast, colon, endometrium (referring to the lining of the uterus), and kidney), and non-alcoholic steatohepatitis, all of which reduce quality of life and life expectancy.

There is now agreement among health experts around the world that the global obesity epidemic will be one of the leading causes of death for present and future generations unless the steady increase in the prevalence of this disorder is stopped. Although this problem was previously considered mainly in Western cultures, now developing countries have joined the ranks of countries suffering from obesity. In 1999, the OA found that obesity is present and growing rapidly in all developing regions, even in countries where hunger still exists. Obesity is defined by the World Health Organization (WHO) as a subject having a body mass index value (BMI=body weight in kg/height in m ² ) >30 kg⋅m ^-2 (normal BMI=20-25 kg⋅m ^-2 ).

Overweight and obesity occur when there is an energy imbalance. The body needs a certain amount of energy (calories) from food to maintain basic vital functions. Body weight remains unchanged when the number of calories eaten is equal to the number of calories the body uses or "burns". During the time when people eat and drink more calories than they burn, the energy balance shifts towards weight gain, overweight and obesity.

A possible explanation for the rapid increase in obesity is that it is driven by a combination of genetic, social and environmental factors. While a significant proportion of people manage to maintain a healthy body weight very effectively through careful nutrition and a reasonable level of exercise, for many others, this plan does not achieve the desired healthy state. For a subset of obese people, pharmacotherapy is required to provide the necessary additional support for nutrition, exercise, and lifestyle changes to achieve a clinically beneficial weight loss of >5%.

There is no single cause for all cases of overweight and obesity. There is no single approach that will help prevent or treat overweight and obesity. Treatment may include a combination of behavioral changes, diet, exercise, and sometimes weight loss medications. In some cases of extreme obesity, options may be surgery for weight loss. Only four new medicines have been registered in the last 15 years, i.e. dexfenfluramine (Redux®), sibutramine (Meridia®, Reductil®), orlistat (Xenical®), and rimonabant (Acomplia®), for the treatment of obesity. Of these medicines, only three, dexfenfluramine, sibutramine and orlistat, have received worldwide (except Japan) registration. There is a huge need to develop additional anti-obesity drugs that are safe and effective.

SUMMARY OF THE INVENTION

Presented herein are, inter alia , compositions containing the compounds described herein and methods for their use.

In various embodiments, the compounds provided herein contain structural modifications comparable to Celastrol.

In one aspect, the compositions may promote weight loss, reduce body fat, reduce food intake, improve homeostasis, or a combination thereof. The compounds have the structure of formula (I):

where

dotted lines between C ₁ and C ₂ , C ₂ and R ₃ , C ₃ and R ₄ , C ₅ and C ₆ , C ₅ and C ₇ , C ₁ and C ₆ and C ₃ and C ₄ indicate that a single or a double bond, as valence permits;

R ₁ is -CN, -COOH, -COOCH ₂ CH ₃ , -CONHR ₅ , -CONR ₅ R ₅ , -COOR ₅ , -COOCH _3, -CH ₂ NR ₅ R ₅ , -CH ₂ OCONR ₅ R ₅ , - CH ₂ NR ₅ COOR ₅ , -CH ₂ R ₅ , -CH ₂ NR ₅ CONR ₅ R ₅ , -CH ₂ OH, -CH ₂ OR ₅ , alkyl sulfate, alkyl sulfonate, alkyl phosphate, -CH ₂ OSO ₃ R ₅ , -CH ₂ OSO ₂ R ₅ , -CH ₂ OPO ₃ R ₅ R ₅ , -CH ₂ OPO ₃ HR _{5 ,} -CH ₂ OPO ₃ H _{2 ,} -C(=NR ₅ )NR ₅ R ₅ , -NR ₅ C(= NR ₅ )NR ₅ R ₅ , -CONH ₂ , -CH ₂ CONR ₅ R ₅ , -SR ₅ , -SO ₃ R ₅ , -SO ₂ R ₅ , -CH ₂ NHCOR ₅ , -CH ₂ NHCNR ₅ NR ₅ R ₅ , -CH ₂ COSR ₅ , CH ₂ NR ₅ COR ₅ , -CH ₂ NR ₅ CNR ₅ NR ₅ R ₅ , -CH ₂ NR ₅ COSR ₅ , -CH ₂ NHSO ₂ R ₅ , -CH ₂ NR ₅ SO ₂ R ₅ , -CHNR ₅ , -CHNOR ₅ , -H, -NH ₂ , -NHR ₅ , -NR ₅ R ₅ , -OH, -OR ₅ , phosphate, -OPO ₃ R ₅ R ₅ , -OPO ₃ HR _{5 ,} -OPO ₃ H ₂ , -NCO, -NCS, -N ₃ , -R ₅ , -C≡CR ₅ , -(CH=CH)R ₅ , -SH, -SR ₅ , -SO ₂ H, -SO ₃ H, -SO ₂ NR ₅ R ₅ , -SO ₃ R ₅ , -NHCOR ₅ , -NHCNR ₅ NR ₅ R ₅ , -NHCOSR ₅ , secondary amide, tertiary amide, -NR ₅ COR ₅ , -NR ₅ C( =NH)NR ₅ R ₅ , -NR ₅ COSR ₅ , -NHC(=NR ₅ )R ₅ , -NR ₅ C( =NR ₅ )R ₅ , -NHSO ₂ (NH ₂ ), -NHSO ₂ R ₅ , -NR ₅ SO ₂ R ₅ , -NR ₅ SO ₂ NR ₅ R ₅ , -OCOR ₅ , -OCONR ₅ R ₅ , - O(C=O)OR ₅ , -SCOR ₅ , -O(C=NH)NR ₅ R ₅ , -OCSNHR ₅ , -OS(=O ₂ )R ₅ , -OS(=O ₂ )NR ₅ R ₅ , -SCONR ₅ R ₅ , -CH ₂ -aryl, -CH ₂ -heteroaryl,

R ₂ is -H, -CH ₃ , -SCH(CH ₃ ) ₂ , -SC(=O)CH ₃ , -SC(=O)R ₅ , -SCH ₂ CH ₂ OCOCH _{3 ,} -SR ₅ , -SOR ₅ , -SOOR ₅ , -SCONR ₅ R ₅ ,

R ₃ is -OCOCH ₃ , -OCOOCH ₂ CH ₃ , -OR ₇ , -R ₇ or -NR ₅ R ₅ if a double bond is present between C ₁ and C ₂ , C ₃ and C ₄ and C ₅ and C ₆

R ₄ is -OCOCH ₃ , -OCOOCH ₂ CH ₃ , -OR ₇ , -R ₇ or -NR ₅ R ₅ if a double bond is present between C ₁ and C ₂ , C ₃ and C ₄ , and C ₅ and C ₆ ;

R ₃ is O if R ₄ is O and a double bond is present between C ₂ and R ₃ and C ₃ and R ₄ ;

R ₄ is -OCH _{3 ,} -OP(=O)(OCH ₃ ) ₂ , -OH, -OCOOCH ₂ CH ₃ , -OCONHCH ₂ CH ₃ , -OCOOCH(CH ₃ ) ₂ , -OR ₇ , -R ₇ or -NR ₅ R ₅ if R ₃ is O and a double bond is present between C ₂ and R ₃ ; R ₃ and R ₄ can also be combined to form a heterocyclic or carbocyclic ring;

R ₅ is independently selected at each occurrence from hydrogen, alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, amine, or heteroaryl, optionally substituted with substituents individually selected from alkyl, alkoxy, cycloalkyl, ether, amine, optionally substituted one or more alkyl, halogen, hydroxyl, ether, cyano, nitrile, CF ₃ , ester, amide, cycloalkylamide, sugar, heteroarylamide optionally substituted with alkyl and/or alkoxy, urea, carbamate, thioether, sulfate, sulfonyl, sulfonic acid , carboxylic acid and aryl, or two R ₅ groups taken together form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group optionally substituted with substituents individually selected from alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, heteroaryl, amine, halogen, hydroxyl, ether, nitrile, cyano, nitro, CF ₃ , ester, amide, m ocheva, carbamate, thioether or carboxylic acid group; and

R ₇ is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl or heteroaryl, optionally substituted with substituents individually selected from alkyl, cycloalkyl, ether, amine, halogen, hydroxyl, ether, nitrile, cyano, nitrile, CF ₃ ester, amide, urea, carbamate, thioester or carboxylic acid,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments, R ₁ is -NR ₅ C(=NR ₅ )NR ₅ R ₅ , -SR ₅ , -SO ₃ R ₅ , -SO ₂ R ₅ , -NH ₂ , -NHR ₅ , -NR ₅ R ₅ , -OH, -OR ₅ , -NCO, -NCS, -N ₃ , -SH, -SR ₅ , -SO ₂ H, -SO ₃ H, -SO ₂ NR ₅ R ₅ , -SO ₃ R ₅ , - NHCOR ₅ , -NHCNR ₅ NR ₅ R ₅ , -NHCOSR ₅ , -NR ₅ COR ₅ , -NR ₅ C(=NH)NR ₅ R ₅ , -NR ₅ COSR ₅ , -NHC(=NR ₅ )R ₅ , -NR ₅ C(=NR ₅ )R ₅ , -NHSO ₂ (NH ₂ ), -NHSO ₂ R ₅ , -NR ₅ SO ₂ R ₅ , -NR ₅ SO ₂ NR ₅ R ₅ , -OCOR ₅ , -OCONR ₅ R ₅ , -O(C=O)OR ₅ , -SCOR ₅ , -O(C=NH)NR ₅ R ₅ , -OCSNHR ₅ , -OS(=O ₂ )R ₅ , -OS(=O ₂ )NR ₅ R ₅ or -SCONR ₅ R ₅ .

In some embodiments, R ₂ is H.

In some embodiments, R ₄ is -OH, -OR ₇ or -R ₇ if R ₃ is O and a double bond is present between C ₂ and R ₃ .

In one aspect, the compositions contain compounds of the structure of formula (II):

where R ₁ is OR _a or NR _a R _b where R _a and R _b are each independently hydrogen, R ₅ , C(=NR ₅ )NR ₅ R ₅ , -CO, -CS, -COR ₅ , -CNR ₅ NR ₅ R ₅ , -COSR ₅ , -C(=NH)NR ₅ R ₅ , -C(=NR ₅ )R ₅ , -SO ₂ (NH ₂ ), -SO ₂ R ₅ , -SO ₂ R ₅ , -SO ₂ NR ₅ R ₅ , -COR ₅ , -CONR ₅ R ₅ , -(C=O)OR ₅ , -(C=NH)NR ₅ R ₅ , -CSNHR ₅ , -S(=O ₂ )R ₅ or -S(=O ₂ )NR ₅ R ₅ , and

R ₅ each as described in formula (I), or

a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments, R ₁ is NR _a R _b , and may be represented in formula (II)-a:

In certain embodiments, R ₁ is NH(CO)R ₅ where R ₅ is preferably alkyl, cycloalkyl, or aryl.

In certain embodiments, R ₁ is NHAc.

Typical connections, but not limited to, include the following connections:

,

,

,

,

,

,

и

.

,

,

,

,

,

,

and

.

In one aspect, a pharmaceutical composition is provided comprising the compounds described herein, for example, compounds of formula (I) and formula (II), including variants thereof, and a pharmaceutically acceptable excipient.

In one aspect, a method for treating obesity in a subject in need thereof is provided. The method includes administering to the subject a composition that contains an effective amount of a compound of formula (I), including variants thereof. Alternatively, a method for treating obesity in a subject in need thereof comprises administering to the subject a composition that contains an effective amount of compounds of formula (II), including variants thereof. Additionally, a method for treating obesity in a subject in need thereof comprises administering to the subject a composition that contains an effective amount of compounds of formula (I), formula (II), or combinations thereof, including variants thereof.

In some embodiments, the subject is leptin resistant. In some embodiments, the subject has elevated levels of leptin in, for example, blood. In certain embodiments, the subject has no response ( eg, decreased appetite, improved BMI, and/or weight loss of at least about 5%, 4%, 3%, 2%, or 1%) to leptin administration, and/ or the effectiveness of leptin administration declines over time ( eg, this is determined by the abolition of weight loss or more frequent hunger in the subject). In some embodiments, the subject has a blood or serum leptin concentration of about 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 ng/ml or more. In some embodiments, the subject is male and has a blood or serum leptin concentration of about 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50 ng/mL or greater. In some embodiments, the subject is a female and has a blood or serum leptin concentration of about 30, 35, 40, 45, 50, 75, 100 ng/mL, or greater.

In one aspect, administration of the composition includes oral administration, intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intra-arterial administration, intra-articular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppositories, inhaled administration or subcutaneous administration.

In some embodiments, a method for treating obesity in a subject in need thereof comprises orally administering a composition containing an effective amount of one or more of the compounds described herein.

In some embodiments, a method for treating obesity in a subject in need thereof comprises intraperitoneally administering a composition containing an effective amount of one or more of the compounds described herein.

In some embodiments, a method for treating obesity in a subject in need thereof comprises intraperitoneally administering a composition containing an effective amount of one or more of the compounds described herein.

In one aspect, a composition is provided wherein the composition is used to treat an obesity-related disease or disorder. The obesity-related disease or disorder is selected from the group consisting of obesity, pre-obesity, morbid obesity, Prader-Willi syndrome, obesity associated with hypothalamus injury, non-alcoholic steatohepatitis, hyperlipidaemia, hypertension, diabetes, lipodystrophy, hepatic steatosis, Bardet-Biedl syndrome, Cohen's syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome and MOMO syndrome (ocular anomalies of macrosomia, obesity, macrocephaly).

In one aspect, a method is provided for treating an obesity-related disease or disorder, comprising administering to a subject suffering from or susceptible to an obesity-related disease or disorder, one or more compositions of formula (I), formula (II), or combinations thereof. The obesity-related disease or disorder is selected from the group consisting of obesity, pre-obesity, morbid obesity, Prader-Willi syndrome, obesity associated with hypothalamus injury, non-alcoholic steatohepatitis, hyperlipidaemia, hypertension, diabetes, lipodystrophy, hepatic steatosis, Bardet-Biedl syndrome, Cohen's syndrome, cardiovascular disease, arthritis, stroke, metabolic syndrome, and MOMO syndrome.

In one aspect, the composition is administered in combination with another therapy.

In some aspects, administration also includes oral administration, intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intra-arterial administration, intra-articular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppositories, inhaled administration or subcutaneous administration.

In one aspect, the composition is administered in a form selected from the group consisting of pills, capsules, tablets, granules, powders, salts, crystals, liquid, serums, syrups, suspensions, gels, creams, pastes, films, patches, and vapors.

In one aspect, the subject is a mammal. Moreover, the subject is a person. In yet another aspect, the subject is a human with a body mass index (BMI) greater than 30 kg/m ² .

In one aspect, a method is provided for treating cancer in a subject in need thereof. The method includes administering an effective amount of one or more of the compounds described herein.

In one aspect, a composition is provided wherein the composition is used to treat a cancer-related disease or disorder. The cancer-related disease or disorder is selected from the group consisting of gastric cancer, multiple myeloma, melanoma, leukemia, lymphoma, renal cell carcinoma, renal cell carcinoma, breast cancer, prostate cancer, head and neck cancer, non-small cell lung cancer , brain cancer, and glioblastoma multiforme (GBM).

In one aspect, a method of treating a cancer-related disease or disorder is provided, comprising administering to a subject suffering from or susceptible to a cancer-related disease or disorder, one or more compositions of formula (I). The cancer-related disease or disorder is selected from the group consisting of gastric cancer, multiple myeloma, melanoma, leukemia, lymphoma, renal cell carcinoma, renal cell carcinoma, breast cancer, prostate cancer, head and neck cancer, non-small cell lung cancer , brain cancer, and glioblastoma multiforme (GBM).

In one aspect, a kit is provided that includes the compositions used in the treatment of obesity described herein and instructions for use in the treatment of obesity. In some embodiments, the kit may be used for oral administration or intraperitoneal administration of compositions for the treatment of obesity.

Each option described here is considered applicable to each of the other options described. Thus, all combinations of various elements described herein are included within the scope of the invention. Other aspects are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

On FIG. 1 is a graph of daily body weights in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 2 is a plot of area under the curve (AUC) of daily change in body weight in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 3 is a graph of the cumulative change in body weight in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 4 is a plot of area under the curve (AUC) of cumulative change in body weight in alimentary obese (AO) mice during oral treatment with compounds at 2,000 μg/kg as shown in Table 13 for 11 days.

In FIG. 5 is a graph of the cumulative change in body weight in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 6 is a graph of cumulative food intake in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 7 is a plot of area under the curve of cumulative body weight change (AUC) in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 8 is a plot of percent whole blood glucose between days 1 and 11 in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 9 is a graph of glucose values between days 1 and 11 in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 10 shows daily body weight data in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 11 shows daily weight change data in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 12 shows cumulative body weight change data in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 13 shows daily food intake data in alimentary obese (AO) mice following treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 14 shows cumulative food intake data in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 15 shows day 1 glucose data in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 16 shows day 11 glucose data in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

In FIG. 17 shows data on glucose changes in alimentary obese (AO) mice during oral treatment with compounds at 2000 μg/kg as shown in Table 13 for 11 days.

DETAILED DESCRIPTION OF THE INVENTION

I. DEFINITIONS

Abbreviations used here have the usual meaning used in the field of chemistry and biology. The chemical structures and formulas presented here are created according to the standard rules of chemical valency known in the field of chemistry.

As used herein, the term "about" in the context of numerical values or ranges means ±10% of the numerical value or range cited or claimed unless the context requires a more limited range.

It will be appreciated that if an interval of a parameter is presented, all integers in that interval and their tenths are also included in this invention. For example, "0.2-5 mg" includes 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, etc. up to and including 5.0 mg.

When substituent groups are defined by their usual left-to-right chemical formulas, they equally cover identical substituents that would result from right-to-left writing of the structure, eg, -CH ₂ O- is equivalent to -OCH ₂ -.

Certain compounds of this invention may exist in non-solvated forms as well as solvated forms, including hydrogenated forms. In general, solvated forms are equivalent to non-solvated forms and are included within the scope of this invention. Certain compounds in accordance with this invention may exist in a variety of crystalline or amorphous forms. In general, all physical forms are equivalent in their use contemplated in this invention and are included within the scope of this invention.

In this description, the term "salt" refers to ionic compounds that are obtained by the reaction of neutralization of an acid and a base. They consist of a conjugate number of cations (positively charged ions) and anions (negative ions) so that the product is electrically neutral (no full charge). These constituent ions may be inorganic, such as chloride (Cl ^- ), or organic, such as acetate (C ₂ H ₃ O ₂ ^- ); and may be monatomic, such as fluoride (F ^- ) or polyhydric, such as sulfate (SO ₄ ^2- ).

Certain compounds in accordance with this invention have asymmetric carbon atoms (optical or chiral centers) or double bonds; enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisomeric forms, which can be defined, in terms of absolute chemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids, and individual isomers are also included in the scope of this invention. The compounds of this invention do not include those known in the art to be too unstable to synthesis and/or isolation. This invention includes compounds in racemic and optically pure forms. Optically active (R)- and (S)- or (D)- and (L)- isomers can be obtained using chiral synthons or chiral reagents, or separated using conventional methods. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless otherwise indicated, the compounds are considered to include both E and Z geometric isomers.

As used herein, the term "isomers" refers to compounds having the same number and type of atoms, and therefore the same molecular weight, but differing in the structural arrangement or configuration of the atoms.

The term "tautomer" as used herein refers to one or more structural isomers which exist in equilibrium and which are easily converted from one isomeric form to another.

One skilled in the art will recognize that certain compounds of this invention may exist in tautomeric forms, where all such tautomeric forms of the compounds are included within the scope of the invention.

Unless otherwise indicated, structures depicted herein also include all stereochemical forms of the structure; those. , R and S configurations for each asymmetric center. Therefore, individual stereochemical isomers as well as enantiomeric and diastereomeric mixtures of these compounds are included within the scope of the invention.

Unless otherwise indicated, structures depicted herein also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the structures shown except for deuterium or tritium replacement of hydrogen, or replacement of carbon with ¹³ C- or ¹⁴ C-rich carbon, are included within the scope of this invention.

The compounds of this invention may also contain unnatural proportions of atomic isotopes on one or more of the atoms that make up such compounds. For example, compounds can be radiolabeled with radioactive isotopes such as, for example, tritium ( ³ H), iodine-125 ( ¹²⁵ I) or carbon-14 ( ¹⁴ C). All isotopic variants of the compounds of this invention, whether radioactive or not, are included within the scope of this invention.

It should be noted that in the application those alternatives are written in Markush groups, for example, each amino acid position that contains more than one possible amino acid. It is especially noted that each member of the Markush group should be considered separately, thereby constituting another option, and the Markush group should not be considered as one unit.

The single number used here means one or more. In addition, the phrase "substituted" in this description means that the specified group may be substituted by one or more of any or all of the listed substituents. For example, if a group such as an alkyl or heteroaryl group is "substituted with an unsubstituted C1-C20 alkyl or an unsubstituted _2-20 membered heteroalkyl", the group may contain _one or more unsubstituted C1-C20 alkyls and/or one or more unsubstituted 2 -20-membered heteroalkyls. Moreover, if a moiety is substituted with an R substituent, the moiety may be referred to as "R-substituted". If a moiety is R-substituted, the moiety is substituted with at least one R substituent, and each R substituent is optionally different from the other.

The term "alkyl", by itself or as part of another substituent, means, unless otherwise indicated, a straight (i.e., non-branched) or branched non-cyclic carbon chain (or carbon), or a combination thereof, which may be fully saturated, mono- or polyunsaturated, and may include di- and multivalent radicals having the indicated number of carbon atoms (ie, C1-C10 means from one to ten carbon atoms). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologues and isomers, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. An unsaturated alkyl group is one that has one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadianyl), 2,4-pentadianyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl and higher homologues and isomers. Alkoxy is alkyl attached to the rest of the molecule through an oxygen linker (-O-). The alkyl portion may be an alkenyl portion. The alkyl portion may be an alkynyl portion. The alkyl portion may be fully saturated.

The term "alkyl sulfate", by itself or as part of another substituent, means, unless otherwise indicated, alkyl substituted with O(SO ₂ )O ^- sulfate or a salt thereof.

The term "alkylsulfonate", by itself or as part of another substituent, means, unless otherwise indicated, alkyl substituted with (SO ₂ )O ^- sulfonate or a salt thereof.

The term "alkyl phosphate", by itself or as part of another substituent, means, unless otherwise indicated, alkyl substituted with phosphate PO ₄ ^- or a salt thereof.

The term "cycloalkyl", by itself or as part of another substituent, means, unless otherwise indicated, a monocyclic or polycyclic (e.g., bicyclic or tricyclic) saturated hydrocarbon that consists of hydrogen and carbon atoms arranged in a structure containing one ring or multiple rings. where all carbon-carbon bonds are single bonds. Examples of monocyclic alkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, and examples of polycyclic alkyl include norbornyl, adamantyl and the like.

The term "carbocyclic", by itself or as part of another substituent, means, unless otherwise indicated, a cyclic carbon chain (or carbon) which may be fully saturated, mono- or polyunsaturated and may include di- and polyvalent radicals having the indicated number of atoms carbon (i.e., C1-C10 means from one to ten carbon atoms). The carbocycle may have a structure containing one ring or multiple rings without limitation. Examples of saturated cyclic alkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, and examples of unsaturated carbocyclic groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl and the like.

The term "heteroalkyl", by itself or in combination with another term, means, unless otherwise indicated, a stable straight or branched non-cyclic chain, or a combination thereof, comprising at least one carbon atom and at least one heteroatom selected from a group comprising O, N, P, Si and S, and where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si, but not limited to, can be placed at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to: -CH ₂ -CH ₂ -O-CH ₃ , -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ -CH ₂ -N(CH ₃ )-CH ₃ , -CH ₂ -S-CH ₂ -CH ₃ , -CH ₂ -CH ₂ , -S(O)-CH ₃ , -CH ₂ -CH ₂ -S(O) ₂ -CH ₃ , -CH=CH-O-CH3 , -Si(CH ₃ ) ₃ , -CH ₂ -CH=N-OCH ₃ , -CH=CH-N(CH ₃ )-CH ₃ , -O-CH ₃ , -O-CH ₂ -CH ₃ and - CN. Up to two or three heteroatoms may be consecutive, as in, for example, -CH ₂ -NH-OCH ₃ and -CH ₂ -O-Si(CH ₃ ) ₃ . The heteroalkyl moiety may include one heteroatom (eg O, N, S, Si, or P). The heteroalkyl moiety may include two optionally different heteroatoms (eg O, N, S, Si, or P). The heteroalkyl portion may include three optionally different heteroatoms (eg O, N, S, Si, or P). The heteroalkyl moiety may include four optionally different heteroatoms (eg O, N, S, Si, or P). The heteroalkyl moiety may include five optionally different heteroatoms (eg O, N, S, Si, or P). The heteroalkyl portion may include up to 8 optionally different heteroatoms (eg O, N, S, Si or P).

The term "heterocyclic", by itself or in combination with another term, means, unless otherwise indicated, a cyclic chain containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and where the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S and Si can be placed at any internal position of the cyclic heteroalkyl group or at the position where the heterocyclic group is attached to the remainder of the molecule. Examples include, but are not limited to: -CO-, -OCOO-, -CH ₂ -CH ₂ -O-CH ₃ , -CH ₂ -CH ₂ -NH-CH ₃ , -CH ₂ -CH ₂ -N(CH ₃ )-CH ₃ , -CH ₂ -S-CH ₂ -CH ₃ , -CH ₂ -CH ₂ , -S(O)-CH ₃ , -CH ₂ -CH ₂ -S(O) ₂ -CH ₃ , -CH=CH-O-CH ₃ , -Si(CH ₃ ) ₃ , -CH ₂ -CH=N-OCH ₃ , -CH=CH-N(CH ₃ )-CH ₃ , -O-CH ₃ , - O-CH ₂ -CH ₃ and -CN. Up to two or three heteroatoms can be consecutive, such as -CH ₂ -NH-OCH ₃ and -CH ₂ -O-Si(CH ₃ ) ₃ . The heteroalkyl moiety may include one heteroatom (eg O, N, S, Si, or P). The heteroalkyl moiety may include two optionally different heteroatoms (eg O, N, S, Si, or P). The heteroalkyl moiety may include three optionally different heteroatoms (eg O, N, S, Si, or P). The heteroalkyl moiety may include four optionally different heteroatoms (eg, O, N, S, Si, or P). The heteroalkyl moiety may include five optionally different heteroatoms (eg O, N, S, Si, or P). The heteroalkyl portion may include up to 8 optionally different heteroatoms (eg O, N, S, Si or P).

The term "secondary amide", by itself or as part of another substituent, means, unless otherwise indicated, an amide in which the nitrogen atom is bonded directly to two carbon atoms.

The term "tertiary amide", by itself or as part of another substituent, means, unless otherwise indicated, an amide in which the nitrogen atom is bonded directly to three carbon atoms.

Description of the compounds in accordance with this invention is limited to the principles of chemical bonding known to experts in this field of technology. Therefore, if a group can be substituted with one or more of a variety of substituents, such substitution is chosen to follow the principles of chemical bonding and yield compounds that are not inherently unstable and/or that are known to those skilled in the art to be likely to be unstable under ambient conditions. such as aqueous, neutral and some known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in accordance with chemical bonding principles known to those skilled in the art to avoid compounds with intrinsic instability.

The terms "treat" or "treatment" refer to any indication of success in treating or alleviating an injury, disease, pathology, or condition, including any objective or subjective measure, such as a reduction in the severity of a symptom; remission; reduction in symptoms or greater tolerance by the patient to the injury, pathology, or condition; slowing down the rate of degeneration or deterioration in health; less debilitating end-stage degeneration; improvement in the physical or mental condition of the patient. Treatment or relief of symptoms may be based on objective or subjective parameters; including the results of a physical examination, neuropsychiatric studies, and/or a psychiatric evaluation. For example, certain methods in accordance with this invention treat diseases associated with weight gain, such as obesity.

An "effective amount" is an amount sufficient for a compound to accomplish its intended purpose, as compared to no compound (e.g., achieving the effect for which it is administered, treating a disease, reducing enzyme activity, increasing enzyme activity, reducing a signaling pathway, reducing one or more symptoms of a disease or condition An example of an "effective amount" is an amount sufficient to promote the treatment, prevention, or reduction of a symptom or symptoms of a disease, which may also be referred to as a "therapeutically effective amount". (s) or elimination of the symptom(s). the onset (or recurrence) of an injury, disease, pathology, or condition, or reduce the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition, or symptoms thereof. The full prophylactic effect is not necessarily achieved with a single dose and may only occur after multiple doses. Thus, a prophylactically effective amount may be administered in one or more administrations. "Amount that reduces activity", as used herein, refers to the amount of antagonist required to reduce the activity of an enzyme relative to the absence of an antagonist. The exact amounts depend on the goal of treatment and are determined by those skilled in the art using known methods (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding ( 1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

The term "associated" or "associated with" in the context of a substance or activity or function of a substance associated with a disease (e.g., obesity) means that the disease is caused (in whole or in part), or a symptom of the disease is caused (in whole or in part) by the substance or the activity or function of the substance. In this description, what is described as being associated with a disease, if a causative factor, may be the goal of treating the disease. For example, a disease associated with weight gain, such as obesity, may be treated with an agent (eg, a compound described herein) effective in reducing weight gain.

A "control" or "control experiment" or "standard control" is used according to its usual meaning, and refers to an experiment in which subjects or experimental reagents are treated as in a parallel experiment, except for the omission of a method, reagent, or experimental variable. In some cases, a control is used as a standard or for comparison in evaluating the effects of an experiment.

As defined herein, the term "inhibiting", "inhibits", "inhibiting", and the like, in relation to an interaction with a protein inhibitor (e.g., an antagonist), means a negative effect (e.g., reduction) on the level of activity or function of the protein compared to the level of activity or function of the protein in the absence of the inhibitor. In some embodiments, inhibition refers to the reduction of a disease or symptoms of a disease. Thus, inhibition may include at least partially, partially or completely blocking stimulation, reduction, prevention or delay of activation or inactivation, desensitization or suppression of signal transduction or enzyme activity or amount of protein.

As defined herein, the term "activation", "activate", "activating" and the like in relation to the interaction of a protein activator (e.g., agonist) means a positive effect (e.g., increase) on the activity or function of a protein compared to the activity or function of a protein in the absence of an activator (e.g., described here connections). Thus, activation may include at least partially, partially or completely up-stimulating, increasing or resolving activation, or activating, sensitizing or activating signal transduction or enzyme activity, or reducing the amount of a deleterious mediator/substance in disease. Activation may include at least partial, partial or complete up-stimulation, increase or resolution of activation, or activation, sensitization or activation of signal transduction or enzyme activity, or reduction of a deleterious mediator/substance.

The term "modulator" refers to a composition that increases or decreases the level of a target molecule or the function of a target molecule. In some embodiments, the modulator is an anti-inflammatory agent. In some embodiments, the modulator is a leptin inhibitor. In some embodiments, the modulator is a leptin ligand.

An "anti-obesity agent" refers to the property of a substance or treatment to reduce weight gain and promote weight loss. Examples of anti-obesity agents include Sibutramine, Phentermine, Mazindol, Diethylpropion, Leptin, Orlistat, Beta-3 agonists and Rimonabant.

The term "obese" in this description refers to a patient having a body mass index of more than 30 kg/m ² . "Overweight" and "pre-obese" as used herein refers to patients having a body mass index greater than 25 kg/m ² . "Morbid obesity" as used herein refers to a patient having a BMI greater than 40 kg/m ² , a BMI greater than 35 kg/m ² in association with one or more comorbidities, a BMI greater than 30 kg/m ² in association with uncontrolled diabetes, or a combination thereof .

The term "prodrug" refers to a pharmacological agent, such as a drug, that is administered to a subject in an inactive (or significantly less active) form. Upon administration, the prodrug is metabolized in the body ( in vivo ) to a compound having the desired pharmacological activity.

The terms "patient", "subject", "individual", and the like, refer to a living organism suffering from or susceptible to a disease or condition that can be treated by administering a compound or pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, cats, primates, monkeys, goats, sheep, cows, deer, and other non-mammals. In some embodiments, the subject is a pet such as a dog or cat. In some embodiments, the patient is a human. In some embodiments, the patient is preobese, obese, or morbidly obese. In certain embodiments, the patient is not pre-obese, obese, or morbidly obese, but has previously been pre-obese, obese, or morbidly obese. In some embodiments, the patient desires to reduce body weight or has a reduced appetite. Alternatively, or in addition, the patient has an obesity-related disease or disorder. These examples are non-limiting. The terms "patient", "subject", "individual", and the like as used herein are not limiting and, in general, can be used interchangeably. That is, an individual described as a "patient" does not necessarily have the disease or is under the care of a medical professional, but may simply be seeking or desiring treatment in the absence of medical advice (eg, self-medication). A "disease" or "condition" refers to a condition or health condition of a patient or subject that can be treated with a compound, pharmaceutical composition, or method provided herein. In some embodiments, the disease is a disease that causes weight gain. In some embodiments, the disease is obesity. Obesity may be the primary cause of the disease and/or disorder being treated, or may also be the result of a primary disease and/or disorder.

A "pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refers to a substance that aids administration of the active agent and absorption by the subject and can be incorporated into the compositions of this invention without causing significant toxicological side effects in the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline solutions, normal lactated sucrose Ringer's solution, normal glucose solution, carriers, fillers, disintegrants, lubricating agents, coatings, sweeteners, flavoring agents, saline solutions (such as Ringer's solution), alcohols, oils, gelatins, hydrocarbons such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine and dyes, and the like. Such preparations may be sterilized and, if desired, mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, colorants and/or flavors and the like, which do not react adversely. with compounds according to the invention. The person skilled in the art will recognize that other pharmaceutical excipients may be used in accordance with this invention.

The term "preparation" includes the formulation of the active compound with an encapsulating material as a carrier to obtain a capsule in which the active ingredient, with or without other carriers, is surrounded by a carrier that is associated with it. Also included are starch capsules and lozenges. Tablets, powders, capsules, pills, cachets and lozenges can be used as solid dosage forms suitable for oral administration.

As used herein, the term "administration" means oral administration, suppository administration, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intracranial, intranasal, or subcutaneous administration, or implantation of a slow-release device such as a mini-osmotic pumps, subject. Administration is by any route, including parenteral and transmucosal (eg, buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other delivery routes include, but are not limited to, liposomal formulations, intravenous infusion, transdermal patches, and the like. By "co-administration" is meant that the composition described herein is administered simultaneously, immediately before or immediately after the administration of one or more additional therapies (eg, an anti-obesity agent). The compound may be administered alone, or may be administered to the patient together with another. Co-administration includes the simultaneous or sequential administration of a compound alone or in combination (more than one compound or agent). Thus, the formulations can also be combined, if desired, with other active agents (eg, to reduce metabolic degradation, to increase prodrug degradation and drug release, analyte). The compositions can be delivered transdermally, topically, formulated as applicators, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders and aerosols. Oral preparations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, suspensions, etc., suitable for ingestion by a patient. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. Liquid form preparations include solutions, suspensions and emulsions, for example solutions in water or water/propylene glycol. The compositions may further include components that provide delayed release and/or comfort. Such components include high molecular weight anionic flour-simulating polymers, gelling polysaccharides, and finely divided drug carrier substrates. These components are discussed in more detail in US Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes. The compositions can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug-containing microspheres that are slowly released under the skin (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations (see, for example, Gao Pharm. Res. 12:857-863, 1995), or as microspheres for oral administration (see, for example, Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). compositions can be delivered by liposomes that are fused to the cell membrane or endocytosed, i.e., using receptor ligands attached to a liposome that binds to cell surface membrane protein receptors, causing endocytosis.When using liposomes, especially when the surface of the liposome has receptor ligands specific to target cells, or otherwise preferentially targeted to a specific organ, can focus on delivering compositions to target cells in vivo (See, for example, Al-Muhammed, J. Microencapsul. 13:2 93-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989). The compositions can also be delivered in the form of nanoparticles.

II. CONNECTIONS

Provided herein are, inter alia, compositions that promote weight loss, body fat reduction, food intake reduction, homeostasis improvement, or a combination thereof.

In one preferred aspect, the composition may include a compound having the structure of formula (I):

where

dotted lines between C ₁ and C ₂ , C ₂ and R ₃ , C ₃ and R ₄ , C ₅ and C ₆ , C ₅ and C ₇ , C _{1 and} C _{6 ,} and C ₃ and C ₄ indicate that may be present single or double bond, as valency permits;

R ₁ is -CN, -COOH, -COOCH ₂ CH ₃ , -CONHR ₅ , -CONR ₅ R ₅ , -COOR ₅ , -COOCH ₃ , -CH ₂ NR ₅ R ₅ , -CH ₂ OCONR ₅ R ₅ , - CH ₂ NR ₅ COOR ₅ , -CH ₂ R ₅ , -CH ₂ NR ₅ CONR ₅ R ₅ , -CH ₂ OH, -CH ₂ OR ₅ , alkyl sulfate, alkyl sulfonate, alkyl phosphate, -CH ₂ OSO ₃ R ₅ , -CH ₂ OSO ₂ R ₅ , -CH ₂ OPO ₃ R ₅ R ₅ , -CH ₂ OPO ₃ HR _{5 ,} -CH ₂ OPO ₃ H ₂ -C(=NR ₅ )NR ₅ R ₅ , -NR ₅ C(=NR ₅ )NR ₅ R ₅ , -CONH ₂ , -CH ₂ CONR ₅ R ₅ , -SR ₅ , -SO ₃ R ₅ , -SO ₂ R ₅ , -CH ₂ NHCOR ₅ , -CH ₂ NHCNR ₅ NR ₅ R ₅ , -CH ₂ COSR ₅ , CH ₂ NR ₅ COR ₅ , -CH ₂ NR ₅ CNR ₅ NR ₅ R ₅ , -CH ₂ NR ₅ COSR ₅ , -CH ₂ NHSO ₂ R ₅ , -CH ₂ NR ₅ SO ₂ R ₅ , -CHNR ₅ , -CHNOR ₅ , -H, -NH ₂ , -NHR ₅ , -NR ₅ R ₅ , -OH, -OR ₅ , phosphate, -OPO ₃ R ₅ R ₅ , -OPO ₃ HR ₅ , -OPO ₃ H ₂ , -NCO, -NCS, -N ₃ , -R ₅ , -C≡CR ₅ , -(CH=CH)R ₅ , -SH, -SR ₅ , -SO ₂ H, -SO ₃ H, -SO ₂ NR ₅ R ₅ , -SO ₃ R ₅ , -NHCOR ₅ , NHCNR ₅ NR ₅ R ₅ , -NHCOSR ₅ , secondary amide, tertiary amide, -NR ₅ COR ₅ , -NR ₅ C(=NH )NR ₅ R ₅ , -NR ₅ COSR ₅ , -NHC(=NR ₅ )R ₅ , -NR ₅ C (=NR ₅ )R ₅ , -NHSO ₂ (NH ₂ ), -NHSO ₂ R ₅ , -NR ₅ SO ₂ R ₅ , -NR ₅ SO ₂ NR ₅ R ₅ , -OCOR ₅ , -OCONR ₅ R ₅ , -O(C=O)OR ₅ , -SCOR ₅ , -O(C=NH)NR ₅ R ₅ , -OCSNHR ₅ , -OS(=O ₂ )R ₅ , -OS(=O ₂ )NR ₅ R ₅ , -SCONR ₅ R ₅ , -CH ₂ -aryl, -CH ₂ -heteroaryl,

;

;

R ₂ is -H, -CH ₃ , -SCH(CH ₃ ) ₂ , -SC(=O)CH ₃ , -SC(=O)R ₅ , -SCH ₂ CH ₂ OCOCH _{3 ,} -SR ₅ , -SOR ₅ , -SOOR ₅ , -SCONR ₂ ,

R ₃ is -OCOCH ₃ , -OCOOCH ₂ CH ₃ , -OR ₇ , -R ₇ or -NR ₅ R ₅ if a double bond is present between C ₁ and C ₂ , C ₃ and C _{4 ,} and C ₅ and C ₆ ;

R ₄ is -OCOCH ₃ , -OCOOCH ₂ CH ₃ , -OR ₇ , -R ₇ or -NR ₅ R ₅ if a double bond is present between C ₁ and C ₂ , C ₃ and C ₄ , and C ₅ and C ₆ ;

R ₃ is O if R ₄ is O and a double bond is present between C ₂ and R ₃ and C ₃ and R ₄ ;

R ₄ is -OCH _{3 ,} -OP(=O)(OCH ₃ ) ₂ , -OH, -OCOOCH ₂ CH ₃ , -OCONHCH ₂ CH ₃ , -OCOOCH(CH ₃ ) ₂ , -OR ₇ , -R ₇ or -NR ₅ R ₅ if R ₃ is O and a double bond is present between C ₂ and R ₃ ; R ₃ and R ₄ can also be combined to form a heterocyclic or carbocyclic ring;

R ₅ is independently selected at each occurrence from hydrogen, alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, amine, or heteroaryl, optionally substituted with substituents individually selected from alkyl, alkoxy, cycloalkyl, ether, amine, optionally substituted one or more alkyl, halogen, hydroxyl, ether, cyano, nitrile, CF ₃ , ester, amide, cycloalkylamide, sugar, heteroarylamide optionally substituted with alkyl and/or alkoxy, urea, carbamate, thioether, sulfate, sulfonyl, sulfonic acid , carboxylic acid and aryl, or two R ₅ groups taken together form a cycloalkyl, heterocycloalkyl, aryl or heteroaryl group optionally substituted with substituents individually selected from alkyl, cycloalkyl, alkoxy, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl, heteroaryl, amine, halogen, hydroxyl, ether, nitrile, cyano, nitro, CF ₃ , ester, urea, a carbamate, thioether or carboxylic acid group;

and

R ₇ is hydrogen, alkyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkenyl, alkynyl, aryl or heteroaryl, optionally substituted substituents individually selected from alkyl, cycloalkyl, ether, amine, halogen, hydroxyl, ether, nitrile, cyano, nitrile, CF ₃ ester, amide, urea, carbamate, thioester or carboxylic acid,

or a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments, the compounds of formula (I) include those that are prodrugs.

In some embodiments, compounds of formula (I) include those in which R ₁ is -CONH ₂ , which can be represented in formula (I)-a:

where each R ₂ , R ₃ and R ₄ is defined in formula (I).

In some embodiments, R ₂ is -H, -SCH(CH ₃ ) ₂ , -SC(=O)CH ₃ ,

In some embodiments, R ₃ is -OC(=O)CH ₃ or -OC(=O)OCH ₂ CH ₃ .

In some embodiments, R ₄ is -OC(=O)CH ₃ or -OC(=O)OCH ₂ CH ₃ .

One subset of the compounds of formula (I) includes the compounds shown below:

.

.

In another embodiment, compounds of formula (I) include those in which R ₁ is -COOH or -COOCH ₃ .

In some embodiments, R ₂ is -CH ₃ , -SC(=O)CH ₃ , -SCH(CH ₃ ) ₂ , or -SCH ₂ CH ₂ OCOCH ₃ .

In some embodiments, R ₃ is -OCOCH ₃ or -OH.

In some embodiments, R ₄ is -OCOCH ₃ or -OH.

One subset of the compounds of formula (I) includes the compounds shown below:

.

.

In some embodiments, compounds of formula (I) include those in which R ₁ is -CN or -CH ₂ NR ₅ R ₅ , such as -CH ₂ N(CH ₃ ) ₂ , which can be represented as formula (I)- b or formula (I)-c, respectively:

where each R ₂ , R ₃ , R ₄ and R ₅ is defined in formula (I).

In some embodiments, R ₂ is -SCH(CH ₃ ) ₂ , -SC(=O)CH ₃ , -H,

In some embodiments, R ₃ is -OCOCH ₃ .

In some embodiments, R ₄ is -OCOCH ₃ .

In some embodiments, R ₃ and R ₄ form a five-membered heterocycle containing -OCOO-.

In some embodiments, R ₅ is alkyl, preferably CH ₃ .

One subset of the compounds of formula (I) includes the compounds shown below:

In some embodiments, compounds of formula (I) include those in which R ₃ is O and a double bond is present between C ₂ and R ₃ and C ₃ and C ₄ .

In some embodiments, R ₁ is -C(=O)OCH ₂ CH ₃ , -CN, -CONH ₂ , -CH ₂ N(CH ₃ ) ₂ ,

.

.

In some embodiments, R ₂ is -H.

In some embodiments, R ₄ is -OCH ₃ , -OP(=O)(OCH ₃ ) ₂ , -OCH ₂ CH ₃ , -OH, -OCONHCH ₂ CH ₃ , -OCH ₂ COOCH ₃ , -OCOOCH ₂ CH ₃ , -OCH ₂ CH ₂ OH, -OCOOCH(CH ₃ ) ₂ or -OCH(CH ₃ ) ₂ .

One subset of the compounds of formula (I) includes the compounds shown below:

In some embodiments, compounds of formula (I) include those in which R ₃ and R ₄ are O and a double bond is present between C ₂ and R ₃ and C ₃ and R ₄ .

In some embodiments, R ₁ is COOH, COOCH ₃ or

.

.

In some embodiments, R ₂ is CH ₃ .

One subset of the compounds of formula (I) includes the compounds shown below:

.

.

In some embodiments, R ₁ is -NR ₅ C(=NR ₅ )NR ₅ R ₅ , -SR ₅ , -SO ₃ R ₅ , -SO ₂ R ₅ , -NH ₂ , -NHR ₅ , -NR ₅ R ₅ , -OH, -OR ₅ , -NCO, -NCS, -N ₃ , -SH, -SR ₅ , -SO ₂ H, -SO ₃ H, -SO ₂ NR ₅ R ₅ , -SO ₃ R ₅ , - NHCOR ₅ , -NHCNR ₅ NR ₅ R ₅ , -NHCOSR ₅ , -NR ₅ COR ₅ , -NR ₅ C(=NH)NR ₅ R ₅ , -NR ₅ COSR ₅ , -NHC(=NR ₅ )R ₅ , -NR ₅ C(=NR ₅ )R ₅ , -NHSO ₂ (NH ₂ ), -NHSO ₂ R ₅ , -NR ₅ SO ₂ R ₅ , -NR ₅ SO ₂ NR ₅ R ₅ , -OCOR ₅ , -OCONR ₅ R ₅ , -O(C=O)OR ₅ , -SCOR ₅ , -O(C=NH)NR ₅ R ₅ , -OCSNHR ₅ , -OS(=O ₂ )R ₅ , -OS(=O ₂ )NR ₅ R ₅ , -SCONR ₅ R ₅ .

In some embodiments, R ₂ is H.

In some embodiments, R ₄ is OH, -OR ₇ or -R ₇ if R ₃ is O and a double bond is present between C ₂ and R ₃ .

One subset of the compounds of formula (I) includes the compounds shown below:

,

,

,

,

,

,

или

.

,

,

,

,

,

,

or

.

In some embodiments, the compounds may include:

.

.

In another preferred aspect, the composition may include a compound having the structure of formula (II):

where R ₁ is OR _a or NR _a R _b where R _a and R _b are each independently hydrogen, R ₅ , -C(=NR ₅ )NR ₅ R ₅ , -CO, -CS, -COR ₅ , -CNR ₅ NR ₅ R ₅ , -COSR ₅ , -C(=NH)NR ₅ R ₅ , -C(=NR ₅ )R ₅ , -SO ₂ (NH ₂ ), -SO ₂ R ₅ , -SO ₂ R ₅ , -SO ₂ NR ₅ R ₅ , -COR ₅ , -CONR ₅ R ₅ , -(C=O)OR ₅ , -(C=NH)NR ₅ R ₅ , -CSNHR ₅ , -S(=O ₂ ) R ₅ or -S(=O ₂ )NR ₅ R ₅ ,

R ₅ is described in formula (I), or

a pharmaceutically acceptable salt or prodrug thereof.

In some embodiments, R ₁ is NR _a R _b , which can be represented in formula (II)-a:

where each R _a or R _b is defined in formula (II).

In certain embodiments, R ₁ is -NH(CO)R ₅ where R ₅ is preferably alkyl, cycloalkyl, or aryl, which may be represented in formula (II)-b:

where each R _a or R _b is defined in formula (II).

Examples of compounds of formula (II) include the compounds shown below:

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or

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The compounds described by formula (I) and formula (II) can be obtained using methods known in the art and described here. For example, Celastrol can be obtained from commercial sources or isolated from plants, Tripterygium, by methods known in the art (Kutney et al, Can. J. Chem . 59:2677, 1981) and Zhang et al, Acta Pharm. Sin . 212:592, 1986). Celastrol may be modified to be a compound of formula (I) or formula (II). The resulting compounds are purified using conventional methods to obtain compounds free of impurities. The resulting compounds are >75, >80, >85, >90, >95, >96, >97, >98, >99, >99.5% pure. Optionally, preferred compounds are >99% pure.

Also provided are compounds of formula (I) and formula (II) which have properties for increased or substantially increased oral bioavailability.

In some embodiments, the compounds of formula (I) and formula (II) may have greater or lesser solubility in water, in aqueous solution and/or saline than Celastrol obtained from commercial sources or isolated from plants. For example, compounds may have solubility in the range of about 0.001 μM to about 150 μM, 0.01 μM to about 100 μM, 0.1 μM to about 100 μM, 1 μM to about 100 μM, 10 μM to about 100 µM, 1 µM to about 50 µM, 10 µM to about 50 µM, 10 µM to about 80 µM, 10 µM to about 25 µM, 25 µM to about 50 µM, 50 µM to about 100 µM , 50 µM to about 75 µM, 25 µM to about 75 µM, or a solubility that is at least about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 µM, or less than about 0.1, 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 µM in an aqueous solution (such as phosphate buffered saline (PBS), e.g. at pH about 7, 7.1, 7.2, 7.3, 7.4, 7.5, or 7-8).

In some embodiments, the compounds of formula (I) and formula (II) have increased or substantially increased stability or half-life in water, aqueous solution or saline. For example, the compounds may have substantially improved stability or resistance under various pH conditions from 2 to 8 in the upper or middle gastrointestinal (GI) or digestive tracts.

In some embodiments, the compounds of formula (I) and formula (II) have increased or substantially increased absorption when administered to a subject. For example, the compounds may have significantly improved permeability across biological membranes. Compounds can show a suitable balance between hydrophobicity (lipophilicity) and hydrophilicity with the help of local ionic charges.

In one aspect, compositions in accordance with this invention contain at least one compound with oral efficacy for the treatment of obesity.

Exemplary compounds with oral efficacy in the treatment of obesity may include the following compounds:

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In another aspect, the composition contains compounds having intraperitoneal efficacy in the treatment of obesity.

Examples of these compounds with intraperitoneal efficacy in the treatment of obesity include the following compounds:

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III. METHODS OF TREATMENT AND DIAGNOSIS

The compounds described above may be used in the treatment of obesity in a subject in need thereof, which comprises administering to the subject an effective amount of compounds of formula e(I). Obesity may be the primary cause of the disease and/or disorder or may be caused by the disease and/or disorder.

In some instances, an effective amount of a compound of formula (I) may be administered as a method of treating weight gain in preobese, obese, or morbidly obese patients.

In some instances, a method for reducing body fat in preobese, obese, or morbidly obese patients comprises administering an effective amount of a compound of formula (I). Body weight or body fat can be reduced by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%, about 5-10%, 5-25% , 10-25%, 10-50%, 25-50% or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50 %.

In some instances, a method of reducing food intake in preobese, obese, or morbidly obese patients comprises administering an effective amount of a compound of formula (I). Average daily food intake (in calories) may be reduced by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more, or about 5-10%, 5-25%, 10-25%, 10-50%, 25-50%.

In some cases, an effective amount of a compound of formula (I) may be administered to reduce the body mass index (BMI) of an obese patient. The patient's BMI can be reduced to <30 kg•m ^-2 (normal BMI=20-25 kg•m ^-2 ).

Finally, a method for improving glucose homeostasis in preobese, obese or morbidly obese patients comprises administering a compound of formula (I). Mean fasting blood glucose levels may be reduced by at least 10%, 12%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or more, or about 5-10%, 5-25%, 10-25%, 10-50%, 25-50%.

Obesity

Obesity is a medical condition in which excess body fat accumulates to such an extent that it can have negative health effects, resulting in reduced life expectancy and/or increased health problems (Haslam et al., Lancet (Review) 366 ( 9492): 1197-209, 2005). In Western countries, people are considered obese if their BMI, an indicator obtained by dividing a person's body weight by the square of a person's height, exceeds 30 kg/m ² , where the interval 25-30 kg/m ² is defined as being overweight. Obesity increases the likelihood of various diseases, especially heart disease, type 2 diabetes, obstructive sleep apnea syndrome, certain types of cancer, and osteoarthritis (Haslam et al., Lancet (Review) 366 (9492): 1197-209, 2005). Obesity is most commonly caused by a combination of excessive food intake, physical inactivity, and genetic predisposition, although some causes are predominantly caused by genes, endocrine disorders, drugs, or psychiatric illnesses. Evidence that some obese people eat little but gain weight due to slow metabolism is limited. On average, obese people have higher energy expenditure than lean people due to the energy required to maintain their increased body weight (Kushner, Treatment of the Obese Patient , 2007).

Obesity is a medical condition in which excess body fat accumulates to the point where it can have adverse health effects. BMI is determined, and fat distribution is assessed through the waist/hip ratio and general cardiovascular risk factors (Sweeting et al., Nutr. J. 6 (1): 32, 2007). BMI is closely related to percentage of body fat and total body fat (Gray et al., J. Clin. Epidemiol . 44 (6): 545-50, 1991).

BMI is defined as the patient's body weight divided by the square of their height. BMI is usually expressed in kilograms per square meter, based on the fact that body weight is measured in kilograms and height is measured in meters. Some modifications to the definitions were made when the surgical literature divided obesity into several categories, the exact meanings of which are still debated (Sturm et al., Public Health 121 (7): 492-6, 2007). Any BMI ≥35 or 40 kg/m ² means severe obesity. BMI ≥35 kg/m ² and tolerable obesity-related health problems or ≥40-44.9 kg/m ² means morbid obesity. BMI ≥45 or 50 kg/m ² means superobese. The World Health Organization (WHO) considers a BMI less than 18.5 to be underweight and can mean malnutrition, eating disorders or other health problems, while a BMI equal to or greater than 25 is considered overweight and above 30 is considered obese (World Health Organization, Global Database on Body Mass Index (2006)). A summary WHO scheme for classifying BMI is provided in Table 1 below.

Table 1: Classification scheme for body mass index

CATEGORY BMI interval - kg / m ² Very strong underweight less than 15 heavy underweight from 15.0 to 16.0 Underweight from 16.0 to 18.5 Normal (healthy weight) from 18.5 to 25 Excess weight from 25 to 30 Obesity type I (moderate obesity) from 30 to 35 Obesity type II (severe obesity) from 35 to 40 Obesity type III (very severe obesity) over 40

Obesity associated with damage to the hypothalamus

Hypothalamic obesity is a complicated medical condition that can occur with the growth of rare brain tumors and other types of damage to the hypothalamus. Craniopharyngioma is one of the tumors that causes obesity associated with damage to the hypothalamus. Damage to the hypothalamus disrupts communication between the intestines and the brain, which causes a constant feeling of hunger.

The hypothalamus and pituitary gland are closely integrated. Damage to the hypothalamus will affect the receptivity and normal functioning of the pituitary gland. Hypothalamic disease can cause deficiency or inhibition of signaling to the pituitary gland, resulting in a deficiency of one or more of the following hormones: thyroid-stimulating hormone, adrenocorticotropic hormone, beta-endorphin, luteinizing hormone, follicle-stimulating hormone, and melanocyte-stimulating hormones. Treatment for hypopituitarism includes hormone replacement therapy (Pinkney, Pituitary News 17, 2000).

Thyroid hormones are responsible for metabolic activity. Insufficient production of thyroid hormones causes suppression of metabolic activity and weight gain. Hypothalamic disease can therefore have the complications of obesity (Pinkney, Pituitary News 17, 2000); (Ling, Trends in Obesity Research , 2004).

Liver steatosis/NASH

Non-alcoholic fatty liver disease (NAFLD) is one of the causes of hepatic steatosis, which occurs when fat (steatosis) is deposited in the liver due to causes other than excessive alcohol consumption. NAFLD is associated with insulin resistance and metabolic syndrome, and may respond to treatments originally developed for other insulin resistant conditions (eg, type 2 diabetes), such as weight loss, metformin, and triazolidinediones (Adams et al. Postgrad. Med. J. 82 (967): 315-22, 2006). Non-alcoholic steatohepatitis (NASH) is the most extreme form of NAFLD and is considered a major cause of liver cirrhosis of unknown etiology (Clark et al. JAMA 289(22):3000-4, 2003).

Most people with NAFLD have few or no symptoms. Patients may complain of fatigue, discomfort, and dull discomfort in the right upper quadrant of the abdomen. Mild jaundice may occur, although rarely. Most often, NAFLD is diagnosed by analyzing abnormal liver function during a blood test. NAFLD is associated with insulin resistance and metabolic syndrome (obesity, associated hyperlipidemia, diabetes mellitus (type II) and high blood pressure) (Adams et al. Postgrad. Med. J. 82 (967): 315-22, 2006).

The general description includes elevated liver enzymes and liver ultrasound showing steatosis. Ultrasound may also be used to rule out gallstone problems (cholelithiasis). Liver biopsy (tissue examination) is the only test accepted as a test that accurately distinguishes NASH from other forms of liver disease, and it can be used to assess the severity of inflammation and resulting fibrosis (Adams et al. Postgrad. Med. J. 82 (967) : 315-22, 2006).

Other diagnostic tests are available. Relevant blood tests include erythrocyte sedimentation rate, glucose, albumin, and renal function. Since the liver is important for the production of proteins used for clotting, certain clotting studies are often performed, especially the INI (International Normalization Index). Blood tests (serology) are commonly used to rule out viral hepatitis (hepatitis A, B, C and herpes viruses such as EBV or CMV), rubella, and autoimmune diseases. Hypothyroidism is most common in patients with NASH, as measured by TSH (Liangpunsakul et al. J. Clin. Gastroenterol . 37(4):340-3, 2003).

metabolic syndrome

Metabolic syndrome is an energy utilization and storage disorder diagnosed when three of five of the following medical conditions coexist: abdominal (central) obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low high-density lipoprotein (HDL) levels. Metabolic syndrome increases the risk of developing cardiovascular disease and diabetes (Kaur, Cardiology Research and Practice, 2014) (Felizola, "Ursolic acid in experimental models and human subjects: potential as an anti-obesity/overweight treatment?" ResearchGate, 2015).

The main feature of the metabolic syndrome is central obesity (also known as visceral, male-pattern, or malnutrition), excess weight in which adipose tissue accumulates primarily around the waist and torso. Other signs of the metabolic syndrome include high blood pressure, low fasting serum HDL cholesterol, elevated fasting serum triglyceride (VLDL triglyceride), poor fasting glucose, insulin resistance, or prediabetes. Associated conditions include hyperuricemia, hepatic steatosis (especially with accompanying obesity) progressing to non-alcoholic fatty liver disease, polycystic ovary syndrome (in women), erectile dysfunction (in men), and acanthocheratoderma.

In the joint interim report of the task force on epidemiology and prevention of the International Diabetes Federation; National Institute of Heart, Lung and Blood Diseases; American Heart Association; World Heart Federation; International Society of Atherosclerosis; and the International Association for the Study of Obesity published guidelines for harmonizing the definition of metabolic syndrome (Alberti et al., Circulation 120 (16): 1640-5, 2009). This definition recognizes that the risk associated with a particular waist measurement is different for different populations.

Hit

Stroke, also known as acute cerebrovascular accident (CVA), cerebrovascular stroke (CVI), or stroke, occurs when there is poor blood flow to the brain, causing cell death. There are two main types of stroke: ischemic, due to lack of blood flow, and hemorrhagic, due to bleeding. They partially lead to the fact that the brain does not function correctly. Signs and symptoms of a stroke may include an inability to move or feel on one side of the body, problems with understanding and speaking, a sensation of spinning around oneself, or loss of vision on one side, among others (Donnan et al., Lancet 371 (9624): 1612-23, 2008) . Signs and symptoms often occur after a stroke occurs. If symptoms last less than one or two hours, it is called a transient ischemic attack (TIA). Hemorrhagic strokes can also be associated with severe headache.

The main risk factor for stroke is high blood pressure. Other risk factors include tobacco smoking, obesity, high blood cholesterol, diabetes mellitus, previous PIA, and atrial fibrillation, among others (Donnan et al., Lancet 371 (9624): 1612-23, 2008). Ischemic stroke is usually caused by blockage of a blood vessel. A hemorrhagic stroke is caused by bleeding either directly into the brain or into the space surrounding the brain. (Feigin et al., Stroke 36 (12): 2773-80, 2005). Bleeding can occur due to a brain aneurysm. Diagnosis is usually made by medical imaging, such as a CT scan or MRI scan, along with a physical examination. Other tests, such as an electrocardiogram (ECG) and blood tests, are done to identify risk factors and rule out other possible causes.

Stroke is diagnosed by several methods: neurological examination (such as NIHSS), CT scans (most often without contrast) or MRI scans, Doppler sonography, and arteriography. The stroke diagnosis itself is clinical, using imaging techniques. Imaging techniques also help determine the subtypes and cause of stroke. There are still no generally accepted blood tests to diagnose the stroke itself, although blood tests can help determine the most likely cause of the stroke (Hill et al., Clin. Chem. 51 (11): 2001-2, 2005).

Cardiovascular disease

Cardiovascular disease (CVD) is a class of diseases that involve the heart or blood vessels. Cardiovascular disease includes coronary heart disease (CHD) such as angina pectoris and myocardial infarction (known as heart attack) (Shanthi et al., Global Atlas on Cardiovascular Disease Prevention and Control 3-18, 2011). Other CADs include stroke, hypertensive cardiopathy, rheumatic heart disease, cardiomyopathy, atrial fibrillation, congenital heart disease, endocarditis, aortic aneurysm, and peripheral arterial disease.

The underlying mechanisms vary depending on the disease in question. Ischemic heart disease, stroke, and peripheral arterial disease include atherosclerosis. They can be caused by high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet, and excessive alcohol consumption, among others. High blood pressure causes 13% of CVD deaths, tobacco causes 9%, diabetes 6%, lack of exercise 6% and obesity 5%. Rheumatic heart disease can occur after untreated angina (Shanthi et al., Global Atlas on Cardiovascular Disease Prevention and Control 3-18, 2011).

Standard tests for cardiovascular disease include: coronary calcinosis, carotid total platelet area, elevated low-density lipoprotein-p, and elevated blood levels of brain natriuretic peptide (also known as B-type) (BNP) (Bertazzo et al., Nat. Mat. 12 , 576-583, 2013) (Inaba et al., Atherosclerosis 220 (1): 128-33, 2012) (J. Clin. Lipidol. Dec;1(6) 583-92, 2007) (Wang et al., N. Engl. J. Med. 350(7): 655-63, 2004).

Diabetes

Diabetes mellitus (DM), commonly referred to as diabetes, is a group of metabolic diseases in which there are high blood sugar levels for a long period. Symptoms of high blood sugar include frequent urination, increased thirst, and increased hunger. If left untreated, diabetes can cause many complications (Diabetes Fact sheet N 312". WHO, 2013). Acute complications include diabetic ketoacidosis and hyperosmolar non-ketoacidotic coma (Kitabchi, et al., Diabetes Care 32 (7): 1335-43, 2009) Serious long-term complications include cardiovascular disease, stroke, chronic kidney failure, leg ulcers, and eye damage.

Diabetes occurs because either the pancreas does not produce enough insulin, or the cells of the body do not respond properly to the insulin produced (Shoback, Greenspan's Basic & Clinical Endocrinology (9th ed.) (2011)).

Diabetes mellitus is characterized by recurrent or persistent high blood sugar and is diagnosed through any of the following: fasting blood glucose ≥7.0 mmol/l (126 mg/dl); plasma glucose ≥11.1 mmol/l (200 mg/dl) two hours after oral administration of 75 g glucose, as in the glucose tolerance test; symptoms of high blood sugar and occasional plasma glucose ≥11.1 mmol/L (200 mg/dL); and glycated hemoglobin (HbA1C) ≥48 mmol/mol (≥6.5 DCCT%) (National Diabetes Clearinghouse (NDIC): National Diabetes Statistics 2011" U.S. Department of Health and Human Services, 2011) (ʺDiabetes Careʺ American Diabetes Association, 2010 ).

A positive result, in the absence of unequivocal high blood sugar, must be confirmed by repeating any of the above methods on another day. It is preferable to measure fasting glucose because of the ease of measurement and significant time to validate a formal glucose tolerance test, which takes two hours to complete and offers no predictive advantage over fasting assays (Saydah et al., Diabetes Care 24(8): 1397-402, 2001). According to the current definition, two measurements of fasting glucose above 126 mg/dL (7.0 mmol/L) are considered a diagnosis of diabetes mellitus.

According to the World Health Organization, people with a fasting glucose level of 6.1 to 6.9 mmol/L (110 to 125 mg/dL) are considered to have impaired fasting glucose; people with plasma glucose at or above 7.8 mmol/L (140 mg/dL) but not reaching 11.1 mmol/L (200 mg/dL) two hours after oral administration of 75 glucose are considered to have impaired tolerance glucose (Definition and diagnosis of diabetes mellitus and intermediate hyperglycemia: report of a WHO/IDF consultation. World Health Organization p. 21, 2006). Of these two predictable conditions, the latter is especially a major risk factor for the development of severe diabetes mellitus as well as cardiovascular disease. The American Diabetes Association has used a slightly different range for impaired fasting glucose since 2003, 5.6 to 6.9 mmol/L (100 to 125 mg/dL) (Bartoli et al., Eur. J. Int. Med. 22 (1 ): 8-12, 2011). Glycated hemoglobin is better than fasting glucose for determining the risk of cardiovascular disease and death from any cause (Selvin et al

A rare disease, diabetes insipidus, has similar symptoms to diabetes mellitus but without disturbances in sugar metabolism (insugar means "no taste" in Latin) and does not involve the same disease mechanisms. Diabetes is part of a broader condition known as the metabolic syndrome.

Hypertension

Hypertension is diagnosed on the basis of persistently high blood pressure. Traditionally, the National Institute for Health and Care Excellence recommends three separate sphygnomanometric measurements at monthly intervals. The American Heart Association recommends at least three measurements in at least two separate wellness visits (Aronow et al., J. Am. Soc. Hypertension : JASH 5 (4): 259-352, 2011). The exceptions are those who have very high blood pressure, especially with poor organ function. Initial evaluation of people with hypertension should include a complete history and physical examination. With the availability of 24-hour ambulatory blood pressure monitoring and home blood pressure machines, it is important not to misdiagnose those who suffer from viewing hypertension, which could lead to changes in protocols. In the United Kingdom, current best practice is to follow a single elevated clinical indication for outpatient measurement or, less preferably, for 7 days of monitoring with a home blood pressure monitor. Pseudohypertension in the elderly or non-compressible arterial syndrome may also require consideration. This condition is believed to be due to calcification of the arteries, which causes abnormally high air cuff blood pressure readings, although intra-arterial blood pressure measurements are normal (Franklin et al., Hypertension 59 (2): 173-8, 2012). Orthostatic hypertension occurs when blood pressure rises while standing.

Hyperlipidemia

Hyperlipidemia includes an abnormal increase in the levels of any or all fats and/or lipoproteins in the blood (Dorland's Medical Dictionary for Health Consumers, 2007). It is the most common form of dyslipidemia (which includes any abnormal fat levels). Fats (fat-soluble molecules) are carried in a protein capsule. The size of this capsule or lipoprotein determines its density. The density of a lipoprotein and the type of apolipoproteins it contains determines the particle's metabolic pathway and its effect on metabolism.

Hyperlipidemias are divided into primary and secondary subtypes. Primary hyperlipidemia usually occurs due to genetic causes (such as a mutation in the receptor protein), while secondary hyperlipidemia occurs due to other underlying causes such as diabetes. Abnormalities of fat and lipoprotein are widespread in the population and are considered a modifiable risk factor for cardiovascular disease due to their effect on atherosclerosis. In addition, some forms may predispose to pancreatitis.

Hyperlipidemia is a group of disorders characterized by an excess of serum cholesterol, especially an excess of LDL-C and/or an excess of triglycerides. Hypercholesterolemia usually resolves without symptoms. Hypertriglyceridemia usually resolves without symptoms until triglyceride levels are above 1000 mg/dL - at which time symptoms include dermatological manifestations such as xanthomas presenting with a rash and gastrointestinal manifestations such as pancreatitis. Hyperlipidemias are most often genetically determined, but may be caused or exacerbated by abnormal diets, drugs, and certain disease states. Drugs that cause hyperlipidemia include: immunosuppressive therapy, thiazide diuretics, progestins, retinoids, anabolic steroids, glucocorticoids, HIV protease inhibitors, alcohol, retinoic acid, and beta-blockers. Diseases associated with secondary hyperlipidaemias include: diabetes mellitus (type I and II), hypothyroidism, Cushing's syndrome, chronic kidney disease, nephrotic syndrome, and cholestatic disorders. Hyperlipidemia is a major avoidable risk factor for atherosclerosis and cardiovascular disease, including coronary heart disease (Dorland's Medical Dictionary for Health Consumers, 2007).

Prader-Willi syndrome

Prader-Willi syndrome affects approximately 1 in 10,000 to 1 in 25,000 newborns (Killeen, Principles of Molecular Pathology 2004). There are over 400,000 people living with Prader-Willi syndrome worldwide (Tweed, AOL Health, September 2009). It is traditionally characterized by hypotonia, short stature, hyperphagia, obesity, behavioral disorders (especially ACS-like behaviour), small hands and feet, hypogonadism, and mild mental retardation (Killeen, Principles of Molecular Pathology 2004). Like autism, Prader-Willi syndrome is a spectrum disorder and symptoms can range from moderate to severe and may change over the life of the patient.

Traditionally, Prader-Willi syndrome is diagnosed based on the clinical picture. Currently, the syndrome is diagnosed using genetic analysis; the analysis is recommended for newborns with severe hypotension. Early diagnosis of Prader-Willi syndrome allows for earlier intervention. The basis of diagnosis is genetic analysis, especially DNA-based methylation analysis, to determine the absence of a parentally derived Prader-Willi syndrome/Angelman syndrome (PWS/AS) region on chromosome 15q11-q13. Such an analysis determines more than 97% of cases. A methylation-specific assay is important to confirm the diagnosis of PWS in all patients, but especially in those who are too small to show clinical diagnostic characteristics or who have atypical findings (Buiting et al., Nat. Genet. 9(4):395-400, 1995).

Bardet-Biedl syndrome

Bardet-Biedl Syndrome (BBS) is a human ciliopathic genetic disorder that causes multiple effects and affects multiple body systems. It is characterized mainly by obesity, retinitis pigmentosa, polydactyly, hypogonadism and renal failure in some cases (Beales et al., J. Med. Genet. 36(6):437-46, 1999).

Bardet-Biedl syndrome is a pleiotropic disorder with variable expressivity and a wide range of clinical variability observed within and between families. The main clinical signs are cone-rod dystrophy, with loss of vision occurring in childhood, preceded by night blindness; posterior axial polydactyly; abdominal obesity, which manifests itself during infancy and remains problematic during adulthood; certain learning disabilities in some, but not all, individuals; hypogonadism in men and complex urogenital deformities in women; and renal dysfunction, a major cause of morbidity and mortality. There are many secondary characteristics that are sometimes associated with SBS, including: speech disorder/slowness, strabismus/cataract/astigmatism, brachydactyly/syndactyly of hands and feet, partial syndactyly (most commonly between the second and third toes), developmental delay, polyuria/polydipsia ( nephrogenic diabetes insipidus), ataxia/poor coordination/imbalance, moderate hypertension (especially of the lower extremities), diabetes mellitus, crowded teeth/hypodontia/small tooth root; high domed palate, cardiovascular abnormalities, liver involvement, anosmia, hearing loss, and Hirschsprung disease (Ross et al. The Clinical, Molecular, and Functional Genetics of Bardet-Biedl Syndrome, in Genetics of Obesity Syndromes, 2008).

Cohen syndrome

This syndrome is believed to be a genetic mutation on chromosome 8 at the 8q22 locus of the COH1 gene (Kolehmainen et al, Am. J. Hum. Genet . 72(6):1359-69, 2003). Cohen's syndrome has several characteristics such as obesity, mental retardation, and craniofacial dysmorphia. It has an autosomal recessive transmission with variable expression (Kivitie-Kallio et al. Am. J. Med. Genet. 102(2):125-35, 2001).

Cohen's syndrome is diagnosed on clinical examination, but the diagnosis is often made difficult by expression variability. Eye complications, although rare, are listed as optic nerve atrophy, ophthalmomycria, chorioretinitis pigmentosa, day blindness (reduced vision in bright light), myopia, strabismus, nystagmus, and iris/retinal coloboma. The general appearance is characterized by obesity with thin/elongated arms and legs. Micrognathia, a short philtrum, and a high-arched palate are common. Variable mental retardation with occasional seizures and deafness are also characteristics of Cohen's syndrome.

MOMO syndrome

MOMO syndrome is an extremely rare genetic disorder that belongs to hypertrophies and is diagnosed in only six cases worldwide, and occurs in 1 in 100 million births. The name is an acronym for four primary aspects of the disorder: macrosomia (overweight at birth), obesity, macrocephaly (excess head size), and ocular abnormalities (Moretti-Ferreira et al. Am. J. Med. Genet. 46(5):555-8 , 1993). There are also common symptoms: sloping forehead, delayed bone maturation, mental retardation. Ocular anomalies mainly include retinal coloboma and nystagmus.

Crayfish

Cancer, also known as malignancy, malignant neoplasm and malignant tumor, is a group of diseases involving abnormal growth of cells with the potential to invade or spread to other parts of the body (Cancer Fact sheet N 297. World Health Organization. February 2014; Defining Cancer. National Cancer Institute. 2014). Not all tumors are cancerous as benign tumors do not spread (Defining Cancer. National Cancer Institute. 2014). Possible signs and symptoms include: new bumps, abnormal bleeding, persistent cough, unexplained weight loss, and changes in intestinal motility, among others (Cancer - Signs and symptoms. NHS Choices. 2014). While these symptoms may indicate cancer, they also occur for other reasons (Cancer - Signs and symptoms. NHS Choices. 2014). There are over 100 different known cancers that affect humans (Defining Cancer. National Cancer Institute. 2014). Cancers are a large family of diseases that involve abnormal cell growth with the potential to invade or spread to other parts of the body (Cancer Fact sheet N 297. World Health Organization. February 2014; Defining Cancer. National Cancer Institute. 2014). A neoplasm or tumor is a group of cells that have undergone unregulated growth, and often form a mass or lump, but may spread diffusely (Cancer Glossary. cancer.org. American Cancer Society. 2013; What is cancer? cancer. gov. National Cancer Institute, 2013 ). Suggested characteristics of cancer include: 1) intensity of anti-growth signals; 2) self-sufficiency in signaling growth; 3) induction and persistence of angiogenesis; 4) avoidance of apoptosis; 5) activation of unlimited replicative potential; and 6) activation of metastasis and tissue invasion (Hanahan, Douglas; Weinberg, Robert A. (January 7, 2000). "The hallmarks of cancer." Cell 100 (1): 57-70). Malignancy is a multi-step process that transforms normal cells into cells that form a visible pre-cancer mass (Hanahan, Douglas; Weinberg, Robert A. (January 7, 2000) "The hallmarks of cancer" Cell 100 (1): 57 -70; Hanahan, Douglas; Weinberg, Robert A. (2011) "Hallmarks of Cancer: The Next Generation" Cell 144 (5): 646-74.

Cancer is a disease of failure to regulate tissue growth. Genes that regulate cell growth and differentiation must be changed in order for normal cells to become cancerous (Croce CM (January 2008). "Oncogenes and cancer". N. Engl. J. Med. 358 (5): 502-11 ). Affected genes fall into two broad categories - tumor suppressor genes and oncogenes. Tumor suppressor genes inhibit cell division and survival. Oncogenes promote cell growth and reproduction. Tumor suppressor genes are genes that inhibit cell division and survival. Malignant transformation can occur through: underexpression or damage of tumor suppressor genes, abnormal overexpression of normal oncogenes, or generation of new oncogenes (Knudson AG (November 2001). "Two genetic hits (more or less) to cancer". Nature Reviews Cancer 1 ( 2): 157-62). Cancer is driven by progressive genetic abnormalities that include mutations in oncogenes, chromosomal abnormalities in tumor suppressor genes, and epigenetic changes (Baylin SB, Ohm JE (February 2006). "Epigenetic gene silencing in cancer - a mechanism for early oncogenic pathway addiction?" Nature Reviews Cancer 6 (2): 107-16).

Most cancers are initially recognized either by the appearance of signs or symptoms or by screening. The final diagnosis requires examination of a tissue sample by a pathologist. Patients with suspected cancer are given diagnostic tests, which include CT scans, blood tests, endoscopy, and x-rays.

Malignant cancers treated with the methods and compositions described herein include gastric cancer, multiple myeloma, leukemia, lymphoma, hepatocellular carcinoma, renal cell carcinoma, prostate cancer, brain cancer, glioblastoma, melanoma, breast cancer, head and neck cancer. and non-small cell lung carcinoma.

Glioblastoma, also known as glioblastoma multiforme (GBM) and grade IV atrocytoma, is the most common and most aggressive malignant primary brain tumor. It includes glial cells and accounts for 52% of all brain tissue tumors and 20% of all intracranial tumors ("Glioblastoma and Malignant Astrocytoma". American Brain Tumour Association (ABTA) 2014). About 50% of people diagnosed with MGD die within a year, while 90% die within three years. Treatment may include chemotherapy, radiation, and surgery. Median survival with standard radiation and chemotherapy with temozolomide is 15 months (Johnson, Derek R.; O'Neill, Brian Patrick (2011). "Glioblastoma survival in the United States before and during the temozolomide era." Journal of Neuro-Oncology 107 ( 2): 359-64). The median survival without treatment is 4½ months. Although no randomized controlled trials have been conducted, surgery remains the standard treatment (Van Meir, E. G.; Hadjipanayis, C. G.; Norden, A. D.; Shu, H. K.; Wen, P. Y.; Olson, J. J. (2010). "Exciting New Advances in Neuro-Oncology : The Avenue to a Cure for Malignant Glioma". CA: A Cancer Journal for Clinicians 60 (3): 166-93).

Although common symptoms of the disease include seizures, nausea and vomiting, headache, memory loss, and hemiparesis, the single most prevalent symptom is a progressive deficit in memory, personality, and neurology due to involvement of the temporal and frontal lobes. The type of symptoms you get depends highly on the location of the tumor, rather than on its pathological properties. The tumor may begin to show symptoms quickly, but in some cases is asymptomatic until it reaches an abnormal size.

When viewed on MRI, glioblastomas often appear as lesions with enlarging rings. Appearance is not specific, however, as other lesions such as abscess, metastases, tumor-like multiple sclerosis, and other cases may have a similar appearance (Smirniotopoulos, J. G.; Murphy, F. M.; Rushing, E. J.; Rees, J. H.; Schroeder, J. W. (2007) "From the Archives of the AFIP: Patterns of Contrast Enhancement in the Brain and Meninges" Radiographics 27 (2): 525-51. The final diagnosis of presumptive MGD by CT and MRI requires a stereotactic biopsy or craniotomy with resection of the tumor and confirmation of the pathology. Since tumor grade is based on the larger malignant part of the tumor, biopsy or intermediate resection of the tumor may underestimate the lesion. Tumor flow imaging using perfusion MRI and measurement of tumor metabolite concentration by MR spectroscopy complements standard MRI in the diagnosis of glioblastoma, showing increased relative cerebral blood volume and elevated choline peak, respectively, but pathology remains the gold standard (Weerakkody, Yuranga; Gaillard, Frank." Glioblastoma". Radiopaedia.org. 2014).

Diagnosis of glioblastoma depends on distinguishing primary glioblastoma from secondary glioblastoma. These tumors arise spontaneously (relapse) or develop from low-grade glioma, respectively (Bleeker, FE; Molenaar, RJ; Leenstra, S (May 2012). "Recent advances in the molecular understanding of glioblastoma." Journal of neuro-oncology 108 (1): 11-27). Primary glioblastomas have a worse prognosis, different tumor biology can give different response to therapy, making it a critical evaluation to determine the prognosis and therapy for the patient (Weerakkody, Yuranga; Gaillard, Frank. "Glioblastoma". Radiopaedia.org 2014). More than 80% of secondary glioblastomas have a mutation in IDH1, while this mutation is rare in primary glioblastoma (5-10%). Thus, IDH1 mutations may be a useful tool for separating primary and secondary glioblastomas in the future, as they are histopathologically very close and recognition without molecular biomarkers is unreliable (The driver and passenger effects of isocitrate dehydrogenase 1 and 2 mutations in oncogenesis and survival prolongation." Biochim Biophys Acta 1846 (2): 326-41 Dec 2014).

IV. PHARMACEUTICAL COMPOSITIONS

The body weight loss agents described above may be formulated into pharmaceutical compositions suitable for use in the methods presented. Such compositions include the active agent (compounds of formula (I)) together with a pharmaceutically acceptable carrier or diluent.

In some cases, the pharmaceutical composition includes compounds of formula (I), its pharmaceutically acceptable salt or prodrug in combination with one or more pharmaceutically acceptable excipients.

In some cases, a pharmaceutical composition containing compounds of formula (I) is administered orally and exhibits higher bioavailability compared to Celastrol. Oral bioavailability may be at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 80%, 90% or 95% or higher compared to Celastrol. Moreover, oral bioavailability can be at least 1%, 2%, 3%, 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40 %, 45%, 50%, 60%, 70%, 80%, 90% or 95% higher than the intravenous bioavailability of the compound of formula (I), and/or at least 1%, 2%, 3% , 4%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 95 % of the level of bioavailability, if the compound is administered intravenously.

The pharmaceutical compositions provided herein include compositions wherein the active ingredient is contained in a therapeutically effective amount, i.e., in an amount effective to achieve the intended purpose. the actual amount effective for a particular application will depend, inter alia, on the condition being treated. When administered in methods of treating a disease, such compositions will contain an amount of the active ingredient effective to achieve the desired result, eg, weight loss. Determination of a therapeutically effective amount of compounds is within the skill of the art, especially in light of the detailed description provided.

Pharmaceutically acceptable salts may be prepared by reacting the free acid or base of the compound described above with a stoichiometric amount of the appropriate base or acid in water, in an organic solvent, or in a mixture thereof. Lists of suitable salts are provided in Remington's Pharmaceutical Sciences, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD, 2000, p. 704; and Handbook of Pharmaceutical salts; Properties, Selection, and Use, P. Heinrich Stahl and Camille G. Wermuth, Eds., Wiley-VCH, Weinheim, 2002.

The body weight loss agent may also be a pharmaceutically acceptable prodrug of any of the compounds described above. Prodrugs are compounds which, when metabolized in vivo , are converted to compounds having the desired pharmacological activity. Prodrugs can be prepared by substitution of suitable functional groups present in the compounds described above with "pro-parts" as described, for example, in H. Bundgaar, Design of Prodrugs (1985). Examples of prodrugs include ester, ether or amide derivatives of the compound described above. Further description of prodrugs is provided in Rautio, J. et al. Nat. Rev. drug disc. 7:255-270, 2008.

For the preparation of pharmaceutical compositions from the compounds described herein, pharmaceutically acceptable carriers may be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. The solid carrier may be one or more substances, which may also be diluents, flavoring agents, binding agents, preservatives, disintegrating agents, or an encapsulating material.

In powders, the carrier is a finely divided solid in admixture with a finely divided active ingredient (eg, the compound provided here). In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compressed into the desired shape and size. Powders and tablets preferably contain 5% to 70% active compound.

Suitable solid fillers include, but are not limited to, magnesium carbonate; magnesium stearate; talc; pectin; dextrin; starch; tragacanth; low melting wax; cacao butter; hydrocarbons; sugars, including but not limited to lactose, sucrose, mannitol or sorbitol, starch from corn, wheat, rice, potatoes or other plants; cellulose such as methylcellulose, hydroxypropylmethylcellulose or sodium carboxymethylcellulose; and gums, including arabic and tragacanth; as well as proteins, including but not limited to gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

The dragee cores are coated with a suitable coating such as concentrated sugar solutions which may also contain gum arabic, talc, polyvinipyrrolidone, carbomer gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to tablet or dragee coatings to identify the product or to indicate the amount of active compound (ie, dose). Pharmaceutical preparations can also be administered orally, using, for example, hard gelatin capsules as well as soft, sealed gelatin capsules and a coating such as glycerol or sorbitol.

To prepare suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active ingredient is homogeneously dispersed therein, for example by stirring. The molten homogeneous mixture is then poured into conveniently sized molds and cooled to solidify.

Liquid preparations include solutions, suspensions, and emulsions, such as water or water/propylene glycol solutions. For parenteral injection, liquid preparations may be formulated in solution in an aqueous solution of polyethylene glycol.

If parenteral administration is necessary or desired, particularly suitable mixtures for the salts described herein are sterile injectable solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories. In particular, carriers for parenteral administration include aqueous dextrose solutions, saline, pure water, ethanol, glycerin, propylene glycol, peanut oil, hemp oil, polyethylene block copolymers, and the like. Ampoules are a convenient dosage form. Salts can also be incorporated into liposomes or administered via transdermal pumps or patches. Pharmaceutical mixtures suitable for use in the various embodiments described herein are well known to those skilled in the art and are described, for example, in Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, PA) and WO 96/05309, ideas which are included here by reference.

Aqueous solutions suitable for oral administration can be prepared by dissolving the active salt (eg, compounds described herein, including variants and examples) in water and adding suitable colorants, flavors, stabilizers, and thickeners, if desired. Aqueous suspensions suitable for oral administration can be prepared by dispersing the finely divided active ingredient in water with a viscous material such as natural or synthetic gums, gums, methylcellulose, cyclodextrin, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth, and dispersing and wetting agents. , such as naturally occurring phosphatides (e.g. lecithin), a condensation product of alkylene oxide and a fatty acid (e.g. polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g. heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexite ( eg polyoxyethylene sorbitan monooleate) or the condensation product of ethylene oxide with a partial ester derived from fatty acid and hexitan anhydride (eg polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweeteners such as sucrose, aspartame or saccharin. The compositions can be adjusted for osmolarity.

Also included are solid preparations which are intended to be converted, shortly before use, to liquid preparations for oral administration. Such liquid forms include solutions, suspensions and emulsions. These preparations may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

Oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweeteners may be added to form an edible oral preparation, such as glycerol, sorbitol, or sucrose. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid. Examples of an oily injectable carrier are provided by Minto, J. Pharmacol. Exp. Ther. 281:93-102, 1997. Pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oil phase may be vegetable oil or mineral oil as described above, or a mixture thereof. Suitable emulsifying agents include natural gums such as gum arabic and tragacanth, natural phosphatides such as soy lecithin, esters or partial esters derived from fatty acids, and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents, such as in the composition of syrups and elixirs. Such compositions may also contain an emollient, preservative or coloring agent.

The compounds described herein may be administered alone or may be co-administered to a patient. Co-administration includes the simultaneous or sequential administration of compounds, alone or in combination (more than one compound). Thus, the preparations can also be combined, if desired, with other active substances (for example, for weight loss).

Compounds can be administered via a variety of routes and approaches, including, but not limited to: oral administration, intravenous administration, topical administration, parenteral administration, intraperitoneal administration, intramuscular administration, intrathecal administration, intralesional administration, intracranial administration, intranasal administration, intraocular administration, intracardiac administration, intravitreal administration, intraosseous administration, intracerebral administration, intra-arterial administration, intra-articular administration, intradermal administration, transdermal administration, transmucosal administration, sublingual administration, enteral administration, sublabial administration, insufflation administration, suppositories, inhalation administration or subcutaneous administration.

The compounds can be prepared and administered in a variety of oral, parenteral and topical dosage forms. Oral preparations include tablets, pills, powders, dragees, capsules, liquids, lozenges, cachets, gels, syrups, suspensions, etc., suitable for ingestion by a patient. The salts of the compounds described herein can also be administered by injection, ie, intravenously, intramuscularly, transdermally, subcutaneously, intraduodenal or intraperitoneally. The salts described herein can also be administered by inhalation, for example intranasally. Additionally, the salts may be administered transdermally. It is also obvious that a variety of routes of administration (eg, intramuscular, oral, transdermal) can be used to administer the salts of the compounds described here. Therefore, pharmaceutical compositions are also provided containing a pharmaceutically acceptable excipient and one or more salts of a compound or compounds described herein.

Pharmaceutical preparations are preferably in unit dosage form. In this form, the preparation is subdivided into doses containing appropriate amounts of the active ingredient. A unit dosage form may be a packaged preparation, where the package contains individual quantities of the drug, such as packaged tablets, capsules, and powders in vials and ampoules. Also, the unit dosage form may be a capsule, tablet, cachet or lozenge per se, or may be a suitable amount of any of these in packaged form.

The amount of active ingredient in a standard formulation may vary or be adjusted per kilogram of body weight, from about 0.1 µg/kg to about 100,000 µg/kg, from 1.0 µg/kg to 10,000 µg/kg, or from 1 µg/kg to 5000 mcg/kg, according to the specific application and strength of the active ingredient. An example of unit doses would be 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000 or 5000 µg/kg. The composition may, if desired, also contain other compatible therapeutic agents. Multiple unit doses may be administered over a 24 hour period. Doses may be administered orally, but other routes of administration may also be used, depending on the severity of the disease/disorder of the patient.

In some embodiments, the amount of compounds of formula (I) or formula (II) may vary or be adjusted from about 1.0 μg to 10,000 μg, for example, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 , 1000, 2000 or 5000 mcg/kg body weight, for the treatment of obesity by oral administration. In some embodiments, the amount of compounds of formula (I) or formula (II) may vary or be adjusted from about 1.0 μg to 10,000 μg, for example, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 , 1000, 2000 or 5000 mcg/kg body weight, for the treatment of obesity by intraperitoneal administration.

Various embodiments include oral administration of a compound described herein. In some embodiments, the compound is administered at a dose of about 0.05 to about 100 mg/kg, about 0.1 to about 0.5 mg/kg, about 0.1 to about 1 mg/kg, about 0. 1 to about 5 mg/kg, about 0.1 to about 10 mg/kg, about 0.1 to about 25 mg/kg, about 1 to about 5 mg/kg, about 1 to about 25 mg/kg kg, about 5 to about 25 mg/kg, about 10 to about 25 mg/kg, about 10 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 75 mg/kg kg or from about 50 to about 100 mg/kg. In certain embodiments, the compound is administered at a dose of about 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0 .7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg. Doses may be administered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more times per day or per week. For example, the compound may be administered once, twice, or three times per day. In some embodiments, the compound is administered before (eg, about 1, 2, 3, 4, 5, or 6 hours before) or with food. Non-limiting examples of methods for converting animal doses, such as mice, to human equivalent doses are known in the art. See, for example, US Food and Drug Administration Center for Drug Evaluation and Research (CDER) (2005) Guidance For Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (available at www.fda.gov /downloads/drugs/guidances/ucm078932.pdf). For example, a mouse dose in mg/kg can be converted to an equivalent human dose (assuming a 60 kg human) based on body surface area by multiplying the mouse dose by 0.08.

Some compounds may have limited water solubility and therefore may require the presence of a surfactant or other suitable co-solvent in the composition. Such co-solvents include: Polysorbate 20, 60 and 80; Pluronic F-68, F-84 and P-103; cyclodextrin; polyoxyl 35 castor oil; or other agents known to the person skilled in the art. Such co-solvents are typically used in an amount of from about 0.01% to about 2% by weight.

Viscosities greater than those of simple aqueous solutions may be desirable to reduce the dispersion variability of the compositions, to reduce the physical separation of the suspension or emulsion components of the composition, and/or to otherwise improve the composition. Such viscosity modifying agents include, for example, polyvinyl alcohol, polyvinylpyrrolidone, methylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, carboxymethylcellulose, hydroxypropylcellulose, chondroitin sulfate and its salts, hyaluronic acid and its salts, a combination of the above, and other agents known to those skilled in the art. Such agents are typically used in an amount of from about 0.01% to about 2% by weight. Determination of acceptable amounts of any of the above adjuvants is easily carried out by a person skilled in the art.

The compositions may further include components for obtaining a delayed release and/or comfort. Such components include high molecular weight anionic mucomimetic polymers, gelling polysaccharides, and finely divided drug substrates. These components are discussed in more detail in US Pat. Nos. 4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents of these patents are incorporated herein by reference in their entirety for all purposes.

The dose and frequency (single or multiple doses) administered to a mammal may vary depending on a variety of factors, such as whether the mammal is suffering from another disease and the route of administration; size, age, sex, health, body weight, body mass index and nutrition of the recipient; the nature and duration of symptoms of the disease being treated (eg, emphysema, asthma, ARDS including oxygen toxicity, pneumonia, chronic obstructive pulmonary disease (COPD), emphysema, cystic fibrosis, bronchopulmonary dysplasia, chronic sinusitis, pulmonary fibrosis), type of concomitant treatment, complications of the treated disease or other health-related problems. The disease may be the primary cause of the disease and/or weight gain disorder. The disease may be caused by a primary disorder and/or a weight gain disorder. Other therapeutic regimens or agents may be used in combination with the methods and compounds described herein. Adjustment and modification of set doses (eg, frequency and duration) is within the skill of the art.

For any compound described herein, a therapeutically effective amount may initially be determined from cell culture studies. Target concentrations are those concentrations of the active compound that are able to achieve the methods described here, as measured by the methods described here or known in the art.

Target concentrations are those concentrations of the active compound that are capable of achieving the methods described here, as measured by the methods described here or known in the art.

As is well known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, the human dose may be formulated to achieve a concentration that is determined to be effective in animals. The dose in humans can be adjusted by monitoring the effectiveness of the compounds and adjusting the dose downward or upward as described above. Dose adjustment to achieve maximum efficacy in humans is based on the methods described above and others, and is within the skill of the art.

Doses may vary depending on the requirements of the patient and the compound used. The dose administered to the patient, in the context of this description should be sufficient to cause a positive therapeutic response in the patient over time. The size of the dose is also determined by the existence, nature and duration of any unwanted side effects. Determining the appropriate dose for a particular situation is within the purview of the practitioner. In general, treatment is started with small doses that are less than the optimum dose of the compound. The dose is then increased slightly until the optimum effect under the circumstances is reached. In some embodiments, the dose is from 0.001% to 10% wt./about. In other embodiments, the dose is from 0.1% to 5% wt./about.

Dosing amounts and intervals may be adjusted individually to obtain levels of administered compound effective for the particular clinical indication being treated. This provides a therapeutic regimen that is appropriate to the severity of the disease state of the individual.

With the information provided here, an effective prophylactic or therapeutic regimen can be planned that does not cause significant toxicity and is effective in treating the clinical symptoms exhibited by a particular patient. Such planning should include careful selection of the active compound, taking into account factors such as potency of the compound, relative bioavailability, body weight of the patient, the presence and severity of unwanted side effects, the preferred route of administration, and the toxicity profile of the chosen agent.

The ratio between toxicity and therapeutic effect for a particular compound is called its therapeutic index and can be expressed as the ratio between LD50 (amount of a compound lethal in 50% of the population) and ED50 (amount of a compound effective in 50% of the population). Compounds that have a high therapeutic index are preferred. Therapeutic index data obtained from cell culture studies and/or animal studies can be used in formulating a dosage range in humans. Doses of such compounds preferably lie within the range of plasma concentrations that include the ED50 with little or no toxicity. The dose may vary within this range depending on the dosage form used and the route of administration used. See, for example, Fingl et al., In: The Pharmacological Basis of Therapeutics, Ch.1, p.1, 1975. The exact composition, route of administration, and dosage may be chosen by the individual physician, taking into account the condition of the patient and the particular route in which apply connection.

V. SET

In one aspect, a kit is provided that contains a composition for use in the treatment of obesity described herein and instructions for use in the treatment of obesity. In some embodiments, the kit may be used for oral administration of compositions for the treatment of obesity, for example, the kit may also include an oral applicator. In some embodiments, the kit may be used for intraperitoneal administration of compositions for the treatment of obesity.

EXAMPLES

The following examples illustrate certain specific embodiments of the invention and should not be construed as limiting the scope of the invention.

The options presented here are further illustrated by the following examples and detailed protocols. However, the examples are only intended to illustrate the options and should not be considered limiting. The contents of all references and published patents and patent applications cited in this specification are incorporated herein by reference.

CHEMISTRY

The following examples illustrate certain specific embodiments of the invention and do not limit the scope of this invention.

The options presented here are further illustrated by the following examples and detailed protocols. However, the examples are only intended to illustrate the options and should not be considered limiting. The contents of all references and published patents and patent applications cited in this specification are incorporated herein by reference.

Example 1

To a solution of Celastrol (150 mg, 0.33 mmol) in DMF (3 ml) was added EtI (104 mg, 0.054 ml, 0.67 mmol) and Na ₂ CO ₃ (70.6 mg, 0.67 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give the product (70 mg, 0.146 mmol, Yield=44%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.02 (1H, d, J=7.2 Hz), 6.96 (1H, s), 6.54 (1H, s), 6.35 ( 1H, d, J=7.2 Hz), 3.90-4.05 (2H, m), 2.44 (1H, d, J=15.5 Hz), 2.21 (3H, s), 2.13-2.25(2H, m), 2.00-2.10(1H, m), 1.77-1.93(3H, m), 1.62-1.73(3H, m ), 1.47-1.55(2H, m), 1.45(3H, s), 1.32-1.42(1H, m), 1.26(3H, s), 1.21( 3H, t, J=7.2Hz), 1.17(3H, s), 1.10(3H, s), 0.93-1.01(1H, m), 0.56(3H, s ); ¹³ C NMR δ (100 MHz, CDCl ₃ ): 178.34, 178.19, 170.09, 164.71, 146.01, 134.12, 127.37, 119.55, 118.12, 117, 12, 60.26, 45.04, 44.26, 42.93, 40.24, 39.40, 38.22, 36.35, 34.76, 33.51, 32.76, 31.59, 30.69, 30.55, 29.78, 29.60, 28.66, 21.63, 18.44, 14.02, 10.26; LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml/ min Column: Poroshell 120 EC-C18, 4.6×30 mm, 2.7 µm): vv=2.20 min, m/z=479.3 [M+H] ⁺ , purity=100% (214 , 254 nm).

Example 2

To a solution of Celastrol (150 mg, 0.333 mmol) in THF (3 ml) was added EtNH ₂ .HCl (40 mg, 0.50 mmol), GATU (190 mg, 0.5 mmol), then NEt ₃ (101 mg, 0 .14 ml, 1 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give the product (23.5 mg, 0.0492 mmol, Yield=15%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.01 (1H, dd, J=7.2, 0.9 Hz), 6.98 (1H, s), 6.53 (1H, d, J =0.9Hz), 6.34(1H, d, J=7.2Hz), 5.62(1H, t, J=5.0Hz), 3.13-3.20(2H, m ), 2.46 (1H, d, J=15.8 Hz), 2.21 (3H, s), 2.10-2.17 (1H, m), 1.98-2.09 (1H, m), 1.82-1.97(4H, m), 1.47-1.74(7H, m), 1.44(3H, s), 1.26(3H, s), 1.15 (3H, s), 1.13 (3H, s), 1.06 (3H, t, J=7.2 Hz), 0.98-1.05 (1H, m), 0.65 (3H, With); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.7 min; Flow rate: 2.2 ml /min, Column: SunFire C18.4.6×50 mm, 3.5 µm): vv=2.33 min, m/z=478.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 3

To a solution of celastrol (200 mg, 0.44 mmol) in THF (5 ml) was added morpholine (78 mg, 0.88 mmol), GATU (254 mg, 0.66 mmol), then DIPEA (114 mg, 0.16 ml, 0.88 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give the product (74 mg, 0.142 mmol, Yield=32%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.02 (1H, dd, J=7.2, 1.0 Hz), 6.97 (1H, s), 6.54 (1H, d, J =1.0 Hz), 6.36 (1H, d, J=7.2 Hz), 3.50-3.80 (8H, m), 2.28-2.36 (2H, m), 2 .22 (3H, s), 2.16-2.23 (1H, m), 2.03-2.13 (1H, m), 1.49-1.93 (8H, m), 1.46 (3H, s), 1.34-1.40 (1H, m), 1.25-1.33 (1H, m), 1.30 (3H, s), 1.28 (3H, s), 1.15 (3H, s), 0.96-1.02 (1H, m), 0.61 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml /min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vv=2.36 min, m/z=520.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 4

To a solution of Celastrol (150 mg, 0.333 mmol) in THF (3 mL) was added 1-methylpiperazine (50 mg, 0.055 mL, 0.50 mmol), GATU (190 mg, 0.5 mmol), then NEt ₃ (67, 4 mg, 0.093 mmol, 0.666 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=10:1) to give the product (25.2 mg, 0.0473 mmol, Yield=14.2%) as a red solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.02 (1H, dd, J=7.2, 1.0 Hz), 6.98 (1H, br), 6.51 (1H, d, J=1.0 Hz), 6.35(1H, d, J=7.2 Hz), 3.60-3.80(2H, m), 2.31-2.45(5H, m), 2.30(3H, s), 2.21(3H, s), 2.04-2.19(2H, m), 1.65-1.92(8H, m), 1.49-1, 62(3H, m), 1.45(3H, s), 1.33-1.39(1H, m), 1.29(3H, s), 1.28(3H, s), 1.24 -1.30 (1H, m), 1.14 (3H, s), 0.95-1.01 (1H, m), 0.61 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.7 min; Flow rate: 2.2 ml /min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vv=1.88 min, m/z=533.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 5

To a solution of Celastrol (200 mg, 0.44 mmol) and MeNO ₂ (54 mg, 0.88 mmol) in THF (3 mL) was added 1M FTBA in THF solution (0.22 mL, 0.22 mmol). The reaction mixture was stirred at room temperature overnight. The reaction is quenched by adding H ₂ O (50 ml). The solution is then extracted with Et ₂ O (2×50 ml). The combined organic layers are washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=1:1) to give the product (139.6 mg, 0.273 mmol, Yield=62%) as a pale yellow solid, ¹ H NMR: δ (400 MHz, CDCl ₃ ): 6.77 (1H, s), 5.69 (1H, d, J=5.9 Hz), 4.54 (1H, dd, J=11.3, 4.0 Hz), 4, 24-4.31 (1H, m), 4.11 (1H, t, J=11.3 Hz), 2.38 (1H, d, J=15.5 Hz), 2.28 (3H, s ), 1.48-2.14(9H, m), 1.45(3H, s), 1.29-1.39(4H, m), 1.20(3H, s), 1.13( 3H, s), 1.05 (3H, s), 0.84-0.92 (1H, m), 0.57 (3H, s); LC-MS (Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml/min; Column: Poroshell 120 EC-C18.4.6×30 mm, 2.7 µm): w =1.86 min, m/z=451.2 [M-CH ₂ NO ₂ ] ⁺ , purity=100% (214, 254 nm).

Example 6

To a solution of celastrol (225 mg, 0.5 mmol) in DMF (10 ml) was added NH ₄ Cl (80 mg, 1.5 mmol), GATU (209 mg, 0.55 mmol), then DIPEA (129 mg, 0 .18 ml, 1.0 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=1:1) to give the product (200 mg, 0.445 mmol, Yield=89%) as a red solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.01 (1H, s), 7.00 (1H, d, J=7.0 Hz), 6.51 (1H, s), 6.33 (1H, dd, J=7.0, 2.4 Hz), 5.34-5.74 (2H, w), 2.42 (1H, d, J=15.1 Hz), 2.22 ( 3H, s), 2.00-2.16(2H, m), 1.82-1.98(4H, m), 1.46-1.72(7H, m), 1.44(3H, s), 1.26(3H, s), 1.20(3H, s), 1.12(3H, s), 0.98-1.05(1H, m), 0.72-0.73 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml /min; Column: Poroshell 120 EC-C18, 4.6×30 mm, 2.7 µm): w=1.81 min, m/z=450.4 [M+H] ⁺ , purity=100% ( 214, 254 nm).

Example 7

To a solution of Celastrol (100 mg, 0.222 mmol) in CH ₂ Cl ₂ (4 ml) was added NEt ₃ (45 mg, 0.062 ml, 0.444 mmol) followed by AcCl (20.9 mg, 0.019 mmol, 0.266 mmol). The reaction mixture was stirred at 0°C for 1 hour. The solution was then diluted with CH ₂ Cl ₂ (100 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1 then CH ₂ Cl ₂ /ethyl acetate=1:1) to give the product (17.3 mg, 0.0351 mmol, Yield=15.8%) as yellow solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.07 (1H, d, J=7.0 Hz), 6.60 (1H, s), 6.30 (1H, d, J=7, 0 Hz), 2.24 (1H, d, J=15.5 Hz), 2.35 (3H, s), 2.15 (3H, s), 2.10-2.18 (2H, m) , 1.95-2.10(2H, m), 1.78-1.90(3H, m), 1.46-1.72(4H, m), 1.45(3H, s), 1 .28-1.40(2H, m), 1.25(3H, s), 1.18(3H, s), 1.08(3H, s), 0.91-0.97(1H, m ), 0.66 (3H, s); LC-MS (Mobile phase: A: H ₂ O (0.01% TFA); B: MeCN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2 ml Column: SunFire C18 50×4.6 mm, 3.5 µm): vy=2.19 min, m/z=493.2 [M+H] ⁺ , purity=100% (214, 254 nm ).

Example 8

Me ₂ NH.HCl (72.4 mg, 0.888 mmol), GATU (338 mg, 0.888 mmol), then NEt ₃ (134.8 mg, 0.186 mmol, 1.332 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=1:1) to give the product (101.4 mg, 0.212 mmol, Yield=48%) as a red solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.02 (1H, dd, J=7.2, 1.2 Hz), 6.96 (1H, s), 6.53 (1H, d, J=1.2Hz), 6.36(1H, d, J=7.2Hz), 3.00-3.25(3H, w), 2.70-3.00(3H, w), 2.32-2.46(2H, m), 2.22(3H, s), 2.06-2.20(2H, m), 1.48-1.93(9H, m), 1, 46 (3H, s), 1.30-1.36 (1H, m), 1.29 (3H, s), 1.28 (3H, s), 1.14 (3H, s), 0.95 -1.01 (1H, m), 0.54 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml Column: Poroshell 120 EC-C18, 4.6 x 30 mm, 2.7 µm): w=1.96 min, m/z=478.4 [M+H] ⁺ , purity=100% ( 214, 254 nm).

Example 9

To a solution of Celastrol ERX1001 ethyl ester (200 mg, 0.418 mmol) in acetone (4 mL) was added K ₂ CO ₃ (115 mg, 0.836 mmol) followed by MeI (1 mL). The reaction mixture is heated at 40°C overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=2:1) to give the product (120 mg, 0.244 mmol, Yield=58%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 6.98 (1H, d, J=7.2, 1.2 Hz), 6.43 (1H, d, J=1.2 Hz), 6, 30 (1H, d, J=7.2 Hz), 3.90-4.06 (2H, m), 3.85 (3H, s), 2.44 (1H, d, J=15.8 Hz ), 2.22(3H, s), 2.00-2.22(3H, m), 1.77-1.92(3H, m), 1.48-1.72(6H, m), 1.45 (3H, s), 1.32-1.42 (1H, m), 1.26 (3H, s), 1.21 (3H, t, J=7.0 Hz), 1.17 (3H, s), 1.10 (3H, s), 0.93-1.00 (1H, m), 0.58 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml /min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vv=2.78 min, m/z=493.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 10

To a solution of Celastrol ERX1001 ethyl ester (160 mg, 0.33 mmol) in THF (20 ml) was added LiAlH ₄ (50.8 mg, 1.33 mmol) in portions. The reaction mixture was stirred at room temperature overnight. The reaction is quenched by adding H ₂ O (5 ml) and acidified to pH 6-7 by adding 0.1 M HCl. The solution is then diluted with EtOAc (200 ml), filtered to remove a solid. The filtrate was washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo to give the crude intermediate. The crude intermediate was dissolved in MeOH (10 ml) and oxidized with an O ₂ balloon while heating at 40° C. overnight. The solution was concentrated in vacuo and the residue was purified by prep-TLC (petroleum ether/EtOAc=2:1) to give the product (58.5 mg, 0.134 mmol, Yield=41% (2 steps)) as a red solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.03 (1H, dd, J=7.1, 1.2 Hz), 6.97 (1H, s), 6.53 (1H, d, J=1.2 Hz), 6.38(1H, d, J=7.1 Hz), 3.43(1H, dd, J=10.5, 5.5 Hz), 3.22(1H, dd, J=10.5, 4.3 Hz), 2.21(3H, s), 2.09-2.16(1H, m), 2.01(1H, s), 1.54-1 .94 (9H, m), 1.45 (3H, s), 1.38 (3H, s), 1.24-1.50 (4H, m), 1.19 (3H, s), 1. 00 (3H, s), 0.94-1.00 (1H, m), 0.80 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA), B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.7 min; Flow rate: 2.2 ml /min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vv=2.48 min, m/z=437.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 11.

To a solution of Celastrol (500 mg, 1.11 mmol) in EtOH (10 ml) was added NaHSO ₃ (127 mg, 1.22 mmol) in 5 ml H ₂ O solution. The reaction mixture was stirred at room temperature for 3 hours. The solution becomes almost colorless and transparent. The solution is concentrated to dryness in vacuo at 40° C. to give a white powder. Enough EtOH is added to dissolve the crude product. The solution is filtered and the filtrate concentrated in vacuo (<40° C.) to about 10 ml. The solution is stored in the refrigerator overnight. The solid is filtered and dissolved in water. After lyophilization, the pure product (114.7 mg, 0.207 mmol, yield=18.6%) is obtained as a white solid, ¹ H NMR: δ (400 MHz, d6-DMSO): 12.10 (1H, br), 8.84 (1H, w), 7.69 (1H, w), 6.56 (1H, w), 5.78 (1H, d, J=6.3 Hz), 4.44 (1H, d , J=6.3 Hz), 2.30-2.38(1H, m), 2.20(3H, s), 1.75-2.07(4H, m), 1.61(3H, s), 1.20-1.60(9H, m), 1.17(3H, s), 1.08(3H, s), 1.05(3H, s), 0.80-0.88 (1H, m), 0.60 (3H, s); LC-MS (Mobile phase: A: water (10 mM ammonium bicarbonate); B: CAN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4.6×50 mm, 3.5 µm): vy=1.48 min, m/z=530.8 [M-Na] ^- (negative ion), purity=95.47% (214 nm).

Example 12.

To a solution of celastrol ethyl ester (500 mg, 1.045 mmol) in EtOH (10 ml) was added NaHSO ₃ (119.6 mg, 1.149 mmol) in 5 ml H ₂ O solution. The reaction mixture was stirred at room temperature overnight. The solution becomes pale yellow. The solution is concentrated to dryness in vacuo at 40°C. The residue was washed with CH ₂ Cl ₂ (4×10 ml), dissolved in sufficient water, lyophilized overnight to give the product (487.6 mg, 0.837 mmol, Yield=80%) as a pale yellow solid. Product can be gradually oxidized in air, ¹ H NMR: δ (400 MHz, d6-DMSO): 8.78 (1H, s), 7.62 (1H, s), 6.57 (1H, s), 5 .80 (1H, d, J=5.9 Hz), 4.46 (1H, d, J=5.9 Hz), 3.80-3.94 (2H, m), 2.34 (1H, d, J=15.5 Hz), 2.20 (3H, s), 1.76-2.10 (4H, m), 1.62 (3H, s), 1.27-1.65 (9H , m), 1.18 (3H, s), 1.10 (3H, s), 1.08 (3H, t, J=7.2 Hz), 1.06 (3H, s), 0.84 -0.92 (1H, m), 0.45 (3H, s); LC-MS (Mobile phase: A: water (10 mM ammonium bicarbonate); B: ACN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge; C18 , 4.6×50 mm, 3.5 μm): vy=1.83 min, m/z=558.8 [M-Na] ^- (negative ion), purity=95.93% (214 nm).

Example 13

To a solution of Celastrol (200 mg, 0.444 mmol) in acetone (5 ml) was added K ₂ CO ₃ (184 mg, 1.332 mmol) followed by PhCH ₂ Br (83.5 mg, 0.058 ml, 0.488 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=3:1) to give the crude product. But it is not pure enough. The crude product is then further washed with petroleum ether while heating at 60° C. twice to obtain the product (40.6 mg, 0.0751 mmol, Yield=17%) as a yellow solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.27-7.36 (5H, m), 7.01 (1H, d, J=6.9 Hz), 6.98 (1H, s) , 6.48 (1H, s), 6.33 (1H, d, J=6.9 Hz), 5.01 (1H, AB, J=12.3 Hz), 4.94 (1H, AB, J=12.3 Hz), 2.44(1H, d, J=15.7 Hz), 2.20-2.28(1H, m), 2.22(3H, s), 1.82- 2.12(3H, m), 1.46-1.74(8H, m), 1.42(3H, s), 1.35-1.44(1H, m), 1.24(3H, s), 1.21(3H, s), 1.09(3H, s), 0.95-1.01(1H, m), 0.51(3H, s); LC-MS: (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml/min Column: SunFire C18, 4.6×50 mm, 3.5 µm): vv=2.91 min, m/z=541.3 [M+H] ⁺ , purity=97.41% ( 214 nm), 100% (254 nm).

Example 14

To a solution of Celastrol (200 mg, 0.444 mmol) in DMF (2 ml) was added K ₂ CO ₃ (123 mg, 0.888 mmol) followed by MeOCH ₂ Br (61 mg, 0.04 ml, 0.488 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=5:2) to give the product (10.0 mg, 0.0202 mmol, Yield=4.6%) as a yellow solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.02 (1H, d, J=7.0 Hz), 6.97 (1H, s), 6.52 (1H, s), 6.35 (1H, d, J=7.0 Hz), 5.20 (1H, AB, J=5.9 Hz), 5.07 (1H, AB, J=5.9 Hz), 3.44 (3H , s), 2.45 (1H, d, J=15.6 Hz), 2.21 (3H, s), 1.30-2.26 (13H, m), 1.45 (3H, s) , 1.27(3H, s), 1.23(3H, s), 1.11(3H, s), 0.96-1.02(1H, m), 0.60(3H, s); LC-MS (Mobile phase: A: water (10 mM ammonium bicarbonate); B: ACN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4.6×50 mm, 3.5 µm, y=2.75 min, m/z=463.3 [M-OMe] ⁺ , purity=100% (214.254 nm).

Example 15

To a solution of ERX1006 (1.0 g, 2.22 mmol) in CH ₂ Cl ₂ (10 ml) was added NEt ₃ (449 mg, 0.62 ml, 4.44 mmol) followed by AcCl (261 mg, 0.24 ml, 3.33 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction is quenched by adding H ₂ O (5 ml). The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=5:1) to give the intermediate (910 mg, 1.92 mmol, Yield=90%) as a yellow solid.

To a solution of intermediate (590 mg, 1.2 mmol) in CH ₂ Cl ₂ (20 ml) was added ClCO ₂ CCl ₃ (475 mg, 2.4 mmol) followed by NEt ₃ (242 mg, 0.33 ml, 2 .4 mmol) drop by drop. The reaction mixture was stirred at room temperature overnight. The reaction is quenched by adding H ₂ O (5 ml). The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=2:1) followed by reverse phase prep-HPLC to give the intermediate (120 mg, 0.278 mmol, Yield=23%) as a red solid.

To a solution of ERX1006 (650 mg, 1.446 mmol) in CH ₂ Cl ₂ (20 ml) was added (MeO) ₂ P(O)Cl (1044 mg, 0.78 ml, 7.23 mmol) followed by NEt ₃ (732 mg , 0.726 ml, 7.23 mmol). The reaction mixture was stirred at room temperature overnight. The reaction is quenched by adding H ₂ O (5 ml). The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=30:1) to give the product (321 mg, 0.744 mmol, Yield=51%) as a red solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.02 (1H, dd, J=7.1, 1.4 Hz), 6.97 (1H, s), 6.53 (1H, d, J=1.3 Hz), 6.37(1H, d, J=7.5 Hz), 2.22(3H, s), 1.55-2.20(14H, m), 1.47( 3H, s), 1.44 (3H, s), 1.29 (3H, s), 1.09-1.15 (1H, m), 1.09 (3H, s), 1.03 (3H , With); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml Column: SunFire C18, 4.6×50 mm, 3.5 µm): w=2.38 min, m/z=432.3 [M+H] ⁺ , purity=100% (214.254 nm) .

Example 16

To a solution of Celastrol (300 mg, 0.666 mmol) in DMF (4 mL) was added K ₂ CO ₃ (184 mg, 1.332 mmol) followed by carboxylic acid 1-chloroethylcyclohexyl ester (151 mg, 0.134 mL, 0.732 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=5:2) twice to give isomer 1 (less polar, 29.7 mg, 0.0478 mmol, Yield=7.2%) and isomer 2 (more polar, 20, 0 mg, 0.0322 mmol, Yield=4.8%) as a yellow solid. Isomer 1: ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.02 (1H, dd, J=7.3, 1.1 Hz), 6.98 (1H, s), 6.74 (1H , q, J=5.5 Hz), 6.55 (1H, d, J=1.1 Hz), 6.35 (1H, d, J=7.3 Hz), 4.45-4.53 (1H, m), 2.41 (1H, d, J=16.1 Hz), 2.20 (3H, s), 1.00-2.20 (23H, m), 1.46 (3H, s), 1.45 (3H, d, J=5.5 Hz), 1.27 (3H, s), 1.20 (3H, s), 1.09 (3H, s), 0.93- 1.00 (1H, m), 0.61 (3H, s); LC-MS: SP-0012508-089-1-01262-LCMSA043 Mobile phase: A: water (10 mM ammonium bicarbonate); B: ACN; Gradient: 5%-95% B in 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4.6×50 mm, 3.5 µm): vy=3.11 min, m/z=477.4 [MC ₆ H ₁₁ OCO ₂ ] ⁺ , purity=100% (214.254 nm) . Isomer 2: ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.02 (1H, dd, J=7.1, 1.1 Hz), 6.97 (1H, s), 6.67 (1H , q, J=5.4 Hz), 6.52 (1H, d, J=1.1 Hz), 6.35 (1H, d, J=7.1 Hz), 4.52-4.58 (1H, m), 2.41 (1H, d, J=15.9 Hz), 2.21 (3H, s), 1.05-2.25 (23H, m), 1.46 (3H, d, J=5.4 Hz), 1.45 (3H, s), 1.26 (3H, s), 1.19 (3H, s), 1.10 (3H, s), 0.94- 1.01 (1H, m), 0.60 (3H, s); LC-MS (Mobile phase: A: water (10 mM ammonium bicarbonate); B: ACN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4.6×50 mm, 3.5 μm): vy=3.12 min, m/z=477.3 [MC ₆ H ₁₁ OCO ₂ ] ⁺ , purity=94.91% (254 nm).

Example 17.

To a solution of Celastrol (1.8 g, 4.0 mmol) in MeOH (20 ml) was added NaBH ₄ (1.52 g, 40 mmol) in portions at 0°C. The reaction mixture was stirred at room temperature for 30 minutes. The solution turns from reddish to colorless. The reaction mixture is quenched with 0.1 M HCl and neutralized to pH=7. The mixture was then extracted with CH ₂ Cl ₂ (2×100 ml) and the combined organic layers washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo to give the crude intermediate (500 mg, 1.104 mmol, Vd=28% ) as a white solid.

To a solution of the crude intermediate (300 mg, 0.66 mmol) in CH ₂ Cl ₂ (20 mL) was added (MeO) ₂ CMe ₂ (690 mg, 6.6 mmol) followed by TsOH (12 mg, 0.066 mmol). The reaction mixture was stirred at room temperature overnight. The solution was diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over Mg ₂ SO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=10:1) to give the product (230 mg, 0.467 mmol, Yield=71%) as a white solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 6.64 (1H, s), 5.73 (1H, dd, J=6.0, 1.5 Hz), 3.27 (1H, dd, J=20.8, 6.3 Hz), 2.98 (1H, d, J=20.8 Hz), 2.41 (1H, d, J=15.7 Hz), 2.10 (3H, s), 1.95-2.15(4H, m), 1.69(3H, s), 1.64(3H, s), 1.24-1.85(9H, m), 1.30 (3H, s), 1.20 (3H, s), 1.17 (3H, s), 1.05 (3H, s), 0.84-0.91 (1H, m), 0.68 ( 3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml /min Column: Poroshell 120 EC-C18, 4.6x30 mm, 2.7 µm): w=2.34 min, m/z=493 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 18.

To a solution of Celastrol (200 mg, 0.444 mmol) in DMF (10 mL) was added pyrrolidine (63 mg, 0.074 mL, 0.888 mmol), GATU (185 mg, 0.48 mmol), then DIPEA (115 mg, 0.16 mL , 0.88 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with H ₂ O (2×100 ml) then brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give the product (140 mg, 0.278 mmol, Yield=63%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.03 (1H, dd, J=7.2, 1.0 Hz), 6.96 (1H, s), 6.53 (1H, d, J =1.0 Hz), 6.35(1H, d, J=7.2 Hz), 3.56-3.70(2H, m), 3.39-3.50(1H, m), 3 .19-3.29(1H, m), 2.34-2.44(2H, m), 2.22(3H, s), 2.09-2.20(2H, m), 1.47 -1.97(12H, m), 1.46(3H, s), 1.23-1.33(2H, m), 1.27(3H, s), 1.22(3H, s), 1.13 (3H, s), 0.94-1.10 (1H, m), 0.55 (3H, s); LC-MS: Mobile phase: A: water (0.01% TFA): B: ACN (0.01% TFA); Gradient: 5%-95% B in 1.2 min; Flow rate: 2.2 ml/min; Column: Poroshell 120 EC-C18, 4.6×30 mm, 2.7 µm): vv=2.01 min, m/z=504.4 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 19.

To a solution of celastrol (200 mg, 0.444 mmol) in DMF (10 mL) was added piperidine (76 mg, 0.088 mL, 0.888 mmol), GATU (185 mg, 0.48 mmol) followed by DIPEA (115 mg, 0.16 mL , 0.88 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with water (2×100 ml) then brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give the product (95 mg, 0.188 mmol, Yield=42%) as a yellow solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.04 (1H, d, J=7.2 Hz), 6.97 (1H, s), 6.54 (1H, s), 6.36 (1H, d, J=7.2 Hz), 2.90-4.10 (4H, w), 2.28-2.42 (2H, m), 2.22 (3H, s), 2, 05-2.21(2H, m), 1.24-1.92(16H, m), 1.46(3H, s), 1.29(3H, s), 1.27(3H, s) , 1.14 (3H, s), 0.94-1.01 (1H, m), 0.60 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml /min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vv=2.63 min, m/z=518.5 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 20.

To a solution of celastrol (200 mg, 0.444 mmol) in THF (5 ml) was added 3-hydroxyacetidine hydrochloride (97 mg, 0.888 mmol), GATU (338 mg, 0.888 mmol), then NEt ₃ (180 mg, 0.25 ml, 1.776 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:4) and reverse phase prep-HPLC to give the product (80.5 mg, 0.159 mmol, Yield=36%) as a red solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.05 (1H, d, J=7.2 Hz), 6.93 (1H, s), 6.54 (1H, s), 6.36 (1H, d, J=7.2 Hz), 4.50-4.60 (2H, m), 3.55-4.30 (4H, m), 2.23 (3H, s), 1, 82-2.28(6H, m), 1.46-1.74(6H, m), 1.43(3H, s), 1.20-1.34(2H, m), 1.25( 3H, s), 1.14(3H, s), 1.11(3H, s), 0.94-1.01(1H, m), 0.64&0.60(3H, s); LC-MS (Mobile phase: A: water (10 mM ammonium bicarbonate); B: ACN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4.6×50 mm, 3.5 μm): vy=2.34 min, m/z=556.4 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 21.

To a solution of celastrol (300 mg, 0.666 mmol) in THF (7.5 ml) was added 4-hydroxypiperidine (135 mg, 1.33 mmol), HATU (507 mg, 1.33 mmol), then NEt ₃ (202 mg, 0.28 ml, 2.0 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (300 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:3) and reverse phase prep-HPLC to give the product (34.8 mg, 0.0652 mmol, Yield=9.8%) as a yellow solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.03 (1H, d, J=7.1 Hz), 6.96 (1H, s), 6.53 (1H, s), 6.36 (1H, d, J=7.1 Hz), 4.0-4.4 (1H, w), 3.94 (1H, s), 3.0-3.9 (2H, w), 2, 00-2.40(3H, m), 2.22(3H, s), 1.20-1.95(17H, m), 1.46(3H, s), 1.29(3H, s) , 1.28(3H, s), 1.15(3H, s), 0.95-1.02(2H, m), 0.58(3H, s); LC-MS (Mobile phase: A: water (10 mM ammonium bicarbonate); B: ACN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4.6×50 mm, 3.5 µm): vy=2.41 min, m/z=534.3 [M+H] ⁺ , purity=96.7% (214 nm), 100% (254 nm ).

Example 22.

To a solution of celastrol (150 mg, 0.33 mmol) in DMF (10 ml) was added azetidine hydrochloride (62 mg, 0.67 mmol), GATU (139 mg, 0.36 mmol), then DIPEA (86 mg, 0.36 mmol). 12 ml, 0.67 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:4) and reverse phase prep-HPLC to give the product (96 mg, 0.196 mmol, Yield=52%) as a yellow solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.04 (1H, d, J=7.1 Hz), 6.97 (1H, s), 6.56 (1H, s), 6.36 (1H, d, J=7.1 Hz), 4.35-4.45 (2H, m), 3.75-3.95 (2H, m), 2.22 (3H, s), 1, 50-2.30(14H, m), 1.47(3H, s), 1.28(3H, s), 1.24-1.34(2H, m), 1.15(3H, s) , 1.10 (3H, s), 0.93-1.00 (1H, m), 0.66 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml /min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vv=2.38 min, m/z=490.4 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 23.

To a solution of Celastrol (300 mg, 0.666 mmol) in DMF (10 ml) was added 3-hydroxypyrrolidine (116 mg, 1.33 mmol), GATU (279 mg, 0.73 mmol), then DIPEA (172 mg, 0.24 ml, 1.33 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (300 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:5) to give the product (110 mg, 0.212 mmol, Yield=32%) as a yellow solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.04 (1H, d, J=7.0 Hz), 6.95 (1H, s), 6.51&6.46 (1H, s), 6 .35 (1H, d, J=7.0 Hz), 4.33-4.55 (1H, m), 3.15-3.95 (1H, m), 2.50-3.05 (1H , m), 2.20 (3H, s), 1.20-2.50 (16H, m), 1.27 (3H, s), 1.25 (3H, s), 1.20 (3H, s), 1.13 (3H, s), 0.93-1.01 (1H, m), 0.61&0.59&0.53 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml /min Column: SunFire C18, 4.6x50 mm, 3.5 µm): w=2.23 min, m/z=520.3 [M+H] ⁺ , purity=100% (214.254 nm) .

Example 24.

To a solution of Celastrol (200 mg, 0.444 mmol) in DMF (15 mL) was added N-hydroxysuccinimide (153 mg, 1.33 mmol), GATU (185 mg, 0.48 mmol), then DIPEA (115 mg, 0.16 ml, 0.89 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (300 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=40:1) to give the product (80 mg, 0.146 mmol, Yield=33%) as a yellow solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.01 (1H, dd, J=7.2, 1.0 Hz), 6.98 (1H, s), 6.57 (1H, d, J=1.0 Hz), 6.35(1H, d, J=7.2 Hz), 2.70-2.90(4H, m), 2.57(1H, d, J=15.9 Hz), 2.28-2.35(1H, m), 2.21(3H, s), 1.48-2.20(11H, m), 1.46(6H, s), 1.25 -1.30 (1H, m), 1.28 (3H, s), 1.12 (3H, s), 0.99-1.06 (1H, m), 0.71 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml /min; Column: Poroshell 120 EC-C18, 4.6×30 mm, 2.7 µm): vv=1.97 min, m/z=548.3 [M+H] ⁺ , purity=100% ( 214 nm), 97.26% (254 nm).

Example 25.

To a solution of ERX1005 (500 mg, 0.97 mmol) in CH ₂ Cl ₂ (10 ml) was added (MeO) ₂ CMe ₂ (1.01 g, 9.7 mmol) followed by p-TsOH.H ₂ O (18 mg, 0.1 mmol). The reaction mixture was stirred at room temperature overnight. The solution was diluted with CH ₂ Cl ₂ (200 ml), washed with sat. NaHCO ₃ (100 ml) then brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/EtOAc=10:1) to give the intermediate (350 mg, 0.635 mmol, Yield=65%) as a white solid.

To a solution of the intermediate (300 mg, 0.54 mmol) in EtOH (20 ml) was added Pd/C (50 mg) followed by H ₂ NNH ₂ .H ₂ O (270 mg, 5.4 mmol). The reaction mixture is stirred at 75°C overnight. The reaction solution is filtered and concentrated in vacuo. The residue was diluted with CH ₂ Cl ₂ (200 ml), washed with water (2×100 ml) then brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (CH ₂ Cl ₂ /MeOH=30:1) to give the product (160 mg, 0.29 mmol, Yield=57%) as a white solid. ¹ H NMR δ (400 MHz, d4-MeOD): 6.63 (1H, s), 5.90 (1H, d, J=5.9 Hz), 3.53-3.60 (1H, m) , 3.01 (1H, dd, J=12.0, 3.4 Hz), 2.43-2.55 (2H, m), 2.02-2.26 (3H, m), 2.06 (3H, s), 1.79-1.95 (3H, m), 1.54-1.76 (6H, m), 1.67 (3H, s), 1.61 (3H, s), 1.47(3H, s), 1.43-1.51(1H, m), 1.30-1.40(2H, m), 1.29(3H, s), 1.12(3H, s), 1.10 (3H, s), 0.85-0.92 (1H, m), 0.82 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.7 min; Flow rate: 2.2 ml /min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): w=1.98 min, m/z=522.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 26.

To a solution of ERX1025 (90 mg, 0.17 mmol) in CH ₂ Cl ₂ (10 ml) was added NEt ₃ (34 mg, 0.34 mmol) followed by AcCl (20 mg, 0.26 mmol). The reaction mixture was stirred at room temperature for 30 minutes. The solution was diluted with CH ₂ Cl ₂ (100 ml), washed with sat. NaHCO ₃ (50 ml) then brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=10:1) to give the product (60 mg, 0.106 mmol, Yield=63%) as a white solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 6.64 (1H, s), 6.24 (1H, t, J=5.6 Hz), 5.67 (1H, d, J=6, 5 Hz), 3.68-3.75(1H, m), 3.45-3.53(1H, m), 3.05-3.14(1H, m), 2.39(1H, d , J=16.0 Hz), 2.27 (3H, s), 1.98-2.12 (2H, m), 2.05 (3H, s), 1.68 (3H, s), 1 .66 (3H, s), 1.47 (3H, s), 1.04-1.82 (12H, m), 1.16 (6H, s), 1.01 (3H, s), 0. 72-0.80 (1H, m), 0.45 (3H, s); LC-MS (Mobile phase: A: water (10 mM ammonium bicarbonate); B: ACN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4.6×50 mm, 3.5 µm): vy=2.17 min, m/z=564.4 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 27.

To a solution of SM (860 mg, 1.746 mmol) in CH ₂ Cl ₂ (20 ml) was added 3,5-dimethylpyrazole (336 mg, 3.492 mmol) followed by PDC (647 mg, 1.746 mmol). The reaction mixture was stirred at room temperature overnight. The solution is filtered through Celite and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=4:1) to give the product (410 mg, 0.809 mmol, Yield=46%) as a white solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 6.72 (1H, s), 6.24 (1H, s), 2.55 (3H, s), 2.41 (1H, d, J= 15.6 Hz), 1.25-2.20 (13H, m), 1.70 (3H, s), 1.67 (3H, s), 1.52 (3H, s), 1.28 ( 3H, s), 1.16(3H, s), 1.09(3H, s), 0.91-0.99(1H, m), 0.66(3H, s); LC-MS (Mobile phase: A: H ₂ O (0.01% TFA); B: MeCN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2 ml /min Column: SunFire C18 50x4.6 mm, 3.5 µm): vy=2.39 min, m/z=507.3 [M+H] ⁺ , purity=99.28% (214 nm ), 100% (254 nm).

Example 28.

To a solution of ERX1017 (300 mg, 0.61 mmol) in THF (10 ml) was added LiAlH ₄ (70 mg, 1.83 mmol) in portions. The reaction mixture was stirred at room temperature for 1 hour. The reaction is quenched by adding water (5 ml). The solution was diluted with EtOAc (200 ml), filtered. The filtrate is separated and the organic layer is washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/EtOAc=10:1) to give the intermediate (180 mg, 0.376 mmol, Yield=62%) as a white solid.

To a solution of the intermediate (150 mg, 0.31 mmol) in CH ₂ Cl ₂ (10 mL) was added NEt ₃ (63 mg, 0.62 mmol) followed by MsCl (71 mg, 0.62 mmol). The reaction mixture was stirred at room temperature overnight. The solution was diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/EtOAc=10:1) to give the intermediate (160 mg, 0.287 mmol, Yield=93%) as a white solid.

To a solution of intermediate (170 mg, 0.305 mmol) in DMF (5 ml) was added KCN (99 mg, 1.528 mmol) and 18-Crown-6 (403 mg, 1.528). The reaction mixture is heated at 120°C in a microwave reactor for 6 hours. The solution was diluted with EtOAc (200 ml), washed with water (2×100 ml) then brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-HPLC to give the product (110 mg, 0.226 mmol, Yield=74%) as a white solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 6.65 (1H, s), 5.80 (1H, d, J=4.7 Hz), 3.30 (1H, dd, J=20, 8, 6.3 Hz), 3.01 (1H, d, J=20.1 Hz), 2.46 (1H, AB, J=16.5 Hz), 2.19 (1H, AB, J= 16.5 Hz), 2.11(3H, s), 2.00-2.07(2H, m), 1.50-1.85(9H, m), 1.68(3H, s), 1.64 (3H, s), 1.25-1.38 (3H, m), 1.31 (3H, s), 1.29 (3H, s), 1.16 (3H, s), 1 .14 (3H, s), 0.97-1.03 (1H, m), 0.87 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml /min Column: Poroshell 120 EC-C18, 4.6x30 mm, 2.7 µm): w=2.35 min, MS peaks not integrated, purity=100% (214, 254 nm).

Example 29.

To a solution of celastrol (150 mg, 0.33 mmol) in DMF (10 ml) was added 2-amino-1,3-propanediol (91 mg, 1.0 mmol), GATU (139 mg, 0.36 mmol), then DIPEA (129 mg, 0.17 ml, 1.0 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=8:1) to give the product (60 mg, 0.115 mmol, Yield=35%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.03 (1H, d, J=7.1 Hz), 7.00 (1H, s), 6.61 (1H, d, J=3.0 Hz), 6.53(1H, s), 6.35(1H, d, J=7.1Hz), 3.45-3.90(5H, m), 3.00-3.45(2H , m), 2.43 (1H, d, J=15.7 Hz), 2.21 (3H, s), 1.47-2.18 (12H, m), 1.43 (3H, s) , 1.26(3H, s), 1.24-1.28(1H, m), 1.18(3H, s), 1.12(3H, s), 0.95-1.05(1H , m), 0.66 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml/ min; Column: SunFire C18, 4.6×50 mm, 3.5 µm): vy=2.08 min, m/z=524.3 [M+H] ⁺ , purity=95.73% (214 nm ).

Example 30.

To a solution of SM (90 mg, 0.178 mmol) in THF (5 mL) was added conc. HCl (1 ml). The reaction mixture is stirred at 70°C for 2 days. Then most of the solvent THF is removed in vacuo. The residue was diluted with CH ₂ Cl ₂ (200 ml), the aqueous layer was separated, dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=10:1) to give the product (4.4 mg, 0.00943 mmol, Yield=5.3%) as a pale yellow solid. ¹ H NMR δ (400 MHz, CD ₃ OD): 6.86 (1H, s), 6.18 (1H, s), 4.62 (2H, br), 2.55 (3H, s), 1 .40-2.50(14H, m), 1.54(3H, s), 1.35(3H, s), 1.18(3H, s), 1.13(3H, s), 0, 93-1.00 (1H, m), 0.76 (3H, s); LC-MS (Mobile phase: A:H2O (0.01% TFA); B:MeCN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2 ml/min Column: SunFire C18 50×4.6 mm, 3.5 µm): vy=2.05 min, m/z=467 [M+H] ⁺ , purity=97.30% 9214 nm), 97.25 % (254 nm).

Example 31.

To a solution of ERX1006 (60 mg, 0.133 mmol) in acetone (2 ml) was added K ₂ CO ₃ (37 mg, 0.267 mmol) followed by MeI (1 ml). The reaction mixture is stirred at 40°C for overnight. The mixture was diluted with CH ₂ Cl ₂ (300 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:2) to give the product (38.4 mg, 0.0826 mmol, Yield=62%) as a yellow solid.

¹ H NMR δ (400 MHz, CDCl3): 6.96 (1H, dd, J=7.0, 1.0 Hz), 6.40 (1H, d, J=1.0 Hz), 6.27 (1H, d, J=7.0 Hz), 5.72 (1H, w), 5.55 (1H, w), 3.84 (3H, s), 2.40 (1H, d, J= 15.7 Hz), 2.22(3H, s), 1.45-2.13(13H, m), 1.44(3H, s), 1.25(3H, s), 1.20( 3H, s), 1.11(3H, s), 0.97-1.04(1H, m), 0.73(3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml /min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vv=2.16 min, m/z=464.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 32.

To a solution of ERX1006 (100 mg, 0.22 mmol) in THF (15 mL) was added K ₂ CO ₃ (46 mg, 0.33 mmol) followed by BrCH ₂ CO ₂ Me (51 mg, 0.032 mL, 0.33 mmol ). The reaction mixture is stirred at 50°C overnight. The mixture was diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=1:1) to give the product (40 mg, 0.077 mmol, Yield=35%) as a yellow solid. ¹ H NMR δ (400 MHz, CDCl3): 7.01 (1H, d, J=7.1 Hz), 6.36 (1H, s), 6.29 (1H, d, J=7.1 Hz ), 5.70(1H, br), 5.39(1H, br), 4.85(2H, s), 3.77(3H, s), 2.40(1H, d, J=15, 4 Hz), 2.31(3H, s), 1.80-2.12(7H, m), 1.46-1.72(6H, m), 1.43(3H, s), 1, 26 (3H, s), 1.20 (3H, s), 1.11 (3H, s), 0.88-1.05 (1H, m), 0.74 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml Column: SunFire C18, 4.6×50 mm, 3.5 µm): w=2.16 min, m/z=522.3 [M+H] ⁺ , purity=100% (214.254 nm) .

Example 33.

To a solution of ERX1006 (300 mg, 0.667 mmol) in CH ₂ Cl ₂ (5 ml) was added Et ₃ N (135 mg, 0.19 ml, 1.334 mmol) followed by ClCO ₂ Et (145 mg, 0.13 ml, 1.334 mmol) dropwise at 0°C. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with CH ₂ Cl ₂ (300 ml), washed with brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=10:1) to give the product (150.6 mg, 0.289 mmol, Yield=43%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl3): 7.06 (1H, dd, J=7.0, 1.0 Hz), 6.47 (1H, d, J=1.0 Hz), 6.33 (1H, d, J=7.0 Hz), 5.67 (1H, w), 5.26 (1H, w), 4.32 (2H, q, J=7.2 Hz), 2.39 (1H, d, J=15.6Hz), 2.21(3H, s), 1.49-2.04(13H, m), 1.46(3H, s), 1.39(1H, t, J=7.2 Hz), 1.27 (3H, s), 1.21 (3H, s), 1.12 (3H, s), 0.99-1.05 (1H, m), 0.76 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml /min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vv=2.15 min, m/z=522.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 34.

To a solution of ERX1006 (300 mg, 0.667 mmol) in (CH ₂ Cl) ₂ (10 mL) was added Et ₃ N (202 mg, 0.28 mL, 2.0 mmol) followed by EtNCO (142 mg, 0.158 mL, 2 .0 mmol). The reaction mixture is stirred at 55°C overnight. The mixture was diluted with CH ₂ Cl ₂ (300 ml), washed with brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=20:1) to remove unreacted SM. Then the crude product is heated at 80°C with 20 ml of ethyl acetate. The solid is filtered and then purified by prep-TLC (ethyl acetate/CH ₂ Cl ₂ =3:1) to give the product (74.7 mg, 0.143 mmol, Yield=22%) as a yellow solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.03 (1H, d, J=7.2 Hz), 6.45 (1H, s), 6.31 (1H, d, J=7, 2 Hz), 5.70 (1H, w), 5.38 (1H, w), 5.26 (1H, t, J=5.0 Hz), 3.26-3.45 (2H, m) , 2.38 (1H, d, J=15.7 Hz), 2.18 (3H, s), 1.47-2.15 (13H, m), 1.45 (3H, s), 1, 26 (3H, s), 1.22 (3H, t, J=7.2 Hz), 1.20 (3H, s), 1.12 (3H, s), 0.98-1.05 (1H , m), 0.75 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml Column: SunFire C18, 4.6×50 mm, 3.5 µm, w=2.06 min, m/z=521.4 [M+H] ⁺ , purity=100% (214.254 nm).

Example 35.

To a solution of SM (160 mg, 0.345 mmol) in CH ₂ Cl ₂ (5 ml) was added ClCO ₂ CCl ₃ (341 mg, 0.21 ml, 1.725 mmol) followed by NEt ₃ (175 mg, 0.24 ml, 1.725 mmol) dropwise at 0°C. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with CH ₂ Cl ₂ (300 ml), washed with brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=30:1) to give the product (71 mg, 0.159 mmol, Yield=46%) as a yellow solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 6.97 (1H, d, J=6.9 Hz), 6.42 (1H, s), 6.32 (1H, d, J=6.9 Hz), 3.85(3H, s), 2.22(3H, s), 2.09-2.18(3H, m), 1.89-2.01(3H, m), 1.53 -1.81 (8H, m), 1.47 (3H, s), 1.44 (3H, s), 1.29 (3H, s), 1.08-1.15 (1H, m), 1.09 (3H, s), 1.05 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml /min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vv=2.33 min, m/z=446.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 36.

To a solution of SM (100 mg, 0.232 mmol) in acetone (2 ml) was added K ₂ CO ₃ (64 mg, 0.463 mmol) followed by BrCH ₂ CO ₂ Me (71 mg, 0.044 ml, 0.463 mmol). The reaction mixture is stirred at 55°C overnight. The mixture was diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /EtOAc=10:1) to give the crude product. The crude product was then washed with Et ₂ O (2×2 ml) to give the pure product (55 mg, 0.109 mmol, Yield=47%) as a yellow solid.

¹ H NMR δ (400 MHz, CDCl3): 7.03 (1H, dd, J=7.0, 1.0 Hz), 6.38 (1H, d, J=1.0 Hz), 6.33 (1H, d, J=7.0 Hz), 4.85 (1H, AB, J=16.5 Hz), 4.84 (1H, AB, J=16.5 Hz), 3.77 (3H , s), 2.32 (3H, s), 2.08-2.18 (3H, m), 1.89-2.00 (3H, m), 1.52-1.81 (8H, m ), 1.46 (3H, s), 1.44 (3H, s), 1.29 (3H, s), 1.08-1.15 (1H, m), 1.09 (3H, s) , 1.04 (3H, s); LC-MS (Mobile phase: A: water (0.1% TFA); B: ACN (0.1% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml Column: Poroshell 120 EC-C18, 4.6 x 30 mm, 2.7 µm): w=1.94 min, m/z=504.3 [M+H] ⁺ , purity=100% (214.254 nm).

Example 37.

To a solution of ERX1015 (120 mg, 0.278 mmol) in CH ₂ Cl ₂ (5 ml) was added Et ₃ N (84 mg, 0.12 ml, 0.834 mmol) followed by ClCO ₂ Et (91 mg, 0.834 ml, 0.834 mmol) drop by drop at 0°C. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with CH ₂ Cl ₂ (300 ml), washed with brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=50:1) to give the product (97.3 mg, 0.193 mmol, Yield=69%) as a yellow solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.08 (1H, d, J=7.2 Hz), 6.49 (1H, s), 6.36 (1H, d, J=7, 2 Hz), 4.32 (2H, q, J=7.2 Hz), 2.22 (3H, s), 2.09-2.20 (3H, m), 1.89-2.03 ( 3H, m), 1.51-1.81(8H, m), 1.49(3H, s), 1.44(3H, s), 1.39(3H, t, J=7.2Hz ), 1.30(3H, s), 1.07-1.15(1H, m), 1.09(3H, s), 1.05(3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml Column: Poroshell 120 EC-C18, 4.6×30 mm, 2.7 µm): w=1.92 min, m/z=504.4 [M+H] ⁺ , purity=98.02 % (214 nm), 97.42% (254 nm).

Example 38.

To a solution of ERX1015 (100 mg, 0.232 mmol) in (CH ₂ Cl) ₂ (5 ml) was added Et ₃ N (117 mg, 0.16 ml, 1.158 mmol) followed by EtNCO (82 mg, 0.0917 ml, 1.158 mmol). The reaction mixture is stirred at 50°C overnight. The mixture was diluted with CH ₂ Cl ₂ (300 ml), washed with brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=20:1) to give the product (59.6 mg, 0.119 mmol, Yield=51%) as a yellow solid. ¹ H NMR δ (400 MHz, CDCl3): 7.04 (1H, d, J=7.0 Hz), 6.47 (1H, s), 6.34 (1H, d, J=7.0 Hz ), 5.17 & 4.71 (1H, w), 3.19-3.36 (2H, m), 2.19 (3H, s), 2.09-2.17 (3H, m), 1, 89-2.02(4H, m), 1.54-1.81(8H, m), 1.48(3H, s), 1.44(3H, s), 1.29(3H, s) , 1.20-1.27(3H, m), 1.23&1.14(3H, t, J=7.2Hz), 1.09(3H, s), 1.05(3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml Column: Poroshell 120 EC-C18, 4.6 x 30 mm, 2.7 µm): w=1.83 min, m/z=503.4 [M+H] ⁺ , purity=95.00 % (214 nm), 98.55% (254 nm).

Example 39.

To a solution of ERX1015 (100 mg, 0.23 mmol) in CH ₂ Cl ₂ (10 ml) was added NEt ₃ (47 mg, 0.065 ml, 0.46 mmol) followed by (MeO) ₂ P(O)Cl (70 mg , 0.052 ml, 0.46 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=2:1) to give the product (28 mg, 0.0519 mmol, Yield=23%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl ₃ ): 7.06 (1H, d, J=7.0 Hz), 6.47 (1H, s), 6.35 (1H, d, J=7.0 Hz), 4.00 (3H, d, J=11.4 Hz), 3.96 (3H, d, J=11.2 Hz), 2.30 (3H, d, J=1.5 Hz) , 2.10-2.18(3H, m), 1.90-2.00(3H, m), 1.54-1.80(8H, m), 1.47(3H, s), 1 .44 (3H, s), 1.29 (3H, s), 1.09-1.14 (1H, m), 1.09 (3H, s), 1.05 (3H, s); LC-MS (Mobile phase: A: water (0.1% TFA); B: ACN (0.1% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml Column: Poroshell 120 EC-C18, 4.6 x 30 mm, 2.7 µm): w=1.82 min, m/z=540.3 [M+H] ⁺ , purity=94.11 % (254 nm).

Example 40.

To a solution of ERX1006 (250 mg, 0.56 mmol) in THF (15 ml) was added 1M BH ₃ .THF (1.67 ml, 1.67 mmol). The reaction mixture was stirred at room temperature for 1 hour. The reaction is quenched by adding water (5 ml). Most of the solvent is removed in vacuo and the solution is acidified with 0.1 M HCl to pH 5-6. The mixture was diluted with CH ₂ Cl ₂ (200 ml), filtered. The filtrate was washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo to give the crude product (250 mg, 0.554 mmol, Vd=100%) as a white solid. The crude product is used in the next step without further purification.

To a solution of intermediate (250 mg, 0.56 mmol) in CH ₂ Cl ₂ (15 ml) was added NEt ₃ (224 mg, 0.31 ml, 2.21 mmol) followed by AcCl (174 mg, 2.21 mmol ). The reaction mixture was stirred at room temperature overnight. The reaction is quenched by adding water (5 ml). The mixture was diluted with CH ₂ Cl ₂ (200 ml), washed with sat. Na ₂ CO ₃ (2×100 ml) then brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=10:1) to give the product (40 mg, 0.0773 mmol, Vd=14%) as a white solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.00 (1H, s), 5.78 (1H, d, J=6.0 Hz), 3.35 (1H, dd, J=21.0 , 6.0 Hz), 3.07 (1H, d, J=21.0 Hz), 2.31 (3H, s), 2.27 (3H, s), 2.03-2.20 (4H , m), 2.07 (3H, s), 1.85-1.97 (2H, m), 1.48-1.72 (8H, m), 1.43 (3H, s), 1, 36 (3H, s), 1.24 (3H, s), 1.02-1.10 (1H, m), 1.07 (3H, s), 1.06 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA); B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml Column: Poroshell 120 EC-C18, 4.6 x 30 mm, 2.7 µm): w=2.06 min, m/z=518.3 [M+H] ⁺ , purity=97.21 % (214 nm).

Example 41.

To a solution of intermediate (300 mg, 0.66 mmol) in CH ₂ Cl ₂ (10 ml) was added NEt ₃ (134 mg, 0.18 ml, 1.32 mmol) followed by (Cl ₃ CO) ₂ CO (395 mg, 1.32 mmol). The reaction mixture was stirred at room temperature overnight. The reaction is quenched by adding water (5 ml). The mixture was diluted with CH ₂ Cl ₂ (200 ml), washed with sat. Na ₂ CO ₃ (2×100 ml) then brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=10:1) to give the product (70 mg, 0.152 mmol, Vd=18%) as a white solid. ¹ H NMR δ (500 MHz, CDCl ₃ ): 7.11 (1H, s), 5.83 (1H, d, J=6.4 Hz), 3.41 (1H, dd, J=21.0 , 6.4 Hz), 3.09 (1H, d, J=21.0 Hz), 2.28 (3H, s), 2.09-2.18 (4H, m), 1.87-1 .96(2H, m), 1.49-1.72(8H, m), 1.44(3H, s), 1.34(3H, s), 1.25(3H, s), 1, 06-1.11 (1H, m), 1.07 (3H, s), 1.06 (3H, s); LC-MS (Mobile phase: A: water (0.1% TFA); B: ACN (0.1% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml /min Column: Poroshell 120 EC-C18, 4.6x30 mm, 2.7 µm): w=2.23 min, m/z=460.3 [M+H] ⁺ , purity=97.77 % (214 nm).

Example 42.

To a solution of ERX1008 (477 mg, 1.0 mmol) in anhydrous THF (40 ml) was added LiAlH ₄ (2.38 g, 75 mmol). The mixture is refluxed overnight. The reaction mixture was quenched with sat. NH ₄ Cl solution. The mixture is heated at 50°C for 2 hours and filtered through a thin layer of silica gel. The solid is washed with THF (3×50 ml). The combined filtrate is concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ : MeOH=10:1) to give the product (23.9 mg, 0.0515 mmol, Vd=5%) as a red solid. ¹ H NMR δ (500 MHz, CDCl3): 7.03 (1H, d, J=7.1 Hz), 6.96 (1H, s), 6.52 (1H, s), 6.39 (1H , d, J=7.1 Hz), 2.34 (6H, w), 2.22 (3H, s), 1.95-2.25 (4H, m), 1.35-1.88 ( 12H, m), 1.44(3H, s), 1.42(3H, s), 1.24(3H, s), 1.09(3H, s), 0.97-1.03(1H , m), 0.79 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml/ Column: SunFire C18, 4.6×50 mm, 3.5 µm): vy=1.83 min, m/z=480.4 [M+H] ⁺ , purity=98.20% (214 nm ), 99.51% (254 nm).

Example 43.

To a solution of Celastrol (100 mg, 0.222 mmol) in MeOH (3 ml) was added iC ₃ H ₇ SH (84.5 mg, 0.10 ml, 1.11 mmol). The reaction mixture was stirred at room temperature for 3 hours. The solution turns from red to a pale reddish yellow. The solution is then concentrated in vacuo to give a crude mixture which is used in the next step without further purification.

Pyridine (0.5 ml) was added to the crude mixture (117 mg, 0.222 mmol, theoretical) in Ac ₂ O (4 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/EtOAc=5:2) to give the product (85.2 mg, 0.139 mmol, overall yield=63%) as a white solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.01 (1H, s), 5.97 (1H, d, J=6.2 Hz), 4.57 (1H, d, J=6.2 Hz), 3.11-3.22 (1H, m), 2.39 (1H, d, J=15.8 Hz), 2.30 (3H, s), 2.27 (3H, s), 2.26(3H, s), 1.25-2.15(13H, m), 1.58(3H, s), 1.39(1H, d, J=6.5Hz), 1.27 (1H, d, J=6.5Hz), 1.25(3H, s), 1.16(3H, s), 1.07(3H, s), 0.89-0.96(1H, m), 0.67 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.7 min; Flow rate: 2.2 ml/ min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vy=2.68 min, m/z=535.2 [MC ₃ H ₇ S] ⁺ , purity=100% (214.254 nm ).

Example 44.

To a solution of Celastrol (50 mg, 0.111 mmol) in MeOH (10 ml) was added iC ₃ H ₇ SH (44 mg, 0.58 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from red to almost colorless. The solution is then concentrated in vacuo to give a crude mixture which is used in the next step without further purification.

Pyridine (91 mg, 1.2 mmol) was added to the crude mixture (56.3 mg, 0.111 mmol, theoretical) in Ac ₂ O (3 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/EtOAc=4:1) to give the product (35 mg, 0.059 mmol, overall yield=49%) as a white solid. ¹ H NMR δ (500 MHz, CDCl ₃ ): 7.02 (1H, s), 6.01 (1H, d, J=6.2 Hz), 4.59 (1H, d, J=6.2 Hz), 3.13-3.22(1H, m), 2.30(3H, s), 2.28(3H, s), 2.26(3H, s), 1.88-2.18 (6H, m), 1.50-1.74 (8H, m), 1.64 (3H, s), 1.43 (3H, s), 1.40 (3H, d, J=6.7 Hz), 1.29 (3H, d, J=6.7 Hz), 1.27 (3H, s), 1.06-1.11 (1H, m), 1.07 (3H, s), 1.04 (3H, s); LC-MS (Mobile phase: A: water (0.1% TFA) B: ACN (0.1% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml/ min

Column: Poroshell 120 EC-C18, 4.6×30 mm, 2.7 µm): vv=2.11 min, m/z=516.3 [MC ₃ H ₇ S] ⁺ , purity=100% (214.254 nm).

Example 45.

To a solution of SM (50 mg, 0.116 mmol) in THF (3 ml) was added 4-methylbenzenethiol (144 mg, 1.16 mmol). The reaction mixture was stirred at room temperature for 3 hours. The solution turns from dark red to pale red. The solution is then concentrated in vacuo to give a crude mixture which is used in the next step without further purification.

Pyridine (0.25 ml) was added to the crude mixture (64.5 mg, 0.116 mmol, theoretical) in Ac ₂ O (2 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/EtOAc=5:2) to give the product (62.4 mg, 0.0975 mmol, overall yield=84%) as a white solid. ¹ H NMR δ (400 MHz, CDCl3): 7.36 (1H, d, J=8.0 Hz), 7.12 (1H, d, J=8.0 Hz), 7.03 (1H, s ), 5.77 (1H, d, J=6.1 Hz), 4.79 (1H, d, J=6.1 Hz), 2.35 (3H, s), 2.32 (3H, s ), 2.28(3H, s), 1.84-2.15(6H, m), 1.35-1.68(8H, m), 1.51(3H, s), 1.42( 3H, s), 1.26(3H, s), 1.02-1.08(1H, m), 1.06(3H, s), 1.00(3H, s); LC-MS (Mobile phase: A: water (0.1% TFA) B: ACN (0.1% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml/ min Column: Poroshell 120 EC-C18, 4.6 x 30 mm, 2.7 µm): w=2.17 min, m/z=516.3 [MC ₇ H ₇ S] ⁺ , purity=98, 54% (214 nm), 96.35% (254 nm).

Example 46.

To a solution of SM (50 mg, 0.116 mmol) in THF (2 ml) was added 4-acetamidothiophenol (58 mg, 0.348 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from dark red to pale red. The solution is then concentrated in vacuo to give a crude mixture which is used in the next step without further purification.

Pyridine (0.5 ml) was added to the crude mixture (69.4 mg, 0.116 mmol, theoretical) in Ac ₂ O (2 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=1:1) to give the product (45.1 mg, 0.066 mmol, 57% overall yield) as a white solid.

¹ H NMR δ (400 MHz, CDCl ₃ ): 7.47 (1H, d, J=8.6 Hz), 7.40 (1H, d, J=8.6 Hz), 7.20 (1H, s), 7.03 (1H, s), 5.76 (1H, d, J=6.2 Hz), 4.80 (1H, d, J=6.2 Hz), 2.33 (3H, s), 2.31(3H, s), 2.28(3H, s), 2.19(3H, s), 1.82-2.14(7H, m), 1.30-1.66 (7H, m), 1.49 (3H, s), 1.42 (3H, s), 1.26 (3H, s), 1.02-1.09 (1H, m), 1.06 ( 3H, s), 1.00 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml/ Column: SunFire C18, 4.6×50 mm, 3.5 µm): vy=2.26 min, m/z=684.4 [M+H] ⁺ , purity=100% (214, 254 nm ).

Example 47.

To a solution of ERX1006 (90 mg, 0.2 mmol) in MeOH (3 ml) was added iC ₃ H ₇ SH (23 mg, 0.3 mmol). The reaction mixture was stirred at room temperature for 2 hours. The solution turns from red to pale yellow. The solution is then concentrated in vacuo to give a crude mixture.

Pyridine (0.5 ml) was added to the crude mixture obtained above in Ac ₂ O (4 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-HPLC (petroleum ether/acetone=1:1) to give the product (46.6 mg, 0.0764 mmol, overall yield=38%) as a white solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.00 (1H, s), 5.97 (1H, d, J=6.3 Hz), 5.63 (1H, br), 5.24 ( 1H, w), 4.57 (1H, d, J=6.3 Hz), 3.12-3.22 (1H, m), 2.40 (1H, d, J=15.3 Hz), 2.30(3H, s), 2.27(3H, s), 2.26(3H, s), 1.42-2.10(13H, m), 1.57(3H, s), 1 .40 (3H, d, J=6.7 Hz), 1.28 (3H, d, J=6.7 Hz), 1.26 (3H, s), 1.19 (3H, s), 1 .11 (3H, s), 0.95-1.02 (1H, m), 0.74 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.7 min; Flow rate: 2.2 ml/ Column: SunFire C18, 4.6×50 mm, 3.5 µm): vy=2.50 min, m/z=610.3 [M+H] ⁺ , purity=96.87% (214 nm ).

Example 48.

To a solution of Celastrol (200 mg, 0.44 mmol) in MeOH (6 ml) was added p-MeC ₆ H ₄ SH (82 mg, 0.66 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from red to pale yellow. The solution is then concentrated in vacuo to give a crude mixture

Pyridine (1 ml) was added to the crude mixture obtained above in Ac ₂ O (8 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=1:1) to give the product (30 mg, 0.0457 mmol, Yield=10%) as a white solid.

To a solution of compound 2 (100 mg, 0.15 mmol) in DMF (10 ml) was added NH ₄ Cl (18 mg, 0.34 mmol), GATU (65 mg, 0.17 mmol), then DIPEA (39 mg, 0.3 mmol). The reaction mixture was stirred at room temperature overnight. The solution is then diluted with EtOAc (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (EtOAc) to give the product (30 mg, 0.0456 mmol, Yield=30%) as a white solid. ¹ H NMR δ (500 MHz, CDCl ₃ ): 7.35 (1H, d, J=7.7 Hz), 7.12 (1H, d, J=7.7 Hz), 7.02 (1H, s), 5.73 (1H, d, J=6.1 Hz), 5.61 (1H, w), 5.15 (1H, w), 4.76 (1H, d, J=6.1 Hz), 2.38 (1H, d, J=15.2 Hz), 2.35 (3H, s), 2.32 (3H, s), 2.31 (3H, s), 2.28 ( 3H, s), 1.30-2.06(13H, m), 1.48(3H, s), 1.24(3H, s), 1.18(3H, s), 1.09(3H , s), 0.94-0.99 (1H, m), 0.70 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml/ Column: SunFire C18, 4.6 x 50 mm, 3.5 µm: y=2.47 min, purity=98.43% (214 nm), 100% (254 nm).

Example 49.

To a solution of ERX1006 (500 mg, 11.0 mmol) in MeOH (15 mL) was added 4-acetamidothiophenol (250 mg, 16.0 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from dark red to pale red. The solution is then concentrated in vacuo to give a crude mixture.

Pyridine (2.5 ml) was added to the crude mixture obtained above in Ac ₂ O (20 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (EtOAc) to give the product (250 mg, 0.357 mmol, Yield=51%) as a white solid. ¹ H NMR δ (500 MHz, CDCl ₃ ): 7.45 (1H, d, J=8.5 Hz), 7.39 (1H, d, J=8.5 Hz), 7.30 (1H, s), 7.01 (1H, s), 5.71 (1H, d, J=6.3 Hz), 5.63 (1H, w), 5.21 (1H, w), 4.76 ( 1H, d, J=6.3 Hz), 2.39 (1H, d, J=16.1 Hz), 2.32 (3H, s), 2.30 (3H, s), 2.28 ( 3H, s), 2.18(3H, s), 1.30-2.06(13H, m), 1.48(3H, s), 1.23(3H, s), 1.18(3H , s), 1.09 (3H, s), 0.92-0.99 (1H, m), 0.69 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml/ min, Column: SunFire C18, 4.6×50 mm, 3.5 µm): vy=2.13 min, m/z=701.3 [M+H] ⁺ , purity=99.36% (214 nm ), 97.26% (254 nm).

Example 50.

To a solution of SM (50 mg, 0.116 mmol) in THF (3 ml) was added 4-fluorobenzenethiol (74 mg, 0.579 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from dark red to pale red. The solution is then concentrated in vacuo to give a crude mixture which is used in the next step without further purification.

Pyridine (0.5 ml) was added to the crude mixture (74.7 mg, 0.116 mmol, theoretical) in Ac ₂ O (2 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=3:1) to give the product (18.2 mg, 0.0283 mmol, Yield=24%) as a white solid.

¹ H NMR δ (400 MHz, CDCl ₃ ): 7.38-7.43 (2H, m), 6.98-7.04 (3H, m), 5.72 (1H, d, J=6, 0 Hz), 4.81 (1H, d, J=6.0 Hz), 2.33 (3H, s), 2.31 (3H, s), 2.28 (3H, s), 1.84 -2.14(6H, m), 1.32-1.66(8H, m), 1.44(3H, s), 1.42(3H, s), 1.25(3H, s), 1.02-1.10(1H, m), 1.06(3H, s), 1.00(3H, s); LC-MS (Mobile phase: A: water (0.1% TFA) B: ACN (0.1% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml/ min Column: Poroshell 120 EC-C18, 4.6 x 30 mm, 2.7 µm): w=2.10 min, m/z=516.3 [M-FC ₆ H ₄ S] ⁺ , purity= 97.87% (214 nm), 99.10% (254 nm).

Example 51.

To a solution of Celastrol (50 mg, 0.116 mmol) in MeOH (5 ml) was added 4-hydroxybenzenethiol (44 mg, 0.348 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from red to almost colorless. The solution is then concentrated in vacuo to give a crude mixture which is used in the next step without further purification.

Pyridine (0.3 ml) was added to the crude mixture (61.9 mg, 0.116 mmol, theoretical) in Ac ₂ O (3 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/EtOAc=3:1) to give the product (20 mg, 0.0292 mmol, Yield=18%) as a white solid. ¹ H NMR δ (400 MHz, CDCl3): 7.43 (2H, d, J=8.4 Hz), 7.04 (2H, d, J=8.4 Hz), 7.03 (1H, s ), 5.79 (1H, d, J=6.3 Hz), 4.85 (1H, d, J=6.3 Hz), 2.32 (3H, s), 2.30 (6H, s ), 2.28 (3H, s), 1.82-2.14 (6H, m), 1.32-1.66 (9H, m), 1.47 (3H, s), 1.42 ( 3H, s), 1.25(3H, s), 1.02-1.10(1H, m), 1.06(3H, s), 1.00(3H, s); LC-MS (Mobile phase: A: water (0.1% TFA) B: ACN (0.1% TFA); Gradient: 5%-95% B over 1.2 min; Flow rate: 2.2 ml/ min Column: Poroshell 120 EC-C18, 4.6x30 mm, 2.7 µm): vv=2.01 min, m/z=516.3 [M-MeCO ₂ C ₆ H ₄ S] ⁺ , purity=100% (214 nm), 99.7% (254 nm).

Example 52.

To a solution of Celastrol (200 mg, 0.44 mmol) in MeOH (6 ml) was added HSCH ₂ CH ₂ OH (53 mg, 0.67 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from red to pale yellow. The was solution was then concentrated in vacuo to give a crude mixture.

Pyridine (1 ml) was added to the crude mixture obtained above in Ac ₂ O (8 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (EtOAc) to give the product (20 mg, 0.0305 mmol, Yield=7%) as a white solid. ¹ H NMR δ (500 MHz, CDCl3): 7.02 (1H, s), 5.98 (1H, d, J=6.0 Hz), 4.63 (1H, d, J=6.0 Hz ), 4.20-4.34(2H, m), 2.96-3.01(1H, m), 2.74-2.83(1H, m), 2.38(1H, d, J =15.5 Hz), 2.30(3H, s), 2.27(3H, s), 2.07(3H, s), 1.30-2.14(11H, m), 1.58 (3H, s), 1.25 (3H, s), 1.12 (3H, s), 1.07 (3H, s), 0.82-0.94 (3H, m), 0.65 ( 3H, s); LCMS: vy=2.33 min, m/z=535.3 [M-MeCO ₂ CH ₂ CH ₂ S] ⁺ , purity=95.75% (214 nm).

Example 53.

To a solution of Celastrol (300 mg, 0.666 mmol) in DMF (3 mL) was added K ₂ CO ₃ (184 mg, 1.332 mmol) followed by CH ₃ I (141 mg, 0.061 mL, 0.732 mmol). The reaction mixture was stirred at room temperature for 2 hours. A large amount of solid appears. The mixture was diluted with H ₂ O (30 ml), filtered. The solid is dissolved with CH ₂ Cl ₂ (300 ml), washed with H ₂ O (2×100 ml) then brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=3:1) to give the product (87.6 mg, 0.189 mmol, Yield=28%) as a reddish yellow solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.02 (1H, dd, J=7.0, 1.2 Hz), 6.96 (1H, s), 6.53 (1H, d, J =1.2 Hz), 6.35 (1H, d, J=7.0 Hz), 3.55 (3H, s), 2.42 (1H, d, J=15.6 Hz), 2, 18 (3H, s), 2.00-2.20 (3H, m), 1.30-1.93 (10H, m), 1.45 (3H, s), 1.26 (3H, s) , 1.18(3H, s), 1.10(3H, s), 0.94-1.01(1H, m), 0.53(3H, s); LC-MS (Mobile phase: A: water (10 mM ammonium bicarbonate) B: ACN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4 .6×50 mm, 3.5 µm): vy=2.77 min, m/z=465.4 [M+H] ⁺ , purity=97.25% (214 nm), 99.77% (254 nm).

Example 54.

MeCOSH (51 mg, 0.67 mmol) is added to a solution of Celastrol (200 mg, 0.44 mmol) in MeOH (20 ml). The reaction mixture was stirred at room temperature for 0.5 hour. The solution turns from red to pale yellow. The solution is then concentrated in vacuo to give a crude mixture.

Pyridine (1 ml) was added to the crude mixture obtained above in Ac ₂ O (8 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (EtOAc) to give the product (131 mg, 0.214 mmol, Yield=49%) as a white solid. ¹ H NMR δ (500 MHz, CDCl3): 7.02 (1H, s), 5.94&5.95 (1H, d, J=6.4 Hz), 5.36&5.37 (1H, d, J= 6.4Hz), 2.39(1H, d, J=15.4Hz), 2.34(3H, s), 2.303&2.307(3H, s), 2.269&2.273(3H, s) , 2.074&2.077(3H, s), 1.30-2.20(13H, m), 1.438&1.451(3H, s), 1.215&1.253(3H, s), 1.169&1.201(3H , s), 1.062 & 1.083 (3H, s), 0.90-1.00 (1H, m), 0.666 & 0.702 (1H, s); LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA)

Gradient: 5%-95% B in 1.7 min; Flow rate: 2.2 ml/min; Column: SunFire C18, 4.6×50 mm, 3.5 µm): w=2.49 & 2.52 min, two peaks.

Example 55.

To a solution of ERX1006 (200 mg, 0.445 mmol) in MeOH (10 ml) was added NaBH ₄ (168 mg, 4.445 mmol) in portions. The solution turns from reddish to colorless. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture is then quenched with 0.1 M HCl and acidified to pH 5-6 with 0.1 M HCl. The mixture was diluted with CH ₂ Cl ₂ (300 ml), filtered, separated. The organic layer was washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo to give a crude intermediate which was used in the next step without further purification.

To the crude mixture (201 mg, 0.445 mmol, theoretical) in CH ₂ Cl ₂ (10 ml) was added NEt ₃ (180 mg, 0.25 ml, 1.78 mmol) followed by EtCO ₂ Cl (193 mg, 0.17 mmol, 1.78 mmol). The reaction mixture was stirred at room temperature for 1 hour. The mixture was then diluted with EtOAc (200 ml), washed with water (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/EtOAc=2:3) to give the product (89.3 mg, 0.150 mmol, Yield=34%) as a white solid. ¹ H NMR δ (400 MHz, CDCl3): 7.09 (1H, s), 5.75 (1H, d, J=6.1 Hz), 5.65 (1H, br), 5.21 (1H , w), 4.31 (2H, q, J=7.1 Hz), 4.31 (2H, q, J=7.1 Hz), 3.33 (1H, dd, J=20.9, 6.1 Hz), 3.06 (1H, d, J=20.9 Hz), 2.41 (1H, d, J=15.6 Hz), 2.12 (3H, s), 1.40 -2.12 (13H, m), 1.38 (3H, t, J=7.1 Hz), 1.38 (3H, t, J=7.1 Hz), 1.33 (3H, s) , 1.22(3H, s), 1.19(3H, s), 1.10(3H, s), 0.94-1.10(1H, m), 0.77(3H, s); LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml/ Column: SunFire C18, 4.6×50 mm, 3.5 µm): vy=2.39 min, m/z=596.3 [M+H] ⁺ , purity=97.29% (214 nm ).

Example 56.

To a solution of ERX1006 (200 mg, 0.445 mmol) in MeOH (10 ml) was added i-PrSH (101.7 mg, 0.124 ml, 1.335 mmol). The solution turns from reddish to pale yellow. The reaction mixture was stirred at room temperature for 0.5 hour. The solution was diluted with CH ₂ Cl ₂ (200 ml), washed with H ₂ O (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo to give the crude intermediate, which was used in the next step without further purification .

To the crude mixture (234 mg, 0.445 mmol, theoretical) in CH ₂ Cl ₂ (10 mL) was added NEt ₃ (225 mg, 0.31 mL, 2.225 mmol) followed by EtCO ₂ Cl (241 mg, 0.21 mmol, 2.225 mmol). The reaction mixture was stirred at room temperature for 1 hour. The mixture was then diluted with EtOAc (200 ml), washed with water (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/EtOAc=2:3) to give the product (145.6 mg, 0.217 mmol, Yield=49%) as a white solid. ¹ H NMR δ (400 MHz, CDCl3): 7.11 (1H, s), 5.97 (1H, d, J=6.3 Hz), 5.64 (1H, br), 5.18 (1H , w), 4.58 (1H, d, J=6.3 Hz), 3.32 (2H, q, J=7.2 Hz), 3.31 (2H, q, J=7.2 Hz ), 3.14-3.23 (1H, m), 2.40 (1H, d, J=15.0 Hz), 2.34 (3H, s), 1.42-2.12 (13H, m), 1.56(3H, s), 1.38(6H, t, J=7.2Hz), 1.37(3H, d, J=6.9Hz), 1.28(3H, d, J=6.9 Hz), 1.26 (3H, s), 1.19 (3H, s), 1.11 (3H, s), 0.96-1.03 (1H, m), 0.74 (3H, s); LC-MS (Mobile phase: A: water (10 mM ammonium bicarbonate) B: ACN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4 .6×50 mm, 3.5 µm): vy=2.69 min, m/z=594.2 [MC ₃ H ₇ S] ⁺ , purity=100% (214.254 nm).

Example 57.

To a solution of ERX1036 (329 mg, 0.631 mmol) in MeOH (6 ml) was added i-PrSH (144 mg, 0.176 ml, 1.892 mmol). The reaction mixture was stirred at room temperature for 1 hour. The mixture was then diluted with EtOAc (300 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC twice (petroleum ether/EtOAc=1:3) to give a 3:1 unpartitioned mixture of A and B (181.8 mg, 0.304 mmol, Yield=48%) as a yellow solid.

¹ H NMR: C3-OCO ₂ Et isomer:

δ (400 MHz, CDCl3): 6.87 (1H, s), 5.97 (1H, d, J=6.2 Hz), 5.70 (1H, br), 5.70 (1H, s) , 5.26 (1H, w), 4.56 (1H, d, J=6.2 Hz), 4.32 (2H, q, J=7.2 Hz), 3.11-3.22 ( 1H, m), 2.43 (1H, d, J=15.1 Hz), 2.30 (3H, s), 1.42-2.10 (13H, m), 1.57 (3H, s ), 1.39 (3H, t, J=7.2 Hz), 1.39 (3H, d, J=7.0 Hz), 1.27 (3H, d, J=7.0 Hz), 1.26 (3H, s), 1.19 (3H, s), 1.11 (3H, s), 0.95-1.03 (1H, m), 0.74 (3H, s).

C2-OCO ₂ Et isomer:

δ (400 MHz, CDCl3): 7.04 (1H, s), 5.97 (1H, d, J=6.2 Hz), 5.70 (1H, br), 5.38 (1H, s) , 5.26 (1H, w), 4.60 (1H, d, J=6.2 Hz), 4.34 (2H, q, J=7.2 Hz), 3.11-3.22 ( 1H, m), 2.43 (1H, d, J=15.1 Hz), 2.41 (3H, s), 1.42-2.10 (13H, m), 1.57 (3H, s ), 1.39 (3H, t, J=7.2 Hz), 1.39 (3H, d, J=7.0 Hz), 1.27 (3H, d, J=7.0 Hz), 1.26(3H, s), 1.19(3H, s), 1.11(3H, s), 0.95-1.03(1H, m), 0.73(3H, s).

LC-MS (Mobile phase: A: water (10 mM ammonium bicarbonate) B: ACN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4 .6×50 mm, 3.5 μm): vy=2.58 min, m/z=522.3 [MC ₃ H ₇ S] ⁺ , purity=97.47% (214 nm).

Example 58.

To a solution of ERX1090 (277 mg, 0.517 mmol) in MeOH (6 mL) was added i-PrSH (118 mg, 0.144 mL, 1.551 mmol). The reaction mixture was stirred at room temperature for 1 hour. The mixture was then diluted with EtOAc (300 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC twice (petroleum ether/EtOAc=1:3) to give an intact 3:1 mixture of A and B (180.4 mg, 0.295 mmol, Yield=57%) as a yellow solid.

¹ H NMR: C3-OCO ₂ Pr-i isomer:

δ (400 MHz, CDCl3): 6.87 (1H, s), 5.97 (1H, d, J=6.4 Hz), 5.73 (1H, s), 5.69 (1H, br) , 5.29 (1H, w), 4.94-5.02 (1H, m), 4.56 (1H, d, J=6.4 Hz), 3.10-3.22 (1H, m ), 2.42 (1H, d, J=15.6 Hz), 2.30 (3H, s), 1.42-2.10 (13H, m), 1.56 (3H, s), 1 .39 (3H, d, J=6.8 Hz), 1.37 (6H, d, J=6.8 Hz), 1.27 (3H, d, J=6.8 Hz), 1.25 (3H, s), 1.19 (3H, s), 1.11 (3H, s), 0.94-1.02 (1H, m), 0.74 (3H, s).

C2-OCO ₂ Pr-i isomer:

δ (400 MHz, CDCl3): 7.04 (1H, s), 5.97 (1H, d, J=6.4 Hz), 5.69 (1H, br), 5.42 (1H, s) , 5.29 (1H, w), 4.94-5.02 (1H, m), 4.60 (1H, d, J=6.4 Hz), 3.10-3.22 (1H, m ), 2.42 (1H, d, J=15.6 Hz), 2.37 (3H, s), 1.42-2.10 (13H, m), 1.56 (3H, s), 1 .39 (3H, d, J=6.8 Hz), 1.37 (6H, d, J=6.8 Hz), 1.27 (3H, d, J=6.8 Hz), 1.25 (3H, s), 1.19 (3H, s), 1.11 (3H, s), 0.94-1.02 (1H, m), 0.74 (3H, s).

LC-MS (phase: A: water (10 mM ammonium bicarbonate) B: ACN; Gradient: 5%-95% B over 1.5 min; Flow rate: 2.0 ml/min; Column: XBridge C18, 4, 6×50 mm, 3.5 µm, y=2.62 min, m/z=536.4 [M-C3H7S] ⁺ , purity=95.39% (214 nm).

Example 59.

To a solution of ERX1006 (300 mg, 0.667 mmol) in CH ₂ Cl ₂ (6 mL) was added Et ₃ N (135 mg, 0.19 mL, 1.334 mmol) followed by 1M ClCO ₂ Pr-i in PhMe solution (163 mg, 1.33 ml, 1.334 mmol) dropwise at 0°C. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with CH ₂ Cl ₂ (300 ml), washed with brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:3) to give the product (302.6 mg, 0.565 mmol, Yield=85%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl3): 7.06 (1H, dd, J=7.4, 1.3 Hz), 6.47 (1H, d, J=1.3 Hz), 6.32 (1H, d, J=7.4 Hz), 5.67 (1H, w), 5.23 (1H, w), 4.93-5.01 (1H, m), 2.39 (1H, d, J=15.4 Hz), 2.21(3H, s), 1.82-2.14(7H, m), 1.48-1.73(6H, m), 1.46(3H , s), 1.39 (3H, d, J=6.0 Hz), 1.39 (3H, d, J=6.4 Hz), 1.27 (3H, s), 1.21 (3H , s), 0.99-1.05 (1H, m), 0.76 (3H, s); LC-MS (Mobile phase: A: water (0.01% TFA) B: ACN (0.01% TFA); Gradient: 5%-95% B over 1.4 min; Flow rate: 2.3 ml/ min

Column: Hypersil GOLD, 4.6×50 mm, 3 μm): vy=2.11 min, m/z=536.3 [M+H] ⁺ , purity=97.63% (214 nm), 100% (254 nm).

Example 60.

To a solution of ERX1074 (2.05 g, 4.412 mmol) in Me ₂ CO (30 ml) was added K ₂ CO ₃ (3.05 g, 22.06 mmol) followed by PhCH ₂ Br (3.77 g, 2.62 ml, 22.06 mmol). The reaction mixture is heated at 50°C overnight. Most of the acetone is removed in vacuo. The residue was dissolved in EtOAc (300 ml), washed with H ₂ O (200 ml), brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=3:1) to give product 2 (2.45 g, 4.416 mmol, Vd=100%) as a yellow solid.

To a solution of compound 2 (2.5 g, 4.51 mmol) in anhydrous THF (100 ml) was added 3 M MeMgBr in THF solution (7.5 ml, 22.5 mmol) at 0° C. dropwise. The reaction mixture was stirred at 0°C for 1 hour. The reaction is quenched by adding H ₂ O (50 ml) and extracted with CH ₂ Cl ₂ (3×50 ml). The combined organic extracts were washed with brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=2:1) to give the crude product (2.0 g, 3.53 mmol, Vd=78%) as a white solid.

The solution is hydrogenated with a balloon of hydrogen. The solution is filtered with celite. The filtered was concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=2:1) to give the product ERX1095 (1.5 g, 3.26 mmol, Vd=89%) as a white solid.

Ag ₂ CO ₃ (148 mg, 0.54 mmol) was added to a solution of ERX1095 (130 mg, 0.27 mmol) in benzene (5 ml). The reaction mixture is heated at rt overnight. The solution is filtered and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=2:1) to give the final product (80 mg, 0.167 mmol, Vd=62%) as a red solid.

ERX1095 data:

¹ H NMR δ (500 MHz, CDCl ₃ ): 6.78 (1H, s), 5.77 (1H, d, J=6.2 Hz), 5.15 (1H, s), 5.07 ( 1H, s), 3.52 (3H, s), 3.44-3.50 (1H, m), 2.42 (1H, d, J=15.7 Hz), 2.22 (3H, s ), 2.14-2.20(1H, m), 2.01-2.11(2H, m), 1.80-1.89(2H, m), 1.30-1.70(8H , m), 1.47 (3H, s), 1.21 (3H, s), 1.16 (3H, s), 1.16 (3H, d, J=6.8 Hz), 1.08 (3H, s), 0.91-0.97 (1H, m), 0.55 (3H, s); LC-MS: vy=2.19 min, m/z=481 [M+H] ⁺ , purity=97.35% (214 nm).

ERX1096 data:

¹ H NMR δ (500 MHz, CDCl ₃ ): 6.28 (1H, s), 5.70 (1H, d, J=6.0 Hz), 3.58 (3H, s), 3.39- 3.46(1H, m), 2.39(1H, d, J=15.7Hz), 2.15-2.24(1H, m), 2.02-2.10(1H, m) , 2.00(3H, s), 1.78-1.92(3H, m), 1.59(3H, s), 1.24-1.74(8H, m), 1.28(3H , d, J=7.3 Hz), 1.18 (3H, s), 1.17 (3H, s), 1.08 (3H, s), 0.93-0.98 (1H, m) , 0.60 (3H, s); ¹³ C NMR δ (125 MHz, CDCl ₃ ): 181.32, 180.57, 178.83, 167.19, 149.49, 146.44, 132.47, 122.64, 122.32, 51, 58, 44.25, 43.88, 40.39, 38.68, 37.18, 36.59, 36.00, 34.77, 34.71, 32.75, 32.54, 31.57, 30.61, 30.48, 29.90, 28.77, 22.77, 20.50, 18.13, 11.35.

LC-MS: vy=2.21 min, m/z=479 [M+H] ⁺ , purity=100% (214 nm).

Example 61.

To a solution of ERX1018 (503 mg, 1.0 mmol) in EtOH (50 ml) was added K ₂ CO ₃ (276 mg, 2.0 mmol), NaI (6 mg, 0.04 mmol), then PhCH ₂ Br (187 mg, 1.1 mmol). The reaction mixture is heated at 80°C overnight. Most of the EtOH is removed in vacuo. The residue was dissolved in CH ₂ Cl ₂ (200 ml), washed with H ₂ O (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=1:1) to give product 2 (255 mg, 0.43 mmol, Vd=43%) as a yellow solid.

To a solution of compound 2 (100 mg, 0.17 mmol) in anhydrous THF (10 ml) was added 3 M MeMgBr in THF solution (0.57 ml, 1.7 mmol) at 0° C. dropwise. The reaction mixture was stirred at 0°C for 1 hour. The reaction is quenched by adding H ₂ O (50 ml) and extracted with CH ₂ Cl ₂ (3×50 ml). The combined organic extracts were washed with brine (50 ml), dried over MgSO ₄ and concentrated in vacuo to give the crude product (50 mg, 0.0824 mmol, Bd=48%) as a white solid.

To a solution of crude compound 3 (50 mg, 0.0824 mmol) in MeOH (10 ml) was added 10% Pd/C (5 mg). The solution is hydrogenated with a balloon of hydrogen. The solution is filtered with celite. The filter is concentrated in vacuo and the residue is purified by silica gel chromatography (petroleum ether/ethyl acetate=1:1) to give product 4 (20 mg, 0.0385 mmol, Vd=47%) as a white solid.

Ag ₂ CO ₃ (43 mg, 0.154 mmol) was added to a solution of compound 4 (40 mg, 0.0771 mmol) in benzene (10 ml). The reaction mixture is heated at 60°C for 1 hour. The reaction is quenched with H ₂ O (50 ml) and extracted with CH ₂ Cl ₂ (3×50 ml). The combined organic extracts were washed with brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ : MeOH=10:1) to give the final product (33 mg, 0.0637 mmol, Vd=83%) as a red solid. ¹ H NMR δ (500 MHz, CDCl ₃ ): 6.27 (1H, s), 5.70 (1H, d, J=6.4 Hz), 3.58-3.68 (2H, m), 3.40-3.51(2H, m), 3.25-3.33(1H, m), 2.34-2.41(2H, m), 2.09-2.18(1H, m ), 2.00 (3H, s), 1.42-1.96 (15H, m), 1.59 (3H, s), 1.26 (3H, d, J=7.1 Hz), 1 .22 (3H, s), 1.19 (3H, s), 1.11 (3H, s), 0.93-0.99 (1H, m), 0.62 (3H, s); LC-MS: vy=2.57 min, m/z=518 [M+H] ⁺ , purity=97.84% (214 nm).

Example 62.

To a solution of ERX1001 (478 mg, 1.0 mmol) in DMF (10 ml) was added K ₂ CO ₃ (276 g, 2.0 mmol) followed by PhCH ₂ Br (187 mg, 1.1 mmol). The reaction mixture is heated at 80°C for 2 hours. The reaction mixture was quenched with ice-H ₂ O (50 ml) and filtered. The solid is dissolved in CH ₂ Cl ₂ (200 ml), washed with H ₂ O (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=3:1) to give product 2 (300 mg, 0.528 mmol, Vd=53%) as a yellow solid.

To a solution of 2 (568 g, 1.0 mmol) in anhydrous THF (20 mL) was added 3 M MeMgBr in THF solution (2.33 mL, 7.0 mmol) at 0° C. dropwise. The reaction mixture was stirred at 0°C for 1 hour. The reaction is quenched by adding H ₂ O (50 ml) and extracted with CH ₂ Cl ₂ (3×50 ml). The combined organic extracts were washed with brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=2:1) to give the crude product (400 mg, 0.685 mmol, Vd=69%) as a white solid.

To a solution of crude product 3 (584 mg, 1.0 mmol) in MeOH (10 ml) was added 10% Pd/C (58 mg). The solution is hydrogenated with a balloon of hydrogen. The solution is filtered with celite. The filtered concentrate in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=3:1) to give the product ERX1100 (450 mg, 0.911 mmol, Vd=91%) as a white solid.

Ag ₂ CO ₃ (548 mg, 2.0 mmol) was added to a solution of ERX1100 (494 mg, 1.0 mmol) in benzene (20 ml). The reaction mixture is heated at 60°C overnight. The solution is filtered and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=3:1) to give the final product (200 mg, 0.813 mmol, Vd=81%) as a red solid.

ERX1100 data:

¹ H NMR: δ (500 MHz, d6-DMSO): 8.85 (1H, s), 7.86 (1H, s), 6.60 (1H, s), 5.73 (1H, d, J =6.2 Hz), 3.82-3.94 (2H, m), 3.32-3.40 (1H, m), 2.34 (1H, d, J=15.2 Hz), 2 .06 (3H, s), 1.91-2.09 (3H, m), 1.20-1.85 (10H, m), 1.39 (3H, s), 1.17 (3H, s ), 1.11 (3H, t, J=7.2 Hz), 1.11 (3H, s), 1.06 (3H, d, J=6.7 Hz), 1.06 (3H, s ), 0.85-0.91 (1H, m), 0.49 (3H, s); LCMS: wy=2.85 min, m/z=495 [M+H] ⁺ , purity=100% (214 nm).

ERX1099 data:

¹ H NMR δ (500 MHz, CDCl ₃ ): 6.28 (1H, s), 5.70 (1H, d, J=5.6 Hz), 3.93-4.08 (2H, m), 3.39-3.46 (1H, m), 2.41 (1H, d, J=15.6 Hz), 2.18-2.23 (1H, m), 2.01-2.09 ( 1H, m), 2.00(3H, s), 1.15-1.92(11H, m), 1.59(3H, s), 1.27(3H, d, J=7.2 Hz ), 1.22 (3H, t, J=7.3 Hz), 1.18 (3H, s), 1.17 (3H, s), 1.08 (3H, s), 0.92-0 .98 (1H, m), 0.63 (3H, s); LC-MS: y=2.81 min, m/z=493 [M+H] ⁺ , purity=100% (214.254 nm).

Example 63.

To a solution of Celastrol (450 mg, 1.0 mmol) in DMF (10 ml) was added K ₂ CO ₃ (276 mg, 2.0 mmol) followed by PhCH ₂ Br (374 mg, 2.2 mmol). The reaction mixture is heated at 80°C for 2 hours. The reaction mixture was quenched with ice-water (50 ml). The mixture is filtered. The solid is dissolved in CH ₂ Cl ₂ (200 ml), washed with H ₂ O (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=3:1) to give product 2 (400 mg, 0.635 mmol, Vd=64%) as a yellow solid.

To a solution of compound 2 (630 mg, 1.0 mmol) in anhydrous THF (20 ml) was added 3 M MeMgBr in THF solution (3.3 ml, 10 mmol) at 0° C. dropwise. The reaction mixture was stirred at 0°C for 1 hour. The reaction is quenched by adding H ₂ O (50 ml) and extracted with CH ₂ Cl ₂ (2×100 ml). The combined organic extracts are washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=2:1) to give the crude product (500 mg, 0.774 mmol, Vd=77%) as a white solid.

To a solution of compound 3 (64.6 mg, 0.1 mmol) in MeOH (10 ml) was added 10% Pd/C (6 mg). The solution is hydrogenated with a balloon of hydrogen. The solution is filtered with celite. The filtered concentrate in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=1:1) to give the product ERX1101 (30 mg, 0.0644 mmol, Vd=64%) as a white solid.

Ag ₂ CO ₃ (55 mg, 0.2 mmol) was added to a solution of ERX1101 (47 mg, 0.1 mmol) in benzene (10 ml). The reaction mixture is heated at 60°C overnight. The solution is filtered and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=1:1) to give the final product ERX1098 (40 mg, 0.0862 mmol, Vd=86%) as a red solid.

ERX1101 data:

¹ H NMR δ (500 MHz, d6-DMSO): 11.9-12.2 (1H, br), 8.80-9.10 (1H, br), 7.80-8.10 (1H, br ), 6.60 (1H, s), 5.72 (1H, d, J=5.6 Hz), 2.35 (1H, d, J=15.3 Hz), 2.05 (3H, s ), 1.94-2.05(3H, m), 1.20-1.85(1H, m), 1.38(3H, s), 1.17(3H, s), 1.08( 3H, s), 1.07(3H, s), 1.04(3H, s), 0.80-0.88(1H, m), 0.65(3H, s); LC-MS: vy=2.03 min, m/z=467.3 [M+H] ⁺ , purity=100% (214 nm).

ERX1098 data:

¹ H NMR δ (400 MHz, CDCl ₃ ): 6.27 (1H, s), 6.65 (1H, d, J=6.5 Hz), 3.36-3.45 (1H, m), 2.38 (1H, d, J=13.9 Hz), 2.07-2.16 (1H, m), 2.00 (3H, s), 1.93-2.03 (1H, m) , 1.32-1.90 (13H, m), 1.58 (3H, s), 1.29 (3H, d, J=7.3 Hz), 1.17 (3H, s), 1, 16 (3H, s), 1.05 (3H, s), 0.86-0.94 (1H, m), 0.69 (3H, s); LCMS: vy=2.04 min, m/z=467 [M+H] ⁺ , purity=100% (214 nm).

Example 64.

To a solution of ERX1006 (200 mg, 0.44 mmol) in MeOH (6 mL) was added MeCOSH (51 mg, 0.67 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from red to pale yellow. The solution is then concentrated in vacuo to give a crude mixture.

Pyridine (1 ml) was added to the crude mixture obtained above in Ac ₂ O (8 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with EtOAc (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (EtOAc) to give the product (100 mg, 0.164 mmol, Yield=37%) as a white solid. ¹ H NMR δ (500 MHz, CDCl3): 7.02 (1H, s), 5.94 (1H, d, J=6.4 Hz), 5.68 (1H, br), 5.45 (1H , w), 5.36 (1H, d, J=6.4 Hz), 2.43 (1H, d, J=15.9 Hz), 2.34 (3H, s), 2.31 (3H , s), 2.28 (3H, s), 2.07 (3H, s), 1.45-2.06 (13H, m), 1.43 (3H, s), 1.21 (3H, s), 1.19 (3H, s), 1.09 (3H, s), 0.96-1.02 (1H, m), 0.73 (3H, s); LCMS: wy=1.90 min, m/z=610 [M+H] ⁺ , purity=97.73% (214 nm).

Example 65.

To a solution of ERX1033 (200 mg, 0.58 mmol) in THF (5.0 ml) was added LiAlH ₄ (28 mg, 0.76 mmol). The reaction mixture is stirred at 0°C for 0.5 hour. The reaction mixture was quenched with sat. NH ₄ Cl solution. The solution is heated at 50° C. in air for 2 hours. The mixture was diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=10:1) to give the product (6 mg, 0.0122 mmol, Yield=3%) as a red solid. ¹ H NMR δ (400 MHz, CDCl3): 7.06 (1H, dd, J=7.1, 0.9 Hz), 6.47 (1H, d, J=0.9 Hz), 6.34 (1H, d, J=7.1 Hz), 5.68 (1H, w), 5.24 (1H, w), 5.09 (1H, t, J=5.2 Hz), 3.98 -4.10 (2H, m), 3.78-3.86 (2H, m), 2.39 (1H, d, J=15.8 Hz), 2.26 (3H, s), 1, 47-2.20(13H, m), 1.45(3H, s), 1.27(3H, s), 1.21(3H, s), 1.12(3H, s), 0.99 -1.06 (1H, m), 0.76 (3H, s); LC-MS: y=2.05 min, m/z=494.3 [M+H] ⁺ , purity=93.7% (214nm), 100% (254 nm).

Example 66.

To a solution of ERX1006 (200 mg, 0.444 mmol) in MeOH (20 ml) was added N-acetyl-L-cysteine ethyl ester (157 mg, 0.888 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from red to pale yellow. The was solution was then concentrated in vacuo to give a crude mixture.

Ac ₂ O (1 ml) was added to the crude mixture obtained above in pyridine (2 ml). The reaction mixture was stirred at room temperature overnight. The mixture is then poured into H ₂ O (100 ml), filtered. The solid is dissolved in EtOAc (200 ml), washed with water (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (EtOAc) to give the product (184.2 mg, 0.259 mmol, 58% overall) as a white solid. ¹ H NMR δ (400 MHz, CDCl3): 7.01 (1H, s), 6.26 (1H, d, J=7.5 Hz), 5.94 (1H, d, J=6.3 Hz ), 5.65 (1H, w), 5.19 (1H, w), 4.88 (1H, q, J=6.0 Hz), 4.63 (1H, d, J=6.3 Hz ), 3.74 (3H, s), 3.31 (1H, dd, J=13.5, 4.8 Hz), 2.93 (1H, dd, J=13.5, 5.9 Hz) , 2.42 (1H, d, J=15.2 Hz), 2.31 (3H, s), 2.27 (3H, s), 2.25 (3H, s), 2.06 (3H, s), 1.45-2.10(13H, m), 1.56(3H, s), 1.26(3H, s), 1.19(3H, s), 1.11(3H, s ), 0.97-1.04 (1H, m), 0.73 (3H, s); LCMS: wy=2.00 min, m/z=534.4 [M-SCH ₂ CH(CO ₂ Me)NHCOMe] ⁺ , purity=100% (214, 254 nm).

Example 67.

To a solution of ERX1033 (52 mg, 0.1 mmol) in MeOH (5.0 ml) was added iC ₃ H ₇ SH (11.4 mg, 0.15 mmol). The reaction mixture was stirred at rt. within 2.0 hours. The solution is concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=10:1) to give the product (16 mg, 0.0268 mmol, Yield=27%) as a yellow solid. ¹ H NMR δ (500 MHz, d4-MeOD): 6.73 (1H, s), 6.06 (1H, d, J=6.2 Hz), 4.61 (1H, d, J=6, 2 Hz), 3.80 (3H, s), 3.14-3.22 (1H, m), 2.47 (1H, d, J=15.7 Hz), 2.39 (3H, s) , 2.04-2.15(3H, m), 1.79-1.95(3H, m), 1.45-1.71(8H, m), 1.54(3H, s), 1 .40 (3H, d, J=6.6 Hz), 1.27 (3H, s), 1.25 (3H, d, J=6.4 Hz), 1.16 (3H, s), 1 .12 (3H, s), 0.92-0.98 (1H, m), 0.77 (3H, s); LC-MS: vy=2.02 min, m/z=522.2 [M-C3H7S] ⁺ , purity=94.01% (214nm), 86.66% (254nm).

Example 68.

To a solution of ERX1006 (45 mg, 0.1 mmol) in DMF (5 ml) was added K ₂ CO ₃ (28 mg, 0.2 mmol) followed by EtI (156 mg, 1.0 mmol). The reaction mixture is stirred at 40°C for 4 hours. The reaction mixture was quenched with ice-water (50 ml) and filtered. The solid is dissolved with CH ₂ Cl ₂ (200 ml), washed with saturated salt solution (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (ethyl acetate) to give the product (25 mg, 0.0523 mmol, Yield=52%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl3): 6.94 (1H, dd, J=7.1, 1.2 Hz), 6.39 (1H, d, J=1.2 Hz), 6.28 (1H, d, J=7.1 Hz), 5.66 (1H, w), 5.20 (1H, w), 4.10 (2H, q, J=7.1 Hz), 2.39 (1H, d, J=16.1 Hz), 2.22 (3H, s), 1.45-2.15 (13H, m), 1.44 (3H, s), 1.34 (3H, t, J=7.0 Hz), 1.26 (3H, s), 1.20 (3H, s), 1.12 (3H, s), 0.99-1.05 (1H, m), 0.76 (3H, s); LC-MS: vy=1.83 min, m/z=478.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 69.

To a solution of ERX1006 (45 mg, 0.1 mmol) in DMF (5 ml) was added K ₂ CO ₃ (28 mg, 0.2 mmol) followed by iC ₃ H ₇ I (170 mg, 1.0 mmol). The reaction mixture is stirred at 40°C for 4 hours. The reaction mixture was quenched with ice-water (50 ml) and filtered. The solid is dissolved with CH ₂ Cl ₂ (200 ml), washed with saturated salt solution (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (ethyl acetate) to give the product (20 mg, 0.0407 mmol, Yield=41%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl3): 6.92 (1H, dd, J=7.1, 1.2 Hz), 6.36 (1H, d, J=1.3 Hz), 6.27 (1H, d, J=7.1 Hz), 5.68 (1H, w), 5.25 (1H, w), 4.69-4.75 (1H, m), 2.39 (1H, d, J=15.9 Hz), 2.20(3H, s), 1.47-2.13(13H, m), 1.44(3H, s), 1.28(3H, d, J =6.0 Hz), 1.26 (3H, d, J=6.0 Hz), 1.26 (3H, s), 1.20 (3H, s), 1.12 (3H, s), 0.99-1.04 (1H, m), 0.77 (3H, s); LC-MS: wy=1.90 min, m/z=492.4 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 70.

To a solution of ERX1015 (80 mg, 0.174 mmol) in EtI (1 ml) and DMF (2 ml) was added K ₂ CO ₃ (72 mg, 0.522 mmol). The reaction mixture is stirred at 50°C overnight. The solution was diluted with CH ₂ Cl ₂ (300 ml), washed with sat. LiCl.H ₂ O (2×100 ml), H ₂ O (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=2:1) to give the product (33.5 mg, 0.0729 mmol, Yield=42%) as a yellow solid. ¹ H NMR: δ (400 MHz, CDCl3): 6.96 (1H, dd, J=7.1, 1.1 Hz), 6.41 (1H, d, J=1.2 Hz), 6, 31 (1H, d, J=7.2 Hz), 4.06-4.16 (2H, m), 2.22 (3H, s), 1.54-2.18 (14, m), 1 .47 (3H, s), 1.44 (3H, s), 1.35 (3H, t, J=7.1 Hz), 1.29 (3H, s), 1.08-1.15 ( 1H, m), 1.09 (3H, s), 1.05 (3H, s); LC-MS: y=1.98 min, m/z=460.2 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 71.

To a solution of ERX1060 (30 mg, 0.0647 mmol) in MeOH (1 ml) was added iC ₃ H ₇ SH (7.6 mg, 0.1 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from reddish to a pale red-yellow. The solution is then concentrated in vacuo to give a crude mixture which is used in the next step without further purification.

Pyridine (0.5 ml) was added to the crude mixture obtained above in Ac ₂ O (4 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with CH ₂ Cl ₂ (100 ml), washed with water (2×50 ml), brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (CH ₂ Cl ₂ /MeOH=10:1) to give the product (5 mg, 0.00801 mmol, overall yield=12%) as a pale yellow solid. ¹ H NMR: δ (500 MHz, CDCl3): 7.02 (1H, s), 6.01 (1H, d, J=6.0 Hz), 4.59 (1H, d, J=6.1 Hz), 3.16-3.23(1H, m), 2.20-2.50(6H, w), 2.31(3H, s), 2.29(3H, s), 2.27 (3H, s), 1.20-2.05(16H, m), 1.57(3H, s), 1.42(3H, s), 1.41(3H, d, J=7.0 Hz), 1.31 (3H, d, J=7.0 Hz), 1.22 (3H, s), 1.10 (3H, w), 0.95-1.01 (1H, m), 0.77 (3H, s); LC-MS: y=1.76 min, m/z=624.3 [M+H] ⁺ , purity=95.93% (214 nm), 95.26% (254 nm).

Example 72.

To a solution of ERX1060 (70 mg, 0.15 mmol) in MeOH (1 ml) was added NaBH ₄ (5.7 mg, 0.15 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from reddish to pale yellow. The solution is then concentrated in vacuo to give a crude mixture which is used in the next step without further purification.

Pyridine (0.5 ml) was added to the crude mixture obtained above in Ac ₂ O (0.5 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with CH ₂ Cl ₂ (100 ml), washed with water (2×50 ml), brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (CH ₂ Cl ₂ /MeOH=10:1) to give the product (10 mg, 0.0182 mmol, Yield=12%) as a pale yellow solid. ¹ H NMR: δ (500 MHz, CDCl3): 7.00 (1H, s), 5.80 (1H, dd, J=6.4, 1.9 Hz), 3.36 (1H, dd, J =20.5, 6.4 Hz), 3.09 (1H, d, J=20.5 Hz), 2.30-2.50 (6H, w), 2.32 (3H, s), 2 .20 (3H, s), 2.08 (3H, s), 1.33-2.07 (16H, m), 1.39 (3H, s), 1.31 (3H, s), 1, 22(3H, s), 1.11(3H, br), 0.95-1.01(1H, m), 0.80(3H, s); LC-MS: vy=1.66 min, m/z=550.4 [M+H] ⁺ , purity=94.27% (214 nm).

Example 73.

To a solution of ERX1060 (20 mg, 0.0431 mmol) in MeOH (1 ml) was added iC ₃ H ₇ SH (5 mg, 0.0647 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from reddish to a pale red-yellow. The solution is then concentrated in vacuo to give a crude mixture which is used in the next step without further purification.

Pyridine (0.5 ml) was added to the crude mixture obtained above in Ac ₂ O (0.5 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with CH ₂ Cl ₂ (100 ml), washed with water (2×50 ml), brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by silica gel chromatography (CH ₂ Cl ₂ /MeOH=10:1) to give the product (10 mg, 0.016 mmol, overall yield=37%) as a pale yellow solid. ¹ H NMR: δ (500 MHz, CDCl3): 7.03 (1H, s), 5.95 (1H, d, J=6.4 Hz), 5.39 (1H, d, J=6.4 Hz), 2.26-2.50(6H, br), 2.36(3H, s), 2.31(3H, s), 2.28(3H, s), 2.08(3H, s ), 1.25-2.06(16H, m), 1.42(3H, s), 1.37(3H, s), 1.21(3H, s), 1.11(3H, br) , 0.94-1.00 (1H, m), 0.77 (3H, s); LC-MS: y=1.65 min, m/z=624.3 [M+H] ⁺ , purity=92.90% (214 nm), 97.84% (254 nm).

Example 74.

To a solution of ERX1003 (90 mg, 0.174 mmol) in anhydrous THF (15 ml) was added LiAlH ₄ (494 mg, 13 mmol). The mixture is refluxed overnight. The reaction mixture was quenched with sat. NH ₄ Cl solution. The mixture is heated at 50°C for 2 hours and filtered through a thin layer of silica gel. The solid is washed with THF (3×50 ml). The combined filtrate is concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ : MeOH=10:1) to give the product (10 mg, 0.0198 mmol, Vd=11%) as a red solid. ¹ H NMR: δ (500 MHz, CDCl3): 7.03 (1H, dd, J=7.3, 1.2 Hz), 6.97 (1H, s), 6.53 (1H, d, J =1.0 Hz), 6.39 (1H, d, J=6.9 Hz), 3.66 (1H, t, J=4.4 Hz), 2.46-2.53 (4H, m ), 2.22 (3H, s), 2.06-2.20 (2H, m), 2.00-2.04 (1H, m), 1.30-1.91 (13H, m), 1.44 (3H, s), 1.40 (3H, s), 1.22 (3H, s), 1.01 (3H, s), 0.94-0.99 (1H, m), 0 .79 (3H, s); LC-MS: vy=1.60 min, m/z=506.3 [M+H] ⁺ , purity=100% (214.254 nm).

Example 75.

To a solution of ERX1004 (150 mg, 0.282 mmol) in anhydrous THF (10 mL) was added LiAlH ₄ (54 mg, 1.5 mmol). The mixture is refluxed overnight. The reaction mixture was quenched with sat. NH ₄ Cl solution. The mixture is heated at 50°C for 2 hours and filtered through a thin layer of silica gel. The solid is washed with THF (3×50 ml). The combined filtrate is concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ :MeOH=10:1) to give the product (10 mg, 0.0193 mmol, Vd=7%) as a red solid. ¹ H NMR: δ (500 MHz, CDCl3): 7.03 (1H, dd, J=7.4, 1.4 Hz), 6.96 (1H, s), 6.53 (1H, d, J =1.0 Hz), 6.39 (1H, d, J=6.9 Hz), 2.42-2.70 (8H, m), 2.37 (3H, s), 2.22 (3H , s), 1.23-2.23 (16H, m), 1.44 (3H, s), 1.40 (3H, s), 1.21 (3H, s), 1.01 (3H, s), 0.93-0.99 (1H, m), 0.78 (3H, s); LC-MS: y=1.69 min, m/z=519.3 [M+H] ⁺ , purity=100% (214 nm), 97.88% (254 nm).

Example 76.

PhCOSH (46 mg, 0.333 mmol) was added to a solution of ERX1006 (100 mg, 0.222 mmol) in MeOH (10 ml). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from red to pale yellow. The solution is then concentrated in vacuo to give a crude mixture.

Pyridine (0.5 ml) was added to the crude mixture obtained above in Ac ₂ O (4 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with CH ₂ Cl ₂ (200 ml), washed with water (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=10:1) to give the product (50 mg, 0.0744 mmol, Yield=34%) as a white solid. ¹ H NMR: δ (500 MHz, CDCl3): 7.95 (2H, t, J=7.4 Hz), 7.56 (1H, t, J=7.4 Hz), 7.44 (2H, t, J=7.4Hz), 7.06(1H, s), 6.06(1H, d, J=6.5Hz), 5.68(1H, br), 5.60(1H, d, J=6.5 Hz), 5.24(1H, br), 2.44(1H, d, J=14.9 Hz), 2.30(3H, s), 2.28(3H, s), 2.12(3H, s), 1.40-2.10(13H, m), 1.51(3H, s), 1.20(3H, s), 1.19(3H, s ), 1.09 (3H, s), 0.96-1.02 (1H, m), 0.76 (3H, s); LC-MS: vy=2.01 min, mass peaks not integrated, purity=99.59% (254 nm).

Example 77.

To a solution of ERX1006 (200 mg, 0.445 mmol) in DMF (5 ml) was added K ₂ CO ₃ (123 mg, 0.890 mmol) followed by CH ₃ CH ₂ CH ₂ I (756 mg, 0.43 ml, 4.45 mmol ). The reaction mixture is stirred at 50°C overnight. The mixture was diluted with EtOAc (200 ml), washed with sat. LiCl.H ₂ O solution (3×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:3, two times) to give the product (101 mg, 0.205 mmol, Yield=46%) as a yellow solid. ¹ H NMR δ (400 MHz, CDCl3): 6.94 (1H, dd, J=7.0, 1.4 Hz), 6.38 (1H, d, J=1.4 Hz), 6.28 (1H, d, J=7.0 Hz), 5.70 (1H, w), 5.35 (1H, w), 4.00 (1H, t, J=6.8 Hz), 2.39 (1H, d, J=15.5 Hz), 2.22 (3H, s), 1.45-2.15 (15H, m), 1.44 (3H, s), 1.26 (3H, s), 1.20 (3H, s), 1.12 (3H, s), 1.00 (3H, t, J=7.6 Hz), 0.98-1.05 (1H, m), 0.75 (3H, s); LC-MS: y=1.89 min, m/z=492.4 [M+H] ⁺ , purity=100% (214 nm), 97.59% (254 nm).

Example 78.

To a solution of ERX1006 (200 mg, 0.445 mmol) in DMF (5 ml) was added K ₂ CO ₃ (123 mg, 0.890 mmol) followed by Me ₂ CHCH ₂ CH ₂ I (881 mg, 0.59 ml, 4.45 mmol ). The reaction mixture is stirred at 50°C overnight. The mixture was diluted with CH ₂ Cl ₂ (200 ml), washed with sat. LiCl.H ₂ O solution (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:3) to give the product (88.8 mg, 0.171 mmol, Yield=38%) as a yellow solid. ¹ H NMR δ (400 MHz, CDCl3): 6.94 (1H, d, J=7.1 Hz), 6.38 (1H, s), 6.28 (1H, d, J=7.1 Hz ), 5.69 (1H, w), 5.30 (1H, w), 4.06 (1H, t, J=6.9 Hz), 2.39 (1H, d, J=15.7 Hz ), 2.21(3H, s), 1.45-2.14(16H, m), 1.44(3H, s), 1.26(3H, s), 1.20(3H, s) , 1.12 (3H, s), 0.98-1.05 (1H, m), 0.94 (6H, d, J=6.4 Hz), 0.76 (3H, s); LC-MS: vy=2.00 min, m/z=520.4 [M+H] ⁺ , purity=98.46% (214 nm), 98.77% (254 nm).

Example 79.

To a solution of ERX1006 (100 mg, 0.222 mmol) in DMF (10 mL) was added K ₂ CO ₃ (61 mg, 0.44 mmol) followed by iodocyclopentane (217 mg, 1.11 mmol). The reaction mixture is stirred at 40°C overnight. The mixture was diluted with CH ₂ Cl ₂ (100 ml), washed with sat. a solution of LiCl.H ₂ O (2×50 ml), brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (ethyl acetate) to give the product (56 mg, 0.108 mmol, Yield=50%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl3): 6.91 (1H, dd, J=7.0, 1.1 Hz), 6.36 (1H, d, J=1.1 Hz), 6.27 (1H, d, J=7.0 Hz), 5.70 (1H, w), 5.36 (1H, w), 5.15-5.19 (1H, m), 2.39 (1H, d, J=15.6 Hz), 2.18 (3H, s), 1.46-2.12 (21H, m), 1.44 (3H, s), 1.26 (3H, s), 1.20 (3H, s), 1.11 (3H, s), 0.98-1.04 (1H, m), 0.76 (3H, s); LC-MS: y=1.44 min, m/z=518.4 [M+H] ⁺ , purity=97.22% (214 nm), 99.30% (254 nm).

Example 80.

To a solution of ERX1006 (180 mg, 0.4 mmol) in DMF (10 mL) was added K ₂ CO ₃ (110 mg, 0.8 mmol) followed by allyl bromide (242 mg, 2.0 mmol). The reaction mixture is stirred at 40°C overnight. The mixture was diluted with CH ₂ Cl ₂ (100 ml), washed with sat. LiCl.H ₂ O solution (2×50 ml), brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (ethyl acetate) to give the product (98 mg, 0.2 mmol, Yield=50%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl3): 6.95 (1H, dd, J=7.0, 1.0 Hz), 6.39 (1H, d, J=1.0 Hz), 6.28 (1H, d, J=7.0 Hz), 6.02-6.12 (1H, m), 5.68 (1H, w), 5.34 (1H, dd, J=17.2, 1 .4 Hz), 5.20 (1H, d, J=10.4 Hz), 4.60 (1H, d, J=5.1 Hz), 2.39 (1H, d, J=16.1 Hz), 2.21(3H, s), 1.45-2.12(13H, m), 1.44(3H, s), 1.26(3H, s), 1.20(3H, s ), 1.12 (3H, s), 0.98-1.05 (1H, m), 0.76 (3H, s); LC-MS: vy=1.84 min, m/z=490.2 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 81.

To a solution of ERX1006 (100 mg, 0.22 mmol) in DMF (10 mL) was added K ₂ CO ₃ (61 mg, 0.44 mmol) followed by 3,3-dimethylallyl bromide (217 mg, 1.11 mmol). The reaction mixture is stirred at 40°C overnight. The mixture was diluted with CH ₂ Cl ₂ (100 ml), washed with sat. LiCl.H ₂ O solution (2×50 ml), brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (ethyl acetate) to give the product (56 mg, 0.11 mmol, Yield=50%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl3): 6.94 (1H, dd, J=7.0, 1.1 Hz), 6.38 (1H, d, J=1.1 Hz), 6.27 (1H, d, J=7.0 Hz), 5.72 (1H, w), 5.52 (1H, tt, J=7.2, 1.3 Hz), 5.46 (1H, w) , 4.54-4.63(2H, m), 2.40(1H, d, J=15.7 Hz), 2.20(3H, s), 1.45-2.12(13H, m ), 1.75(3H, s), 1.69(3H, s), 1.43(3H, s), 1.25(3H, s), 1.20(3H, s), 1.11 (3H, s), 0.98-1.04 (1H, m), 0.75 (3H, s); LC-MS: y=2.43 min, m/z=518.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 82.

To a solution of ERX1006 (200 mg, 0.44 mmol) in DMF (10 mL) was added K ₂ CO ₃ (121 mg, 0.88 mmol) followed by propargyl bromide (263 mg, 2.23 mmol). The reaction mixture is stirred at 40°C overnight. The mixture was diluted with CH ₂ Cl ₂ (100 ml), washed with sat. a solution of LiCl.H ₂ O (2×50 ml), brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (ethyl acetate) to give the product (100 mg, 0.205 mmol, Yield=47%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl3): 7.00 (1H, dd, J=7.0, 1.0 Hz), 6.39 (1H, d, J=1.0 Hz), 6.30 (1H, d, J=7.5 Hz), 5.68 (1H, w), 5.34 (1H, w), 4.88 (1H, AB×d, J=15.9, 2.4 Hz), 4.85 (1H, AB×d, J=15.9, 2.4 Hz), 2.42 (1H, t, J=2.3 Hz), 2.40 (1H, d, J =16.0 Hz), 2.28(3H, s), 1.46-2.13(13H, m), 1.45(3H, s), 1.26(3H, s), 1.20 (3H, s), 1.12 (3H, s), 0.99-1.05 (1H, m), 0.76 (3H, s); LCMS: y=1.78 min, m/z=488.3 [M+H] ⁺ , purity=100% (214 nm), 93.38% (254 nm).

Example 83.

To a solution of NASH (2.0 g, 35.7 mmol) in EtOH (20 ml) was added Me ₂ CHCOCl (4.0 g, 37.6 mmol) dropwise. The solution was stirred at rt for 1 hour. The solution is concentrated in vacuo and used in the next step without further purification.

To a solution of ERX1006 (50 mg, 0.11 mmol) in MeOH (5 ml) was added Me ₂ CHCOSH (23 mg, 0.22 mmol) in EtOH (2 ml). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from red to pale yellow. The solution is then concentrated in vacuo to give a crude mixture (45 mg, 0.081 mmol, Vd=74%).

Pyridine (1 ml) was added to the crude mixture (45 mg, 0.081 mmol) obtained above in Ac ₂ O (1 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with CH ₂ Cl ₂ (50 ml), washed with water (2×30 ml), brine (30 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give the product (10 mg, 0.0157 mmol, Yield=20%) as a white solid. ¹ H NMR δ (500 MHz, CDCl3): 7.01 (1H, s), 5.92 (1H, d, J=6.5 Hz), 5.67 (1H, br), 5.34 (1H , d, J=6.5 Hz), 5.24 (1H, w), 2.68-2.77 (1H, m), 2.30 (3H, s), 2.27 (3H, s) , 2.06(3H, s), 1.30-2.08(13H, m), 1.44(3H, s), 1.20(3H, s), 1.20(3H, d, J =6.7Hz), 1.19(3H, s), 1.19(3H, d, J=5.5Hz), 1.09(3H, s), 0.95-1.01(1H , m), 0.74 (3H, s); LC-MS: y=1.99 min, m/z=638.3 [M+H] ⁺ , purity=96.8% (214 nm), 97.11% (254 nm).

Example 84.

To a solution of ERX1006 (200 mg, 0.445 mmol) in DMF (5 mL) was added K ₂ CO ₃ (123 mg, 0.890 mmol) followed by FCH ₂ CH ₂ I (774 mg, 0.36 mL, 4.45 mmol). The reaction mixture was stirred at 50°C for 1 day. The mixture was diluted with EtOAc (200 ml), washed with sat. LiCl.H ₂ O solution (3×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (ethyl acetate) to give the product (79.4 mg, 0.160 mmol, Yield=36%) as a yellow solid. ¹ H NMR δ (400 MHz, CDCl3): 7.00 (1H, dd, J=7.0, 1.2 Hz), 6.39 (1H, d, J=1.0 Hz), 6.30 (1H, d, J=7.2 Hz), 5.69 (1H, w), 5.31 (1H, w), 4.66 (2H, d×t, J=47.8, 4.0 Hz), 4.37 (2H, d×t, J=31.0, 4.0 Hz), 2.40 (1H, t, J=16.1 Hz), 2.25 (3H, s), 1.46-2.14(13H, m), 1.44(3H, s), 1.26(3H, s), 1.20(3H, s), 1.12(3H, s), 0 .98-1.05 (1H, m), 0.75 (3H, s); LC-MS: y=1.77 min, m/z=496.4 [M+H] ⁺ , purity=98.76% (214 nm), 98.72% (254 nm).

Example 85.

To a solution of ERX1006 (200 mg, 0.445 mmol) in DMF (5 mL) was added K ₂ CO ₃ (123 mg, 0.890 mmol) followed by F ₂ CHCH ₂ I (854 mg, 0.39 mL, 4.45 mmol). The reaction mixture was stirred at 50°C for 1 day. The mixture was diluted with EtOAc (200 ml), washed with sat. LiCl.H ₂ O solution (3×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (ethyl acetate) to give the product (72.2 mg, 0.141 mmol, Yield=32%) as a yellow solid. ¹ H NMR δ (400 MHz, CDCl3): 7.02 (1H, dd, J=7.0, 1.3 Hz), 6.40 (1H, d, J=1.3 Hz), 6.32 (1H, d, J=7.3 Hz), 6.10 (1H, tt, J=55.5, 4.1 Hz), 5.68 (1H, w), 5.28 (1H, w) , 4.31 (1H, td, J=14.0, 4.0 Hz), 2.41 (1H, d, J=15.7 Hz), 2.24 (3H, s), 1.46- 2.15(13H, m), 1.45(3H, s), 1.27(3H, s), 1.21(3H, s), 1.13(3H, s), 0.99-1 .06 (1H, m), 0.76 (3H, s); LC-MS: y=2.19 min, m/z=514.4 [M+H] ⁺ , purity=100% (214.254 nm).

Example 86.

To a solution of ERX1006 (50 mg, 0.11 mmol) in DMF (5 mL) was added K ₂ CO ₃ (45 mg, 0.33 mmol) followed by F ₃ CCH ₂ OSO ₂ CF ₃ (30 mg, 0.13 mmol ). The reaction mixture was stirred at rt. during the night. The mixture was diluted with CH ₂ Cl ₂ (100 ml), washed with sat. LiCl.H ₂ O solution (3×50 ml), brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (ethyl acetate) to give the product (30 mg, 0.0564 mmol, Yield=51%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl3): 7.04 (1H, dd, J=7.5, 1.2 Hz), 6.40 (1H, d, J=1.2 Hz), 6.31 (1H, d, J=7.5 Hz), 5.68 (1H, w), 5.32 (1H, w), 4.57 (1H, q, J=8.8 Hz), 2.40 (1H, d, J=15.9 Hz), 2.24 (3H, s), 1.47-2.14 (13H, m), 1.44 (3H, s), 1.27 (3H, s), 1.20(3H, s), 1.12(3H, s), 0.99-1.05(1H, m), 0.75(3H, s); LC-MS: vy=1.27 min, m/z=532.4 [M+H] ⁺ , purity=100% (214.254 nm).

Example 87.

To a solution of NASH (56 mg, 1.0 mmol) in EtOH (5 ml) was added CF ₃ CH ₂ COCl (161 mg, 1.1 mmol) dropwise at 0°C. The solution was stirred at rt for 1 hour. The solution is concentrated in vacuo and used in the next step without further purification.

To a solution of ERX1006 (50 mg, 0.11 mmol) in MeOH (5 ml) was added Me ₂ CHCOSH (80 mg, 0.55 mmol) in EtOH (2 ml). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from red to pale yellow. The solution is then concentrated in vacuo to give a crude mixture (50 mg, 0.0842 mmol, Vd=77%).

Pyridine (1 ml) was added to the crude mixture (50 mg, 0.0842 mmol) obtained above in Ac ₂ O (1 ml). The reaction mixture was stirred at room temperature overnight. The mixture was then diluted with CH ₂ Cl ₂ (50 ml), washed with water (2×30 ml), brine (30 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (PE:EA=1:1) to give the product (11 mg, 0.0162 mmol, overall yield=20%) as a white solid. ¹ H NMR δ (500 MHz, CDCl3): 7.04 (1H, s), 5.96 (1H, d, J=6.4 Hz), 5.68 (1H, br), 5.49 (1H , d, J=6.4 Hz), 5.31 (1H, w), 3.35 (2H, q, J=10.0 Hz), 2.43 (1H, d, J=15.3 Hz ), 2.31(3H, s), 2.28(3H, s), 2.07(3H, s), 1.44-2.05(13H, m), 1.43(3H, s) , 1.20(3H, s), 1.18(3H, s), 1.09(3H, s), 0.95-1.01(1H, m), 0.73(3H, s); LC-MS: vy=1.88 min, m/z=678.2 [M+H] ⁺ , purity=100% (214.254 nm).

Example 88.

To a solution of Celastrol (200 mg, 0.44 mmol) in DMF (5 ml) was added MeNHOMe.HCl (131 mg, 1.33 mmol), GATU (186 mg, 0.49 mmol), then DIPEA (115 mg, 0 .89 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/EtOAc=1:1) to give the product (150 mg, 0.304 mmol, Yield=69%) as a red solid. ¹ H NMR δ (500 MHz, CDCl ₃ ): 7.03 (1H, dd, J=7.2, 1.1 Hz), 6.96 (1H, s), 6.54 (1H, d, J =1.1 Hz), 6.35(1H, d, J=7.2 Hz), 3.71(3H, s), 3.05(3H, s), 2.81(1H, d, J =16.2 Hz), 2.22(3H, s), 2.04-2.32(3H, m), 1.24-1.90(10H, m), 1.46(3H, s) , 1.26(3H, s), 1.22(3H, s), 1.12(3H, s), 0.94-1.00(1H, m), 0.50(3H, s); LC-MS: y=2.56 min, m/z=494.2 [M+H] ⁺ , purity=96.78% (214 nm), 91.29% (254 nm).

Example 89.

To a solution of Celastrol (500 mg, 1.11 mmol) in MeOH (20 ml) was added N-acetyl-L-cysteine ethyl ester (393 mg, 2.22 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution turns from red to pale yellow. The was solution was then concentrated in vacuo to give a crude mixture.

To the crude mixture obtained above, Ac ₂ O (3 ml) was added to pyridine (5 ml). The reaction mixture was stirred at room temperature overnight. The mixture is then poured into H ₂ O (100 ml), filtered. The solid is dissolved in CH ₂ Cl ₂ (200 ml), washed with water (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (EtOAc) to give the product (408 mg, 0.573 mmol, overall yield=52%) as a white solid. ¹ H NMR δ (400 MHz, CDCl3): 7.02 (1H, s), 6.29 (1H, d, J=6.8 Hz), 5.93 (1H, d, J=6.1 Hz ), 4.89 (1H, q, J=5.8 Hz), 4.61 (1H, d, J=6.0 Hz), 3.73 (3H, s), 3.29 (1H, dd , J=13.6, 4.9 Hz), 2.92 (1H, dd, J=13.6, 6.0 Hz), 2.39 (1H, d, J=15.9 Hz), 2 .31 (3H, s), 2.27 (3H, s), 2.23 (3H, s), 2.05 (3H, s), 1.30-2.16 (13H, m), 1, 56 (3H, s), 1.25 (3H, s), 1.15 (3H, s), 1.07 (3H, s), 0.89-0.97 (1H, m), 0.66 (3H, s); LCMS: wy=1.78 min, m/z=535.2 [M-SCH ₂ CH(CO ₂ Me)NHCOMe] ⁺ , purity=97.44% (214 nm), 100% (254 nm) .

Example 90.

To a solution of ERX1128 (125 mg, 0.252 mmol) in MeOH (10 ml) was added i -PrSH (77 mg, 1.01 mmol). The reaction mixture was stirred at room temperature for 1 hour. The solution is then concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:3) to give the product. The crude product is partially oxidized in air to the starting material.

To a solution of the crude mixture obtained above in MeOH (10 ml) was added K ₂ CO ₃ (70 mg, 0.504 mmol). The reaction mixture was stirred at room temperature overnight. Most of the MeOH is removed in vacuo. The mixture was then dissolved in CH ₂ Cl ₂ (200 ml), washed with water (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether: ethyl acetate=1:2) to give the product (29.4 mg, 0.0533 mmol, overall yield over two steps=21%) as a white solid. ¹ H NMR δ (400 MHz, CDCl3): 6.72 (1H, s), 5.98 (1H, d, J=6.3 Hz), 5.63 (1H, br), 5.16 (1H , w), 4.60 (1H, d, J=6.3 Hz), 4.19-4.28 (4H, m), 3.13-3.23 (1H, m), 2.39 ( 1H, d, J=15.2 Hz), 2.29 (3H, s), 1.43-2.10 (13H, m), 1.56 (3H, s), 1.40 (3H, d , J=6.8 Hz), 1.28 (3H, d, J=6.8 Hz), 1.25 (3H, s), 1.19 (3H, s), 1.11 (3H, s ), 0.96-1.02 (1H, m), 0.74 (3H, s); LC-MS: y=2.18 min, m/z=552.3 [M+H] ⁺ , purity=99.56% (214 nm), 100% (254 nm).

Example 91.

To a solution of ERX1006 (50 mg, 0.11 mmol) in CH ₂ Cl ₂ (10 mL) was added Mg(ClO ₄ ) ₂ (7.4 mg, 0.03 mmol) followed by Boc ₂ O (84 mg, 0.39 mmol). The reaction mixture is stirred at 40°C overnight. The mixture was diluted with CH ₂ Cl ₂ (100 ml), washed with water (3×50 ml), brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (ethyl acetate) to give the product (20 mg, 0.0364 mmol, Yield=33%) as a yellow solid. ¹ H NMR δ (500 MHz, CDCl3): 7.04 (1H, d, J=7.2 Hz), 6.46 (1H, s), 6.31 (1H, d, J=7.2 Hz ), 5.66 (1H, br), 5.22 (1H, br), 2.39 (1H, d, J=15.3 Hz), 2.20 (3H, s), 1.47-2 .15 (13H, m), 1.54 (9H, s), 1.45 (3H, s), 1.27 (3H, s), 1.21 (3H, s), 1.12 (3H, s), 0.99-1.05 (1H, m), 0.76 (3H, s); LC-MS: vy=2.30 min, m/z=550.2 [M+H] ⁺ , purity=100% (214.254 nm).

Example 92.

To a solution of ERX1008 (800 mg, 1.68 mmol) in acetone (5 ml) was added K ₂ CO ₃ (462 mg, 3.35 mmol) followed by MeI (1.2 g, 8.4 mmol). The reaction mixture is heated at 40°C overnight. The mixture is filtered, concentrated in vacuo. The residue was purified by silica gel chromatography (eluant: petroleum ether/ethyl acetate=1:1) to give product 2 (530 mg, 1.078 mmol, yield=64%) as a yellow solid.

To a solution of 2 (530 mg, 1.08 mmol) in THF (10 ml) was added 1.0 M LiAlH ₄ in THF solution (5.4 ml, 5.4 mmol). The reaction mixture is refluxed overnight. The reaction mixture was quenched with sat. NH ₄ Cl solution, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (eluant: petroleum ether/ethyl acetate=1:1) to give product 3 (400 mg, 0.834 mmol, yield=77%) as a white solid.

To a solution of 3 (400 mg, 0.84 mmol) in CH ₂ Cl ₂ (10 ml) was added AcCl (651 mg, 8.4 mmol) and NEt3 (840 mg, 8.4 mmol). The mixture is then stirred at rt. within 2 hours. Water is added to quench the reaction. The mixture is extracted with CH ₂ Cl ₂ (3×20 ml). The combined layers are washed with brine (50 ml), dried over Na ₂ SO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give product 4 (190 mg, 0.364 mmol, yield=43%) as a white solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 6.89 (1H, s), 5.81 (1H, dd, J=6.3, 2.1 Hz), 3.72 (3H, s), 3.35 (1H, dd, J=20.9, 6.3 Hz), 3.06 (1H, dd, J=20.9, 2.1 Hz), 2.87 (6H, w), 2 .33 (3H, s), 2.20 (3H, s), 1.24-2.05 (19H, m), 1.39 (3H, s), 1.29 (3H, s), 1, 23 (3H, s), 1.02-1.08 (1H, m), 0.79 (3H, s); LCMS: y=1.73 min, m/z=522.3 [M+H] ⁺ , purity=100% (214.254 nm).

Example 93.

To a solution of Celastrol (450 mg, 1.0 mmol) in DMF (10 mL) was added Et ₂ NH.HCl (241 mg, 2.2 mmol), GATU (418 mg, 1.1 mmol) followed by DIPEA (645 mg , 5.0 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with sat. LiCl.H ₂ O solution (2×200 ml), brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give the product (101 mg, 0.20 mmol, Yield=20%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.03 (1H, dd, J=7.0, 1.1 Hz), 6.95 (1H, br), 6.52 (1H, d, J =1.1 Hz), 6.36 (1H, d, J=7.1 Hz), 3.62-3.80 (1H, m), 3.10-3.42 (3H, m), 2 .22 (3H, s), 1.98-2.40 (5H, m), 0.95-1.90 (16H, m), 1.45 (3H, s), 1.30 (3H, s ), 1.26 (3H, s), 1.15 (3H, s), 0.63 (3H, s); LC-MS: y=1.98 min, m/z=506.3 [M+H] ⁺ , purity=98.77% (214 nm), 100% (254 nm).

Example 94.

To a solution of ERX1137 (100 mg, 0.198 mmol) in THF (20 ml) was added LiAlH ₄ (38 mg, 1.0 mmol). The reaction mixture is refluxed overnight. The reaction mixture was quenched with sat. NH ₄ Cl solution. The mixture is heated at 60°C for 3 hours. The color turns brown. Then the solution was diluted with CH ₂ Cl ₂ (200 ml), filtered. The filtrate is concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=5:1) to give the product (30 mg, 0.061 mmol, Yield=31%) as a black solid. ¹ H NMR: δ (400 MHz, CDCl ₃ ): 7.04 (1H, d, J=7.0 Hz), 6.98 (1H, s), 6.54 (1H, s), 6.41 (1H, d, J=7.0 Hz), 2.90-3.20 (2H, m), 2.48-2.62 (3H, m), 2.24 (3H, s), 1, 30-2.20(15H, m), 1.46(3H, s), 1.45(3H, s), 1.27(3H, s), 1.26(3H, s), 0, 92-1.04 (7H, m), 0.81 (3H, s); LC-MS: y=1.34 min, m/z=492.3 [M+H] ⁺ , purity=95.35% (214 nm), 100% (254 nm).

Example 95.

To a solution of Celastrol (100 mg, 0.222 mmol) in DMF (5 ml) was added GATU (101 mg, 0.266 mmol) followed by DIPEA (57 mg, 0.076 ml, 0.444 mmol). The solution was stirred at rt for 1 hour. Piperazine (23 mg, 0.266 mmol) is then added. The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with sat. LiCl.H ₂ O solution (2×200 ml), brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=10:1) to give the product (35 mg, 0.0675 mmol, Yield=30%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.02 (1H, d, J=7.2 Hz), 6.54 (1H, s), 6.35 (1H, d, J=7.2 Hz), 3.55-3.75(2H, m), 2.80-2.90(3H, m), 2.28-2.39(2H, m), 2.21(3H, s) , 1.25-2.25(17H, m), 1.46(3H, s), 1.29(3H, s), 1.28(3H, s), 1.14(3H, s), 0.95-1.02 (1H, m), 0.62 (3H, s); LCMS: y=1.21 min, m/z=519.3 [M+H] ⁺ , purity=95.17% (214 nm), 96.74% (254 nm).

Example 96.

To a solution of Celastrol (100 mg, 0.22 mmol) in DMF (5 mL) was added 2,6-dimethylpiperazine (30 mg, 0.26 mmol), HATU (91 mg, 0.24 mmol) followed by DIPEA (57 mg, 0.44 mmol). The reaction mixture was stirred at room temperature overnight. The solution is then diluted with CH ₂ Cl ₂ (100 ml), washed with sat. LiCl.H ₂ O solution (2×50 ml), brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=10:1) to give the product (60 mg, 0.11 mmol, Yield=50%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.03 (1H, d, J=7.1 Hz), 6.97 (1H, br), 6.55 (1H, s), 6.36 ( 1H, d, J=7.1 Hz), 4.00-4.60 (2H, m), 2.60-2.90 (2H, m), 2.28-2.40 (2H, m) , 2.22(3H, s), 1.30-2.20(15H, m), 1.46(3H, s), 1.29(3H, s), 1.27(3H, s), 1.14(3H, s), 1.12(6H, br), 0.94-1.02(1H, m), 0.59(3H, br); LC-MS: vy=1.38 min, m/z=547.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 97.

To a solution of ERX1142 (88 mg, 0.16 mmol) in THF (10 ml) was added LiAlH ₄ (30 mg, 0.8 mmol). The reaction mixture is refluxed overnight. The reaction mixture was quenched with sat. NH ₄ Cl solution. The mixture is heated at 60°C for 3 hours. The color turns brown. Then the solution was diluted with CH ₂ Cl ₂ (200 ml), filtered. The filtrate is concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=5:1) to give the product (20 mg, 0.0375 mmol, Yield=23%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.03 (1H, d, J=7.1 Hz), 6.53 (1H, s), 6.39 (1H, d, J=7.1 Hz), 2.94-3.06 (2H, m), 2.65-2.74 (2H, m), 2.22 (3H, s), 0.92-2.20 (21H, m) , 1.44 (3H, s), 1.40 (3H, s), 1.21 (3H, s), 1.10 (6H, br), 1.00 (3H, s), 0.78 ( 3H, s); LCMS: y=1.25 min, m/z=533.3 [M+H] ⁺ , purity=97.84% (214 nm), 96.36% (254 nm).

Example 98.

To a solution of celastrol (500 mg, 1.11 mmol) in DMF (10 ml) was added MeNH ₂ .HCl (220 mg, 3.3 mmol), GATU (464 mg, 1.2 mmol), then DIPEA (287 mg, 2.2 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with sat. LiCl.H ₂ O solution (2×200 ml), brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give the product (420 mg, 0.906 mmol, Yield=82%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.00 (1H, dd, J=7.2, 1.2 Hz), 6.97 (1H, br), 6.53 (1H, d, J =1.2 Hz), 6.33 (1H, d, J=7.2 Hz), 5.72 (1H, q, J=4.7 Hz), 2.67 (3H, d, J=4 .7 Hz), 2.46 (1H, d, J=15.4 Hz), 2.21 (3H, s), 1.47-2.17 (13H, m), 1.44 (3H, s ), 1.26 (3H, s), 1.15 (3H, s), 1.12 (3H, s), 0.99-1.06 (1H, m), 0.62 (3H, s) ; LC-MS: y=1.66 min, m/z=464.2 [M+H] ⁺ , purity=99.0% (214 nm), 100% (254 nm).

Example 99.

To a solution of Celastrol (450 mg, 1.0 mmol) in DMF (10 mL) was added BnNHMe (363 mg, 3.0 mmol), GATU (420 mg, 1.1 mmol), then DIPEA (260 mg, 2.0 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with sat. LiCl.H ₂ O solution (2×200 ml), brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give the product (370 mg, 0.669 mmol, Yield=67%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.15-7.30 (5H, m), 7.03 (1H, dd, J=7.2, 1.1 Hz), 6.98 (1H , s), 6.53 (1H, s), 6.36 (1H, d, J=7.2 Hz), 4.85 (1H, w), 4.00 (1H, w), 3.08 (3H, w), 2.35-2.48 (2H, m), 2.23 (3H, s), 2.06-2.18 (2H, m), 1.32-1.89 (10H , m), 1.46 (3H, s), 1.31 (3H, s), 1.29 (3H, s), 1.15 (3H, s), 0.97-1.04 (1H, m), 0.55 (3H, s); LC-MS: vy=2.04 min, m/z=554.2 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 100.

To a solution of ERX1150 (100 mg, 0.18 mmol) in THF (5 ml) was added LiAlH ₄ (34 mg, 0.9 mmol). The reaction mixture is refluxed overnight. The reaction mixture was quenched with sat. NH ₄ Cl solution. Then the solution was diluted with CH ₂ Cl ₂ (100 ml), filtered. The filtrate is separated and the organic layer is dried over Na ₂ SO ₄ and concentrated in vacuo. The residue is dissolved in PhMe (5 ml) and Ag ₂ CO ₃ (61 mg, 0.22 mmol) is added. The mixture is stirred at rt. overnight, filtered and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:1) to give the product (15 mg, 0.0278 mmol, Yield=25%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.19-7.36 (5H, m), 7.03 (1H, dd, J=7.1, 0.9 Hz), 6.96 (1H , s), 6.53 (1H, d, J=0.9 Hz), 6.38 (1H, d, J=7.1 Hz), 3.56 (1H, AB, J=13.8 Hz ), 3.55 (1H, AB, J=13.8 Hz), 2.37 (1H, AB, J=13.5 Hz), 2.23 (3H, s), 2.22 (3H, s ), 2.17 (1H, AB, J=13.5 Hz), 2.07-2.13 (1H, m), 1.17-1.90 (15H, m), 1.44 (3H, s), 1.41 (3H, s), 1.23 (3H, s), 1.06 (3H, s), 0.91-0.97 (1H, m), 0.75 (3H, s ); LC-MS: vy=1.48 min, m/z=540.4 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 101.

To a solution of celastrol (200 mg, 0.44 mmol) in DMF (10 ml) was added 2,2-dimethylpiperazine (62 mg, 0.54 mmol), GATU (184 mg, 0.48 mmol), then DIPEA (114 mg , 0.88 mmol). The reaction mixture was stirred at room temperature overnight. The solution was then diluted with CH ₂ Cl ₂ (200 ml), washed with sat. LiCl.H ₂ O solution (2×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ /MeOH=10:1) to give the product (100 mg, 0.183 mmol, Yield=42%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.02 (1H, d, J=7.0 Hz), 6.54 (1H, s), 6.35 (1H, d, J=7.0 Hz), 3.35-4.20 (2H, w), 2.95-3.10 (2H, m), 2.27-2.38 (2H, m), 2.21 (3H, s) , 1.35-2.20(16H, m), 1.45(3H, s), 1.32(3H, s), 1.30(3H, s), 1.19(3H, s), 1.18 (3H, s), 1.15 (3H, s), 0.95-1.02 (1H, m), 0.55 (3H, s); LC-MS: y=1.39 min, m/z=547.3 [M+H] ⁺ , purity=97.82% (214 nm), 98.76% (254 nm).

Example 102.

To a solution of 1 (240 mg, 0.54 mmol) in THF (10 ml) and H ₂ O (1 ml) was added LiOH.H ₂ O (113 mg, 2.68 mmol). The reaction mixture is heated at 50°C for 4 hours. Most of the THF is removed in vacuo. The residue is extracted with CH ₂ Cl ₂ (100×3 ml). The combined extracts were washed with brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ :MeOH=5:1) to give the product (120 mg, 0.284 mmol, Yield=53%) as a brown solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.03 (1H, dd, J=7.2, 1.3 Hz), 6.53 (1H, d, J=1.3 Hz), 6, 37 (1H, d, J=7.2 Hz), 2.22 (3H, s), 2.12-2.18 (1H, m), 1.50-2.05 (16H, m), 1 .46 (3H, s), 1.30 (3H, s), 1.14 (3H, s), 1.10 (3H, s), 0.94-1.01 (1H, m), 0. 96 (3H, s); LC-MS: y=1.43 min, m/z=422.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 103.

To a solution of ERX1146 (80 mg, 0.18 mmol) in MeCN (5 ml) was added N-hydroxysuccinimide ester of acetic acid (112 mg, 0.72 mmol). The reaction mixture was stirred at room temperature overnight. The solution is filtered and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether: EtOAc=1:1) to give the product (32 mg, 0.065 mmol, Yield=36%) as a red solid.

¹ H NMR (two atropisomeric mixtures) i) main isomer δ (400 MHz, d6-DMSO): 8.71 (1H, s), 7.10 (1H, d, J=7.1 Hz), 6.40 (1H, d, J=7.1 Hz), 6.37 (1H, s), 3.61 (1H, AB, J=13.2 Hz), 3.00 (3H, s), 2.79 (1H, AB, J=13.2 Hz), 2.11-2.17 (1H, s), 2.10 (3H, s), 1.98 (3H, s), 1.20-1, 85(13H, m), 1.38(3H, s), 1.35(3H, s), 1.17(3H, s), 0.95(3H, s), 0.84-0.92 (1H, m), 0.74 (3H, s); ii) smaller isomer δ (400 MHz, d6-DMSO): 8.71 (1H, s), 7.10 (1H, d, J=7.1 Hz), 6.40 (1H, d, J=7 .1 Hz), 6.37 (1H, s), 3.30 (1H, AB, J=14.8 Hz), 3.08 (1H, AB, J=14.8 Hz), 2.85 ( 3H, s), 2.11-2.17(1H, s), 2.10(3H, s), 1.99(3H, s), 1.20-1.85(13H, m), 1 .38 (3H, s), 1.35 (3H, s), 1.22 (3H, s), 1.00 (3H, s), 0.84-0.92 (1H, m), 0. 74 (3H, s). LC-MS: y=1.88 min, m/z=492.3 [M+H] ⁺ , purity=100% (214, 254 nm).

Example 104

To a solution of Celastrol (500 mg, 1.11 mmol) in PhMe (25 ml) was added DIPEA (430 mg, 0.57 ml, 3.33 mmol) followed by (PhO) ₂ P(O)N ₃ (458 mg, 0.36 ml, 1.66 mmol). The reaction mixture is heated at 100°C overnight. The solution was diluted with CH ₂ Cl ₂ (200 ml), washed with H ₂ O (200 ml), brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether:ethyl acetate=3:1) to give the crude product (342 mg, 0.764 mmol, Yield=69%) as a red solid.

To a solution of crude 2 (342 mg, 0.764 mmol) in THF (20 ml) was added EtNH ₂ .HCl (312 mg, 3.82 mmol) and DIPEA (494 mg, 0.65 ml, 3.82 mmol). The reaction mixture is heated at 50°C for 3 hours. Most of the THF is removed in vacuo. The residue was dissolved in CH ₂ Cl ₂ (200 ml), washed with H ₂ O (200 ml), brine (200 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ :MeOH=10:1) to give the product (233.6 mg, 0.474 mmol, Yield=62%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.02 (1H, dd, J=7.1, 1.1 Hz), 6.98 (1H, s), 6.53 (1H, d, J =1.0 Hz), 6.36 (1H, d, J=7.3 Hz), 3.96 (1H, t, J=5.5 Hz), 3.85 (1H, s), 2, 95-3.15 (2H, m), 2.89 (1H, d, J=13.8 Hz), 2.21 (3H, s), 1.45-2.20 (13H, m), 1 .44 (3H, s), 1.40 (3H, s), 1.27 (3H, s), 1.12 (3H, s), 1.03 (3H, t, J=7.1 Hz) , 0.94-1.01 (1H, m), 0.77 (3H, s). LCMS: vy=1.79 min, m/z=493.3 [M+H] ⁺ , purity=100% (214.254 nm).

Example 105

To a solution of the isocyanate (50 mg, 0.112 mmol) in THF (2 ml) was added EtONa (38 mg, 0.559 mmol). The reaction mixture is heated at room temperature for 1 hour. The reaction mixture was quenched with NH ₄ Cl (10 ml). The mixture was diluted in CH ₂ Cl ₂ (100 ml), washed with H ₂ O (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ :MeOH=20:1) to give the product (11.8 mg, 0.0239 mmol, Yield=21%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.03 (1H, dd, J=7.2, 0.9 Hz), 6.98 (1H, s), 6.54 (1H, d, J =0.9Hz), 6.37(1H, d, J=7.2Hz), 4.39(1H, s), 3.90-4.07(2H, m), 2.74(1H , d, J=14.8 Hz), 2.22 (3H, s), 2.11-2.18 (1H, m), 1.20-2.05 (12H, m), 1.45 ( 3H, s), 1.37 (3H, s), 1.27 (3H, s), 1.14 (3H, t, J=7.0 Hz), 1.12 (3H, s), 0, 95-1.01 (1H, m), 0.75 (3H, s). LC-MS: y=1.99 min, m/z=494.3 [M+H] ⁺ , purity=100% (214.254 nm).

Example 106

To a solution of isocyanate 1 (478 mg, 1.068 mmol) in THF (20 ml) and H ₂ O (2 ml) was added LiOH.H ₂ O (220 mg, 5.24 mmol). The reaction mixture is heated at 50°C for 4 hours. Most of the THF is removed in vacuo. The residue was diluted in CH ₂ Cl ₂ (100 ml), washed with 0.1 M HCl (100 ml). The entire solution is filtered. The organic layer was washed with H ₂ O (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue and the solid obtained by filtration are dissolved in 200 ml of CH ₂ Cl ₂ :MeOH=10:1 solution, concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ :MeOH=5:1) to give the intermediate (50 mg, 0.119 mmol, Yield=11%) as a brown solid.

To a solution of intermediate 2 (50 mg, 0.119 mmol) in THF (3 ml) was added N-hydroxysuccinimide ester of acetic acid (93 mg, 0.594 mmol) and DIPEA (77 mg, 0.10 ml, 0.594 mmol). The reaction mixture was stirred at room temperature overnight. Most of the THF is removed in vacuo. The residue was diluted in CH ₂ Cl ₂ (100 ml), washed with H ₂ O (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=1:3) to give the product (17.5 mg, 0.0377 mmol, Yield=32%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.01 (1H, dd, J=7.2, 1.2 Hz), 6.98 (1H, s), 6.54 (1H, d, J =1.2 Hz), 6.36 (1H, d, J=7.2 Hz), 5.04 (1H, s), 2.82 (1H, d, J=13.9 Hz), 2, 21(3H, s), 2.12-2.18(1H, m), 1.60-2.05(12H, m), 1.81(3H, s), 1.45(3H, s) , 1.42(3H, s), 1.28(3H, s), 1.12(3H, s), 0.97-1.03(1H, m), 0.77(3H, s). LC-MS: vy=1.81 min, m/z=464.3 [M+H] ⁺ , purity=100% (214.254 nm).

Example 107

PhCOSH (71 mg, 0.06 ml, 0.516 mmol) was added to a solution of ERX1168 (116 mg, 0.258 mmol) in MeOH (5 ml). The mixture immediately turns from reddish to light yellow. The reaction mixture was stirred at rt. within 10 minutes. The solution is concentrated in vacuo. The crude intermediate 2 was used in the next step without further purification.

Ac ₂ O (1 ml) was added to a solution of intermediate 2 (155 mg, 0.258 mmol, theoretical) in pyridine (3 ml). The reaction mixture was stirred at room temperature overnight. The solution is then diluted with CH ₂ Cl ₂ (100 ml), washed with sat. CuSO ₄ ,5H ₂ O (2×100 ml), H ₂ O (100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (petroleum ether/ethyl acetate=2:3), then recrystallized from MeCN (5 mL) to give the product (59.9 mg, 0.0873 mmol, Yield=34%) as a white solid.

¹ H NMR δ (400 MHz, CDCl ₃ ): 7.93-7.97 (2H, m), 7.54-7.60 (1H, m), 7.41-7.48 (2H, m) , 7.07 (1H, s), 6.07 (1H, d, J=6.4 Hz), 5.60 (1H, d, J=6.4 Hz), 5.08 (1H, s) , 2.77-2.86(1H, m), 2.30(3H, s), 2.30(3H, s), 2.12(3H, s), 1.81(3H, s), 1.43-2.10 (13H, m), 1.51 (13H, s), 1.41 (3H, s), 1.20 (3H, s), 1.08 (3H, s), 0 .93-1.00 (1H, m), 0.79 (3H, s). LC-MS: y=2.42 min, m/z=686.4 [M+H] ⁺ , purity=97.26% (214 nm), 100% (254 nm).

Example 108

To a solution of ERX1077 (220 mg, 0.52 mmol) in CH ₂ Cl ₂ (10 ml) was added NEt ₃ (105 mg, 1.04 mmol) followed by Ms ₂ O (109 mg, 0.63 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was quenched with H ₂ O (20 ml). The solution is extracted with CH ₂ Cl ₂ (3×20 ml). The combined extracts were washed with brine (50 ml), dried over MgSO ₄ and concentrated in vacuo. The residue was purified by prep-TLC (hexane:EtOAc=1:3) to give the product (22 mg, 0.044 mmol, Yield=8.4%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.08 (1H, s), 7.02 (1H, d, J=6.2 Hz), 6.51 (1H, s), 6.37 ( 1H, d, J=6.2 Hz), 4.42(1H, s), 2.96(3H, s), 2.21(3H, s), 2.14-2.23(2H, m ), 1.45-2.05(12H, m), 1.49(3H, s), 1.46(3H, s), 1.29(3H, s), 1.12(3H, s) , 1.00-1.06 (1H, m), 1.00 (3H, s). LC-MS: vy=1.52 min, m/z=500.3 [M+H] ⁺ , purity=100% (214.254 nm).

Example 109

To a solution of ERX1077 (880 mg, 2.087 mmol) in MeCN (20 ml) was added a 37% solution of HCHO (7.0 ml) followed by NaBH ₃ CN (656 mg, 10.44 mmol) in portions. The reaction mixture was stirred at room temperature overnight. The solution was diluted with CH ₂ Cl ₂ (200 ml), washed with 0.1 M HCl (320 ml), H ₂ O (3×100 ml), brine (100 ml), dried over MgSO ₄ and concentrated in vacuo to obtain crude intermediate 2 (653 mg, 1.446 mmol, Yield=69%) as a white solid.

¹ H NMR: T242-T242-H1-20160216-SP-0012627-016-CDCL3 δ (400 MHz, CDCl ₃ ): 6.83 (1H, s), 5.77 (1H, d, J=4.5 Hz), 3.29 (1H, dd, J=20.7, 6.2 Hz), 3.01 (1H, dd, J=20.3 Hz), 2.63 (6H, s), 2, 15(3H, s), 2.01(3H, s), 1.25-2.00(17H, m), 1.38(3H, s), 1.23(3H, s), 1.19 (3H, s), 0.67 (3H, s).

To a solution of crude intermediate 2 (600 mg, 1.328 mmol) in CH ₂ Cl ₂ (50 ml) was added Ag ₂ CO ₃ (733 mg, 2.657 mmol). The reaction mixture was stirred at room temperature overnight. The solution is filtered and concentrated in vacuo. The residue was purified by prep-TLC (CH ₂ Cl ₂ :MeOH=5:1) to give the product (86 mg, 0.191 mmol, Yield=14%) as a red solid. ¹ H NMR δ (400 MHz, CDCl ₃ ): 7.02 (1H, dd, J=7.1.1.3 Hz), 6.95 (1H, s), 6.51 (1H, d, J = 7.2 Hz), 6.40 (1H, d, J=1.2 Hz), 2.67 (6H, s), 2.22 (3H, s), 1.47-2.13 (15H , m), 1.46 (3H, s), 1.43 (3H, s), 1.27 (6H, s), 0.85 (3H, s). LC-MS: y=1.27 min, m/z=450.3 [M+H] ⁺ , purity=100% (214.254 nm).

BIOLOGICAL STUDIES: V.B. INTRODUCTION

The following test compounds were tested to determine anti-obesity activity when administered to rodents. Test compounds are first formulated at doses of 50, 100, 200, 400, 500, 1000 and 2000 μg/kg and administered once daily at a dose of 2 ml/kg ip. within a few days. Test compounds are compared with vehicle administration to mice. The experiment consists of two phases: the pre-administration phase and the administration phase. The body weight of the animals in each test group is measured daily. Tables 2-5 summarize the study parameters. Table 6 shows the results of the tested compounds, from the dose and anti-obesity effect.

Table 2: Study parameters

Duration of administration Acclimatization administration - to all 75 animals on days 11-14 of the phase before administration (approximately 100 µl for each animal)
Administration of test compound: daily for 10 days (~15:00) Cooking frequency Daily Verification tube Groups 2-12: prepare 1 day one day before the administration phase (P14) to confirm the dissolution of the compound, this preparation is not administered, but is used to confirm that the compound is suitable for administration on day 1. Test compound storage conditions ≤-60°C, light protection Test dose mixing instructions For test items (groups 2-12):
stock solution: 1:1 solution (v/v) of solutolum in DMAC to dissolve the test compound; the stock solution is stored frozen at -60°C and thawed just before use.
Injection Solution: Dilute the stock solution in an appropriate amount of saline (final vehicle: 10% Solutol, 10% DMAC, 80% saline) on the day of administration. Carrier Groups 1-12 and acclimatization introduction:
Stock: 1:1 solution (v/v) solyutol:DMAC
Solution for injection: 0.9% saline solution
Final vehicle: 10% solyutol, 10% DMAC, 80% saline Dose storage conditions Freezing and protection from light Dose Volume Adjustment Dose calculation based on most typical body weight Covance ACUA Protocol 04811-B View and group Mouse - C57BL/6 diet-induced obesity (AO) Floor Male Source Taconic Vendor nomenclature C57BL/6NTac (AO) Approximate age 20-21 weeks at the start of the study Order Quantity 75 Number of participants in the study 60 Supplier / type of feed TD95217 Feeding details Feed ad libitum, see Lifetime Parameters for fasting details.
Give sufficient food throughout the study on Day 1 of the administration phase. Food fortification Animals do not receive food with special enrichment. Water Greenfield - tap water
Gel plates are not placed with these animals; animals are observed and if they do not take water from the automatic drinker, they are placed on water bottles. Accommodation Individual placement in shoe box type cages with a layer of sawdust and nests. Acclimatization Place in a vivarium for at least 1 week prior to vehicle administration. Environmental conditions Light day: light 05:00-17:00
12 hours light, 12 hours dark (may be interrupted for research activities)
Temperature: 68-79°F
Relative Humidity: 30%-70%

Table 3: pre-administration phase

Acclimatization Introduction Days 11-14 of the pre-administration phase (approximately 15:00).
Note: Inject all 75 animals with vehicle intraperitoneally (approximately 100 µl) autonomously. For intraperitoneal injection, 1/3 cm ³ syringes are used. Clinical signs Mortality is checked twice a day (morning and evening) Non-sampling method On day 14 of the pre-dose phase; animals are randomly selected based on body weight. The BRAT system is used. Body mass Pre-dose body weight is determined daily from day 11 to day 14 of the pre-dose phase for all animals. food intake Determined daily on days 11-14 of the phase before administration
If the intake value is not 0-6 grams within 24 hours, reweigh once and document. Blood glucose level On day 14 of the pre-dose phase (all animals)
Approximately 08:00: Fasting mice in clean shoe box type cages.
Approximately 14:00: All animals have their tail clamped for blood sampling at each time point. Place a drop of blood from each animal into two different Accu Chek Aviva glucometers to assess glucose levels. Record all glucometer readings.

Table 4: Introduction phase

Clinical signs Twice a day (morning and evening) mortality. Record explicit changes Compound introduction Daily at approximately 15:00 every day
(Note: 1/3 cm ³ syringes are used for intraperitoneal injection)
The dose volume is calculated based on the most typical body weight.
Animals are injected one at a time. Body mass Daily food intake Daily (approximately 30 minutes before the start of the dark period)
If the intake value is not 0-6 grams within 24 hours, reweigh once and document. Blood glucose level On Day 11 of the Introduction Phase
Approximately 08:00: Fasting mice in clean shoe box type cages.
Approximately 14:00: Animals have their tail clamped for blood sampling at each time point. Place a drop of blood from each animal into two different Accu Chek Aviva glucometers to assess glucose levels. Record all glucometer readings.

Table 5: Liquid Sample Collection

Sample number Collection type Phase day Time Tube preparation and processing Collection method Sample volume Anticoagulant and/or tube type Storage before processing Centrifuge one Whole blood P 14, 11 After 6 hours of fasting ND tail clip 5 µl Glucometer (twice) ND ND

Abbreviations: P=before administration

Animals are sacrificed with carbon dioxide (or anesthesia), then decapitated, bilateral thoracotomy, phlebotomy, or removal of vital organs to ensure death after the last blood draw or scheduled completion. Displacement of the cervical vertebrae is acceptable in animals that do not require examination or probing.

Any animal that does not survive to the end of the study is discarded without further evaluation.

Carry out descriptive statistics (n, mean, standard error of the mean, standard deviation). Additionally, one-way analysis of variance followed by Dunnett's posterior test is performed for body weight (total change in body weight), glucose (change in percent glucose), and food intake (total food intake). The area under the curve (for body weight, cumulative body weight change, daily body weight change and food intake) is calculated for the treatment groups. As a suitable comparison, a test such as one-way ANOVA followed by Dunnett's post hoc test is performed on group means to calculate area under the curve.

All procedures in this protocol comply with the United States Department of Agriculture (USDA) Animal Welfare Act (9 CFR parts 1, 2, and 3); Guidelines for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, The National Academies Press, Washington, D.C.); and the National Institutes of Health, Laboratory Animal Welfare (for NIH funded research). Where possible, the techniques in this study are designed to avoid or minimize discomfort, stress, and pain in the animals.

This non-clinical laboratory test is not intended to be performed in full compliance with the FDA Code of International Practice for Laboratory Testing (GLP), 21 CFR Part 58, but is performed in accordance with Covance Model Instructions.

This study complies with all applicable sections of the Guidelines for the Care and Use of Laboratory Animals. Where possible, the techniques in this study are designed to avoid or minimize discomfort, stress, and pain in the animals. All methods are described in this study protocol or in written laboratory methods. These techniques are based on most of the current available technologies regarding the correct use and management of laboratory animals.

Table 6-A: in vivo the activity of the compounds (VB introduction).

Compound Structure dose (µg/kg) activity
+++ > Celastrol @ 100 mcg/kg
++=Celastrol @ 100 mcg/kg
+ < Celastrol @ 100 mcg/kg
- inactive
*too active - research completed Oral bioavailability ERX1000

100 ++ ERX1001

200 ++ - ERX1001

100 ++ ERX1002

100 ++ ERX1003

100 ++ + ERX1004

100 - ERX1005

100 - ERX1006

100 +++ ++ ERX1007

100 ++ ERX1008

100 ++ ERX1009

400 ++ ERX1009

1000 * ERX1009

100 - ERX1010

100 +++ - ERX1011

100 - ERX1012

100 - ERX1013

100 - ERX1014

200 ++ ERX1014

100 - ERX1015

100 ++ -- ERX1016-P1

100 - ERX1016-P2

100 - ERX1017

100 - ERX1018

400 ++ ERX1018

100 + ERX1019

100 + ERX1020

100 ++ + ERX1021

100 ++ ERX1022

100 ++ ERX1023

100 ++ ERX1024

100 - ERX1025

100 - ERX1026

100 - ERX1027

100 - ERX1028

100 - ERX1029

100 +++ -- ERX1030

100 - - ERX1031

400 ++ ERX1031

100 - ERX1033

500 - ERX1033

100 - ERX1036

100 ++ + ERX1037

200 ++ - ERX1037

100 + ERX1041

100 - ERX1043

500 - ERX1043

100 - ERX1046

100 ERX1047

400 ++ ERX1047

100 - ERX1050

400 - ERX1050

100 - ERX1056

100 - ERX1058

100 - ERX1060

100 ++ ++ ERX1061

100 - ERX1062

100 - ERX1063

400 ++ ERX1063

100 - ERX1064

400 +++ ERX1064

100 - ERX1066

400 +++ ++ ERX1066

200 ++ ++ ERX1066

100 + ++ ERX1067

100 ++ ERX1068

100 ++ ERX1071

100 - ERX1072

400 +++ ERX1072

200 ++ ERX1072

100 + ERX1073

100 - ERX1076

1000 ++ + ERX1076

400 + + ERX1076

100 - + ERX1077

100 ++ ERX1084

400 - ERX1084

100 - ERX1085

200 - ++ ERX1085

100 - ++ ERX1085

400 ++ ERX1087

400 ++ ERX1088

400 ++ ERX1090

200 - ERX1090

100 - ERX1090

400 ++ ERX1095

2000 - ERX1096

2000 - ERX1096

500 - ERX1097

1500 - ERX1098

2,000 - ERX1098

500 - ERX1101

2000 - ERX1102

fifty + ERX1102

200 +++ ERX1102

100 ++ ERX1102

400 ++ ERX1103

1000 ++ ERX1103

400 - ERX1105

400 +++ ERX1106

2000 - ERX1107

fifty - ERX1107

100 + ERX1107

1000 * ERX1107

400 * ERX1108

100 - ERX1108

1000 * ERX1109

100 - ERX1109

1000 +++ ERX1112

200 - ERX1113

400 - ERX1115

200 + ERX1116

400 ++ ERX1116

100 - ERX1117

400 +++ ERX1119

200 +++ ERX1121

200 ++ ERX1123

200 ++ ERX1124

200 ++ ERX1125

200 - ERX1126

200 ++ ERX1127

200 ++ ERX1129

200 ++ ERX1131

200 ++ ERX1132

200 ++ ERX1136

400 - ERX1138

400 ++ ERX1139

400 ++ ERX1140

400 ++ ERX1142

400 ++ ERX1143

400 - ERX1145

400 +++ ERX1146

400 +++ ERX1147

400 +++ ERX1149

100 +++ ERX1155

400 ++ ERX1166

400 +++ ERX1167

400 +++ ERX1168

100 ++ ERX1168

400 +++ ERX1173

100 ++ ERX1175

400 +++ ERX1177

400 ++ Pristimerin

100 +++

Table 6-B: Compound solubility data.

Connection under test Testing system Solubility (µM) Medium OSO propranolol FRPB (pH 7.4) 97.45 0.01 Ketoconazole FRPB (pH 7.4) 35.70 0.01 Tamoxifen FRPB (pH 7.4) 0.82 0.05 ERX1077 FRPB (pH 7.4) 16.70 0.02 ERX1107 FRPB (pH 7.4) 11.40 0.07 ERX1115 FRPB (pH 7.4) 0.56 0.24 ERX1117 FRPB (pH 7.4) 0.91 0.03 ERX1116 FRPB (pH 7.4) 0.47 0.37 ERX1121 FRPB (pH 7.4) 1.55 0.02 ERX1169 FRPB (pH 7.4) 0.12 0.26 ERX1124 FRPB (pH 7.4) 1.04 0.01 ERX1066 FRPB (pH 7.4) 0.63 0.04 ERX1076 FRPB (pH 7.4) 6.44 0.02 ERX1170 FRPB (pH 7.4) 13.40 0.08 ERX1171 FRPB (pH 7.4) 3.18 0.01 ERX1174 FRPB (pH 7.4) 3.90 0.03 ERX1175 FRPB (pH 7.4) 4.03 0.03 ERX1177 FRPB (pH 7.4) 49.50 0.00 ERX1187 FRPB (pH 7.4) 91.95 0.00 ERX1074(Pristimerin) FRPB (pH 7.4) 4.19 0.07 ERX1188 FRPB (pH 7.4) 3.30 0.05 ERX1037 FRPB (pH 7.4) 23.50 0.02 ERX1047 FRPB (pH 7.4) 1.13 0.09 ERX1060 FRPB (pH 7.4) 1.11 0.03 ERX1006 FRPB (pH 7.4) 1.15 0.02 ERX1015 FRPB (pH 7.4) 0.79 0.06 ERX1020 FRPB (pH 7.4) 0.54 0.45 ERX1029 FRPB (pH 7.4) 1.61 0.11 ERX1031 FRPB (pH 7.4) 8.56 0.03 ERX1000 (Celastrol) FRPB (pH 7.4) 3.89 0.01 ERX1008 FRPB (pH 7.4) 0.08 0.04 ERX1010 FRPB (pH 7.4) 0.64 0.05 ERX1007 FRPB (pH 7.4) 48.75 0.02

N/A: not purchased

Concentration tested: 100 µM (1% DMSO)

Test systems: FRPB (pH 7.4)

Incubation conditions: shaking (1000 rpm) for 1 hour at room temperature

Sample size: duplicates (n=2)

Bioanalytical method: LC-MS/MS

Comments:

As shown in Table 6-B, solubility values less than 10 μM mean that these compounds show low solubility; solubility values from 10 μM to 80 μM mean that these compounds show moderate solubility; solubility values greater than 80 μM mean that these compounds show high solubility.

Table 6-C: Compound solubility data.

Connection under test Testing system Solubility (µM) Medium OSO propranolol FRPB (pH 7.4) >100(107.50) 0.03 Ketoconazole FRPB (pH 7.4) 35.20 0.06 Tamoxifen FRPB (pH 7.4) 1.49 0.16 ERX1168 FRPB (pH 7.4) 5.05 0.06 ERX1172 FRPB (pH 7.4) 0.90 0.27 ERX1173 FRPB (pH 7.4) 1.74 0.01

Concentration tested: 100 µM (1% DMSO)

Test systems: FRPB (pH 7.4)

Incubation conditions: shaking (1000 rpm) for 1 hour at room temperature

Sample size: duplicates (n=2)

Bioanalytical method: LC-MS/MS

As shown in Table 6-B, solubility values less than 10 μM mean that these compounds show low solubility; solubility values from 10 μM to 80 μM mean that these compounds show moderate solubility; solubility values greater than 80 μM mean that these compounds show high solubility.

Table 6-D: Compound Solubility Data

Connection under test Testing system Solubility (µM) OSO propranolol FRPB (pH 7.4) 97.45 0.009432928 Ketoconazole FRPB (pH 7.4) 35.7 0.007922765 Tamoxifen FRPB (pH 7.4) 0.8155 0.047689605 ERX1077 FRPB (pH 7.4) 16.7 0.016936689 ERX1107 FRPB (pH 7.4) 11.4 0.074432293 ERX1115 FRPB (pH 7.4) 0.5575 0.244792123 ERX1117 FRPB (pH 7.4) 0.914 0.030945592 ERX1116 FRPB (pH 7.4) 0.4705 0.368206507 ERX1121 FRPB (pH 7.4) 1.55 0.018247917 ERX1169 FRPB (pH 7.4) 0.1235 0.257650244 ERX1124 FRPB (pH 7.4) 1.035 0.00683195 ERX1066 FRPB (pH 7.4) 0.6275 0.043947672 ERX1076 FRPB (pH 7.4) 6.435 0.023075746 ERX1170 FRPB (pH 7.4) 13.4 0.08443066 ERX1171 FRPB (pH 7.4) 3.175 0.01113554 ERX1174 FRPB (pH 7.4) 3.895 0.03086217 ERX1175 FRPB (pH 7.4) 4.03 0.028073718 ERX1177 FRPB (pH 7.4) 49.5 0.002856997 ERX1187 FRPB (pH 7.4) 91.95 0.003845061 ERX1074(Pristimerin) FRPB (pH 7.4) 4.19 0.067504227 ERX1188 FRPB (pH 7.4) 3.3 0.051425948 ERX1037 FRPB (pH 7.4) 23.5 0.02407172 ERX1047 FRPB (pH 7.4) 1.13 0.08760615 ERX1060 FRPB (pH 7.4) 1.11 0.025481325 ERX1006 FRPB (pH 7.4) 1.145 0.018526815 ERX1015 FRPB (pH 7.4) 0.7915 0.058069413 ERX1020 FRPB (pH 7.4) 0.5405 0.451344754 ERX1029 FRPB (pH 7.4) 1.605 0.110141243 ERX1031 FRPB (pH 7.4) 8.56 0.026433898 ERX1000 (Celastrol) FRPB (pH 7.4) 3.89 0.010906531 ERX1008 FRPB (pH 7.4) 0.0841 0.040358056 ERX1010 FRPB (pH 7.4) 0.644 0.052703611 ERX1007 FRPB (pH 7.4) 48.75 0.01595523

BIOLOGICAL INVESTIGATION: ORAL (PO) INTRODUCTION

1. Essence

The aim of this study is to evaluate test products in alimentary obese (AO) mice.

Male C57BL/6 AO mice are divided into 12 groups and dosed with 2 mg/kg Celastrol (ERX1000-4), ERX1006, ERX1007, ERX1037, ERX1060, ERX1077, ERX1107, ERX1149, ERX1168, ERX1177, and Pristimerin, respectively . Animals are dosed by oral gavage once a day for 10 days in a volume of 2 ml/kg. Two vehicle controls include 1% methylcellulose (400 cps) in citric acid and phosphate buffer.

Parameters assessed include mortality, clinical observations, body weight and food intake, and blood glucose.

All animals survive to the scheduled end of the trial. Statistically significant changes in body weight, food intake and percentage change in glucose values were noted in animals treated with ERX1000-4 and ERX1168. Statistically significant changes in body weight were noted in animals treated with ERX1006.

2. Methods

2.1 Testing system and study plan

2.1.1 Specification and acclimatization of animals

Nutritionally obese (AO) male C57BL/6 mice are obtained from Taconic. Animals are acclimatized to cage conditions for at least 1 week prior to administration of the acclimatizing vehicle. Animals are acclimatized to administration via vehicle oral gavage once daily for 11-14 days of the pre-administration phase.

At the start of administration, the age of the animals ranges from 21 to 22 weeks and the body weight ranges from 29.8 to 40.1 g.

2.1.2 Environmental conditions, food and water

2.1.2.1 Placement in cages

Animals are housed individually in shoebox-type cages with wood chip bedding and nests.

2.1.2.2 Water

Water is given ad libitum .

2.1.2.3 Power

Animals are offered TD95217 ad libitum until fasting is required for study procedures.

2.1.2.4 Environment

Environmental controls are set to maintain the following conditions in animal cages: temperature 68 to 79°F, relative humidity 30 to 70%, and a 12 hour day/night cycle. Any changes to these conditions are stored in the original data and do not affect the result of the study.

2.1.2.5 Food fortification

Animals are not given specially fortified food.

2.1.3 Animal identification and distribution for research

Animals are identified by indelible paint on the tail and a plate on the cage.

Animals are distributed for study using a computerized method designed to achieve a balance of body weight relative to group distribution.

2.1.4 Study design: Table 7

Group Tested product Dose (mg/kg) Animal numbers one Carrier 0 1-5 2 ERX1000-4 2 6-10 3 ERX1006 2 11-15 four ERX1007 2 16-20 5 ERX1037 2 21-25 6 ERX1060 2 26-30 7 ERX1077 2 31-35 eight ERX1107 2 36-40 9 ERX1149 2 41-45 ten ERX1168 2 46-50 eleven ERX1177 2 51-55 12 Pristimerin 2 56-60

2.2 Test product and carrier

2.2.1 Test product

Tested product Storage ERX1000-4 Store at ≤-60°C, Protect from light ERX1006 Store at ≤-60°C, Protect from light ERX1007 Store at ≤-60°C, Protect from light ERX1037 Store at ≤-60°C, Protect from light ERX1060 Store at ≤-60°C, Protect from light ERX1077 Store at ≤-60°C, Protect from light ERX1107 Store at ≤-60°C, Protect from light ERX1149 Store at ≤-60°C, Protect from light ERX1168 Store at ≤-60°C, Protect from light ERX1177 Store at ≤-60°C, Protect from light Pristimerin Store at ≤-60°C, Protect from light

2.2.2 Media

The carrier is 1% methylcellulose (400 cps) in citric acid and phosphate buffer.

2.2.3 Compilation of the product under test

The compositions of the test product are prepared twice by mixing. Dose concentrations are based on the product tested, as indicated.

The dosage compositions are stored frozen and protected from light.

2.3 Lifetime techniques

2.3.1 Dose administration

Dosed compositions are administered via oral gavage once a day for 10 days in a dose volume of 2 ml/kg. Doses are based on the most recently recorded target body weight.

2.3.2 Body weights

Body weights are recorded daily on days 11-14 of the pre-dose phase. Body weights are collected for all animals daily during the administration phase prior to administration.

2.3.3 Food intake

Quantification of food intake is recorded daily on days 11-14 of the pre-administration phase during the administration phase.

2.3.4 Blood glucose level

Blood for blood glucose determination is collected from all animals via a tail clip on day 14 of the pre-administration phase and on day 11 of the administration phase after 6 hours of fasting. A drop of blood from each animal is placed into two different Accu-Chek® Aviva glucometers to assess glucose levels. Take the third value, if necessary.

2.4 Final procedures

All animals are anesthetized with carbon dioxide, sacrificed and discarded without further evaluation on day 11.

2.5 Data evaluation and statistical analysis

Conduct descriptive statistics (n, mean, standard error of the mean, standard deviation). Additionally, one-way analysis of variance followed by Dunnett's posterior test is performed for body weight (total change in body weight), glucose (change in percent glucose), and food intake (total food intake). The area under the curve (for body weight, cumulative body weight change, daily body weight change and food intake) is calculated for the treatment groups. As a suitable comparison, a test such as one-way ANOVA followed by Dunnett's post hoc test is performed on group means to calculate area under the curve. Any additional statistical analysis and interpretation of results is the responsibility of the sponsor.

3. Results

3.1 Lifetime assessments

3.1.1 Animal treatment outcome

The result of treatment of animals is presented in tables 11-1-11-12.

All animals survived to the planned end of the study.

3.1.2 Clinical observations

Changes in clinical conditions were not noted for any of the treated groups.

3.1.3 Body weights

The body weight data are summarized in Tables 9-1-9-12 and in FIG. 1-5; individual data are presented in tables 12-1-12-36 and in figures 10-12 (daily body weight, daily body weight change and total body weight change).

ERX1000-4 has a statistically significant reduction in area under the curve (AUC) for daily and cumulative body weight change compared to controls.

ERX1168 has a statistically significant reduction in area under the curve (AUC) for daily and cumulative body weight change and cumulative body weight change compared to controls.

ERX1008 has statistically significant reductions in total body weight change compared to controls.

3.1.4 Food intake

Food consumption is summarized in tables 10-1-10-6 and in FIG. 6-7; individual data are shown in Fig. 13-14.

ERX1000-4 has statistically significant reductions in total food intake compared to controls.

ERX1168 has a statistically significant reduction in area under the curve (AUC) for total food intake and total food intake compared to controls.

3.1.5 Blood glucose level

The blood glucose data are summarized in FIG. 8-9; individual data are shown in Fig. 15-17.

ERX1000-4 has statistically significant reductions in percentage change in glucose values compared to controls.

ERX1168 has statistically significant reductions in percent change in glucose values compared to controls.

4. Related information about the study

4.1 Deviations from the study

4.1.1 Deviations from the protocol

Table 8

Procedure Protocol deviations Testing system and study plan Dose Administration On day 6, food intake, body weight and dose are determined from 15:53 to 16:24. This departure from the study does not affect the overall interpretation of the study results, nor does it compromise the integrity of the study.

5. RESULTS

Table 9: Results in vivo activity of the compounds (P.O. introduction).

Compound Structure Dose (µg/kg) activity
+++>Celastrol @2000 mcg/kg
++=Celastrol @2000 mcg/kg
+<Celastrol @2000 mcg/kg
- not active ERX1000-4 (Celastrol)

2000 ++ ERX1006

2000 ++ ERX1007

2000 + ERX1037

2000 - ERX1060

2000 - ERX1077

2000 - ERX1107

2000 + ERX1149

2000 + ERX1168

2000 +++ ERX1177

2000 + Pristimerin

2000 -

5.1 TOTAL BODY WEIGHT

Table 9-1

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Data are presented in "d" Phase DSNG Day one 2 3 four 5 6 1M Average 34.3 34.5 33.9 33.4 33.8 33.5 SO 2.33 2.48 2.29 3.01 2.58 2.68 N 5 5 5 5 5 5 2/M Average 34.2 34.0 33.1 32.5 32.1 31.4 SO 2.27 2.09 2.08 2.59 2.79 2.43 N 5 5 5 5 5 5 %-diff. 0% -one% -2% -3% -5% -6%

Table 9-2

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Data are presented in "d" Phase DSNG Day one 2 3 four 5 6 3M Average 34.1 33.9 33.4 32.8 32.6 32.5 SO 1.49 1.91 1.83 2.25 2.11 1.90 N 5 5 5 5 5 5 %-diff. -one% -2% -one% -2% -four% -3% 4/M Average 34.5 34.1 33.3 33.3 33.1 32.4 SO 2.02 2.50 2.25 2.03 2.13 2.39 N 5 5 5 5 5 5 %-diff. one% -one% -2% 0% -2% -3%

Table 9-3

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Data are presented in "d" Phase DSNG Day one 2 3 four 5 6 5/M Average 35.0 34.8 34.6 34.2 34.3 34.5 SO 2.73 2.56 2.77 2.92 2.83 2.81 N 5 5 5 5 5 5 %-diff. 2% one% 2% 2% one% 3% 6/M Average 34.6 34.1 34.4 34.1 34.2 34.4 SO 2.81 3.05 2.54 2.63 2.48 2.75 N 5 5 5 5 5 5 %-diff. one% -one% one% 2% one% 3%

Table 9-4

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Data are presented in "d" Phase DSNG Day one 2 3 four 5 6 7/M Average 34.4 34.1 34.0 33.7 33.7 34.0 SO 2.57 2.72 2.44 2.59 2.60 2.62 N 5 5 5 5 5 5 %-diff. 0% -one% 0% one% 0% one% 8/M Average 34.2 34.3 33.4 33.2 33.1 32.7 SO 2.02 2.31 1.97 2.05 2.29 2.25 N 5 5 5 5 5 5 %-diff. 0% -one% -one% -one% -2% -2%

Table 9-5

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Data are presented in "d" Phase DSNG Day one 2 3 four 5 6 9/M Average 34.9 34.5 34.1 34.0 33.5 33.1 SO 3.10 3.26 3.44 3.63 3.63 3.74 N 5 5 5 5 5 5 %-diff. 2% 0% one% 2% -one% -one% 10/M Average 34.5 34.0 32.2 31.9 31.1 29.5 SO 3.57 4.26 3.58 3.78 3.46 3.13 N 5 5 5 5 5 5 %-diff. one% -one% -5% -four% -eight% -12%

Table 9-6

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Data are presented in "d" Phase DSNG Day one 2 3 four 5 6 11/M Average 34.9 34.4 34.2 33.7 33.8 33.5 SO 3.30 2.61 2.64 2.83 2.86 3.08 N 5 5 5 5 5 5 %-diff. 2% 0% one% one% 0% 0% 12/M Average 34.2 34.0 33.7 33.3 33.3 33.4 SO 3.84 3.53 4.22 4.45 4.63 4.87 N 5 5 5 5 5 5 %-diff. 0% -one% -one% 0% -one% 0%

Table 9-7

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Data are presented in "d" Phase DSNG Day 7 eight 9 ten eleven 1M Average 33.5 33.8 33.2 33.5 34.1 SO 2.51 2.64 3.05 3.15 2.77 N 5 5 5 5 5 2/M Average 30.6 30.4 30.3 30.0 29.1 SO 2.50 2.36 2.41 2.57 2.41 N 5 5 5 5 5 %-diff. -9% -ten% -9% -ten% -fifteen%

Table 9-8

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Data are presented in "d" Phase DSNG Day 7 eight 9 ten eleven 3M Average 32.0 31.7 31.4 31.2 30.9 SO 2.04 2.18 2.05 2.39 2.34 N 5 5 5 5 5 %-diff. -four% -6% -5% -7% -9% 4/M Average 32.4 32.7 32.3 32.1 32.3 SO 2.21 2.35 2.56 2.83 2.98 N 5 5 5 5 5 %-diff. -3% -3% -3% -four% -5%

Table 9-9

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Data are presented in "d" Phase DSNG Day 7 eight 9 ten eleven 5/M Average 35.0 34.8 34.3 34.8 34.9 SO 2.66 3.05 3.45 3.43 3.43 N 5 5 5 5 5 %-diff. four% 3% 3% four% 2% 6/M Average 33.8 33.3 33.1 33.5 34.4 SO 2.90 2.73 3.29 3.26 3.28 N 5 5 5 5 5 %-diff. one% -one% 0% 0% one%

Table 9-10

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/Gender Data are presented in "d" Phase DSNG Day 7 eight 9 ten eleven 7/M Average 33.3 33.7 33.5 33.8 33.9 SO 2.52 2.29 2.13 2.42 2.71 N 5 5 5 5 5 %-diff. -one% 0% one% one% -one% 8/M Average 32.4 32.3 32.2 32.4 32.7 SO 2.53 2.38 2.35 2.85 2.40 N 5 5 5 5 5 %-diff. -3% -four% -3% -3% -four%

Table 9-11

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Data are presented in "d" Phase DSNG Day 7 eight 9 ten eleven 9/M Average 32.8 33.3 33.1 33.5 33.6 SO 3.90 3.69 3.81 3.28 4.01 N 5 5 5 5 5 %-diff. -2% -one% 0% 0% -one% 10/M Average 29.2 29.1 30.0 29.2 29.2 SO 2.26 2.24 2.50 3.56 3.34 N 5 5 5 5 5 %-diff. -13% -fourteen% -ten% -13% -fourteen%

Table 9-12

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Data are presented in "d" Phase DSNG Day 7 eight 9 ten eleven 11/M Average 33.4 33.1 33.0 33.1 33.0 SO 3.08 3.08 3.29 3.17 3.49 N 5 5 5 5 5 %-diff. 0% -2% -one% -one% -3% 12/M Average 33.5 33.1 32.9 33.2 33.9 SO 4.77 4.91 5.05 4.81 5.01 N 5 5 5 5 5 %-diff. 0% -2% -one% -one% -one%

5.2 TOTAL FOOD CONSUMPTION

Table 10-1

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Data are presented in "g/animal/day" X-X interval Phase PRED Day 11-12 12-13 13-14 1M Average 3.5 1.9 1.6 SO 0.74 0.38 0.67 N 5 5 5 2/M Average 3.0 2.0 1.9 SO 1.68 1.25 0.55 N 5 5 5 %-diff. -fifteen% 9% 16%

Table 10-2

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Data are presented in "g/animal/day" X-X interval Phase PRED Day 11-12 12-13 13-14 3M Average 3.8 2.7 1.8 SO 0.36 0.61 0.46 N 5 5 5 %-diff. 9% 45% ten% 4/M Average 2.8 2.4 1.9 SO 0.92 0.80 0.50 N 5 5 5 %-diff. -19% thirty% 17%

Table 10-3

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/Gender Data are presented in "g/animal/day" X-X interval Phase PRED Day 11-12 12-13 13-14 5/M Average 4.1 2.9 2.2 SO 2.19 0.81 0.38 N 5 5 5 %-diff. eighteen% 53% 33% 6/M Average 2.6 1.5 1.2 SO 0.81 1.24 0.52 N 5 5 5 %-diff. -25% -19% -26%

Table 10-4

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Data are presented in "g/animal/day" X-X interval Phase PRED Day 11-12 12-13 13-14 7/M Average 2.9 2.5 2.2 SO 0.27 0.66 0.63 N 5 5 5 %-diff. -17% 32% 38% 8/M Average 3.5 2.9 2.0 SO 1.99 1.28 0.35 N 5 5 5 %-diff. one% 52% 23%

Table 10-5

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Data are presented in "g/animal/day" X-X interval Phase PRED Day 11-12 12-13 13-14 9/M Average 3.3 2.3 1.9 SO 0.62 0.30 0.54 N 5 5 5 %-diff. -5% 21% 16% 10/M Average 2.5 2.5 2.4 SO 1.88 1.05 0.97 N 5 5 5 %-diff. -thirty% 31% 47%

Table 10-6

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Data are presented in "g/animal/day" X-X interval Phase PRED Day 11-12 12-13 13-14 11/M Average 3.9 2.4 1.8 SO 0.82 0.70 0.43 N 5 5 5 %-diff. ten% 27% eleven% 12/M Average 2.6 2.5 2.3 SO 0.67 0.11 0.48 N 5 5 5 %-diff. -25% 32% 40%

5.3 TABLES WITH INDIVIDUAL DATA OF ANIMALS

Table 11-1: Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Result phase Phase Week Phase day Result status Final body weight (g) 1M one Introduction 2 eleven Planned killing and release - 2 Introduction 2 eleven Planned killing and release - 3 Introduction 2 eleven Planned killing and release - four Introduction 2 eleven Planned killing and release - 5 Introduction 2 eleven Planned killing and release -

Table 11-2

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Result phase Phase week Phase day Result status Final body weight (g) 2/M ten Introduction 2 eleven Planned killing and release - 6 Introduction 2 eleven Planned killing and release - 7 Introduction 2 eleven Planned killing and release - eight Introduction 2 eleven Planned killing and release - 9 Introduction 2 eleven Planned killing and release -

Table 11-3

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/Gender Animal number Result phase Phase Week Phase day Result status Final body weight (g) 3M eleven Introduction 2 eleven Planned killing and release - 12 Introduction 2 eleven Planned killing and release - 13 Introduction 2 eleven Planned killing and release - fourteen Introduction 2 eleven Planned killing and release - fifteen Introduction 2 eleven Planned killing and release -

Table 11-4

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Result phase Phase week Phase day Result status Final body weight (g) 4/M 16 Introduction 2 eleven Planned killing and release - 17 Introduction 2 eleven Planned killing and release - eighteen Introduction 2 eleven Planned killing and release - 19 Introduction 2 eleven Planned killing and release - twenty Introduction 2 eleven Planned killing and release -

Table 11-5

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Result phase Phase week Phase day Result status Final body weight (g) 5/M 21 Introduction 2 eleven Planned killing and release - 22 Introduction 2 eleven Planned killing and release - 23 Introduction 2 eleven Planned killing and release - 24 Introduction 2 eleven Planned killing and release - 25 Introduction 2 eleven Planned killing and release -

Table 11-6

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Result phase Phase week Phase day Result status Final body weight (g) 6/M 26 Introduction 2 eleven Planned killing and release - 27 Introduction 2 eleven Planned killing and release - 28 Introduction 2 eleven Planned killing and release - 29 Introduction 2 eleven Planned killing and release - thirty Introduction 2 eleven Planned killing and release -

Table 11-7

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Result phase Phase Week Phase day Result status Final body weight (g) 7/M 31 Introduction 2 eleven Planned killing and release - 32 Introduction 2 eleven Planned killing and release - 33 Introduction 2 eleven Planned killing and release - 34 Introduction 2 eleven Planned killing and release - 35 Introduction 2 eleven Planned killing and release -

Table 11-8

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Result phase Phase week Phase day Result status Final body weight (g) 8/M 36 Introduction 2 eleven Planned killing and release - 37 Introduction 2 eleven Planned killing and release - 38 Introduction 2 eleven Planned killing and release - 39 Introduction 2 eleven Planned killing and release - 40 Introduction 2 eleven Planned killing and release -

Table 11-9

8351983

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Result phase Phase week Phase day Result status Final body weight (g) 9/M 41 Introduction 2 eleven Planned killing and release - 42 Introduction 2 eleven Planned killing and release - 43 Introduction 2 eleven Planned killing and release - 44 Introduction 2 eleven Planned killing and release - 45 Introduction 2 eleven Planned killing and release -

Table 11-10

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Result phase Phase week Phase day Result status Final body weight (g) 10/M 46 Introduction 2 eleven Planned killing and release - 47 Introduction 2 eleven Planned killing and release - 48 Introduction 2 eleven Planned killing and release - 49 Introduction 2 eleven Planned killing and release - fifty Introduction 2 eleven Planned killing and release -

Table 11-11

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Result phase Phase Week Phase day Result status Final body weight (g) 11/M 51 Introduction 2 eleven Planned killing and release - 52 Introduction 2 eleven Planned killing and release - 53 Introduction 2 eleven Planned killing and release - 54 Introduction 2 eleven Planned killing and release - 55 Introduction 2 eleven Planned killing and release -

Table 11-12

Result for an individual animal

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/Gender Animal number Result phase Phase Week Phase day Result status Final body weight (g) 12/M 56 Introduction 2 eleven Planned killing and release - 57 Introduction 2 eleven Planned killing and release - 58 Introduction 2 eleven Planned killing and release - 59 Introduction 2 eleven Planned killing and release - 60 2Introduction 2 eleven Planned killing and release -

Table 12-1: Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 1M one 34.9 34.1 33.5 34.4 33.0 32.1 2 35.3 36.2 35.2 35.5 35.2 34.8 3 38.7 37.6 37.0 37.1 34.4 35.7 four 40.1 38.1 37.9 38.0 37.5 37.9 5 34.3 34.1 33.2 33.0 31.3 32.1

Table 12-2

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 10/M 46 35.6 35.2 35.0 35.1 33.1 32.5 47 39.3 40.3 40.4 41.2 39.5 40.4 48 37.3 37.2 37.2 37.5 36.9 36.2 49 35.3 33.1 32.5 33.3 31.9 31.2 fifty 33.0 31.0 30.6 32.1 31.1 29.9

Table 12-3

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 11/M 51 33.8 32.5 31.8 31.7 32.0 32.2 52 33.3 33.7 32.6 33.1 32.1 31.9 53 37.1 36.7 35.6 36.4 35.5 34.5 54 41.9 41.6 42.2 41.7 40.1 38.4 55 35.5 36.2 36.1 36.2 35.0 34.9

Table 12-4

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 12/M 56 42.4 41.2 41.3 41.9 40.0 39.4 57 35.2 34.5 33.4 35.0 33.3 33.1 58 31.7 31.0 31.4 31.6 29.8 30.2 59 38.2 37.5 37.3 37.6 35.5 35.4 60 33.9 32.5 32.9 33.1 32.3 32.1

Table 12-5

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/Gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 2/M ten 35.9 35.2 35.1 35.5 34.6 34.4 6 35.1 33.5 32.5 32.9 30.8 30.8 7 37.3 37.4 37.7 37.9 37.0 36.4 eight 34.5 35.5 34.0 34.6 33.5 33.5 9 37.5 37.3 36.0 37.2 34.9 35.0

Table 12-6

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/Gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 3M eleven 37.6 38.0 37.4 38.5 35.7 35.0 12 34.5 35.2 35.4 35.4 35.0 35.6 13 32.2 32.7 32.5 32.7 32.7 31.8 fourteen 35.2 34.8 34.1 34.3 32.4 31.8 fifteen 36.7 36.8 36.3 36.9 34.9 35.2

Table 12-7

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 4/M 16 36.8 35.2 34.1 34.2 33.8 33.2 17 39.8 38.9 38.7 38.6 36.3 36.4 eighteen 35.7 35.7 35.3 35.2 34.7 34.2 19 32.5 31.7 32.3 32.7 31.4 30.3 twenty 37.6 36.9 36.5 37.3 36.2 36.2

Table 12-8

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 5/M 21 36.0 36.7 35.4 35.7 35.1 36.4 22 38.7 38.8 38.8 39.2 39.1 38.4 23 31.8 33.1 33.4 32.7 32.0 32.4 24 37.3 36.2 36.9 36.7 35.7 34.1 25 36.1 33.6 33.4 34.0 33.1 32.7

Table 12-9

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 6/M 26 36.2 34.5 33.2 33.7 32.9 33.2 27 38.2 36.6 34.7 35.1 34.3 34.1 28 35.9 34.9 33.5 32.7 31.3 29.8 29 40.9 41.2 39.8 40.6 38.7 38.2 thirty 38.9 39.0 37.1 37.4 35.6 35.2

Table 12-10

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 7/M 31 32.6 33.2 31.6 32.7 31.5 31.2 32 34.8 34.0 33.1 33.6 32.8 32.5 33 36.1 35.8 35.6 35.7 34.5 34.1 34 40.2 40.4 39.7 40.6 38.3 38.4 35 38.0 37.0 36.9 36.6 34.9 34.4

Table 12-11

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 8/M 36 34.1 35.1 34.9 35.1 33.6 33.5 37 36.6 37.5 38.0 37.5 35.8 36.5 38 37.7 37.9 37.6 37.7 36.3 36.5 39 36.2 33.2 31.9 32.5 31.2 31.0 40 34.1 34.6 33.8 34.6 34.1 33.9

Table 12-12

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group/Gender Animal number Phase Data are presented in "d" Day PRED 11 PRED 12 PRED 13 PRED 14 DSNG 1 DSNG 2 9/M 41 33.3 33.3 33.1 33.5 33.3 32.2 42 38.3 37.3 36.8 36.1 35.8 34.8 43 41.4 41.5 41.4 41.0 39.7 39.9 44 33.7 33.4 32.3 32.5 31.5 31.7 45 35.6 36.3 35.2 36.5 34.3 34.1

Table 12-13

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 1M one 32.1 31.5 31.0 30.3 30.9 30.6 2 34.3 34.0 34.5 34.8 35.0 35.6 3 35.6 36.0 36.1 35.3 35.4 36.2 four 36.5 36.2 36.3 36.1 35.7 35.2 5 31.1 29.2 31.2 30.9 30.7 31.2

Table 12-14

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 10/M 46 30.7 30.0 29.2 28.4 28.9 28.7 47 37.3 37.5 36.1 33.9 32.3 32.7 48 34.4 34.2 33.2 31.6 30.7 29.5 49 30.5 29.1 28.8 27.0 27.0 27.7 fifty 28.3 28.9 28.0 26.7 27.3 26.9

Table 12-15

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 11/M 51 31.8 31.2 31.3 31.4 31.4 31.0 52 31.8 30.8 30.9 30.1 29.9 29.8 53 34.6 34.8 34.2 33.7 33.7 33.4 54 38.2 37.7 38.0 38.1 38.0 37.8 55 34.6 34.2 34.4 34.4 34.1 33.7

Table 12-16

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 12/M 56 40.1 40.3 40.3 40.9 40.7 40.3 57 32.6 32.4 32.3 32.2 31.6 31.2 58 29.2 29.0 28.6 28.7 29.1 28.2 59 35.3 34.7 35.1 35.1 35.8 35.8 60 31.2 30.3 30.1 29.9 30.2 30.0

Table 12-17

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 2/M ten 33.3 32.7 31.1 30.6 29.8 30.2 6 30.3 29.2 30.1 29.6 28.4 27.8 7 36.0 36.3 37.0 35.4 34.6 34.2 eight 32.2 31.4 30.7 29.5 28.9 29.4 9 33.5 33.1 31.7 31.8 31.4 30.3

Table 12-18

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 3M eleven 33.4 31.9 32.1 31.5 30.4 29.2 12 35.1 35.7 35.6 34.8 34.3 34.2 13 32.1 32.0 31.7 31.8 31.1 31.5 fourteen 31.2 30.0 30.0 30.3 30.0 30.1 fifteen 35.4 34.4 33.6 34.2 34.0 33.7

Table 12-19

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 4/M 16 32.4 32.9 32.1 31.9 32.0 31.8 17 35.7 35.1 35.4 34.8 34.7 34.8 eighteen 33.1 32.8 32.4 31.7 31.6 32.0 19 30.2 30.4 30.4 29.0 29.4 29.6 twenty 35.3 35.4 35.1 34.6 34.5 35.3

Table 12-20

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 5/M 21 35.5 34.6 34.9 35.8 36.1 36.2 22 39.0 38.9 38.6 38.6 38.8 39.0 23 32.4 31.3 31.5 31.7 32.7 31.3 24 33.3 33.5 34.7 34.4 35.1 34.9 25 32.6 32.5 32.0 32.2 32.3 32.5

Table 12-21

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 6/M 26 32.7 33.4 33.3 33.6 32.2 32.0 27 34.6 33.1 33.5 32.5 32.2 31.9 28 31.3 30.9 31.2 31.5 30.7 30.2 29 37.8 38.0 37.8 38.1 37.3 36.5 thirty 35.7 35.0 35.4 36.4 36.4 35.8

Table 12-22

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 7/M 31 31.3 31.3 31.1 31.9 31.1 32.0 32 32.4 31.5 31.7 32.3 31.3 32.1 33 34.3 33.6 33.4 33.1 33.0 33.1 34 37.7 37.6 37.6 38.4 37.3 37.6 35 34.4 34.7 34.7 34.1 34.0 33.7

Table 12-23

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 8/M 36 31.9 32.0 32.0 31.4 31.2 30.8 37 35.6 35.4 35.2 34.7 34.6 34.9 38 35.0 34.5 34.7 34.7 34.7 33.9 39 31.0 30.3 29.6 29.5 28.7 29.0 40 33.7 33.9 33.9 33.3 33.0 32.8

Table 12-24

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 3 DSNG 4 DSNG 5 DSNG 6 DSNG 7 DSNG 8 9/M 41 31.2 31.0 30.8 30.6 30.3 30.5 42 35.0 35.5 35.1 34.3 34.4 34.5 43 39.5 39.6 39.0 38.9 38.8 38.9 44 31.1 31.1 30.1 29.4 29.0 29.6 45 33.8 32.7 32.5 32.2 31.5 32.8

Table 12-25

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 9 DSNG 10 DSNG 11 1M one 29.8 29.7 30.7 2 35.4 35.8 35.5 3 35.6 35.8 36.7 four 35.4 35.9 36.0 5 30.0 30.5 31.5

Table 12-26

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 9 DSNG 10 DSNG 11 10/M 46 29.5 28.3 28.9 47 34.1 34.6 34.0 48 30.4 30.7 30.9 49 27.8 27.2 26.0 fifty 28.3 25.4 26.3

Table 12-27

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 9 DSNG 10 DSNG 11 11/M 51 31.0 31.2 31.3 52 29.4 29.5 28.8 53 33.3 33.8 34.0 54 38.1 37.9 38.2 55 33.4 33.0 32.6

Table 12-28

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "g" Day DSNG 9 DSNG 10 DSNG 11 12/M 56 40.6 40.2 41.4 57 31.4 31.3 32.3 58 28.2 28.6 29.6 59 35.2 35.9 36.4 60 29.3 29.8 29.8

Table 12-29

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 9 DSNG 10 DSNG 11 2/M ten 30.8 30.1 28.7 6 27.4 27.5 26.9 7 33.9 33.7 32.9 eight 29.0 27.7 27.3 9 30.4 31.1 29.8

Table 12-30

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "g" Day DSNG 9 DSNG 10 DSNG 11 3M eleven 28.9 28.2 27.6 12 33.7 34.4 33.9 13 31.2 31.9 31.4 fourteen 30.1 29.6 29.8 fifteen 33.3 31.9 31.6

Table 12-31

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 9 DSNG 10 DSNG 11 4/M 16 31.2 30.9 30.8 17 34.6 34.7 35.3 eighteen 31.3 30.7 31.3 19 29.1 28.9 28.7 twenty 35.2 35.5 35.5

Table 12-32

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 9 DSNG 10 DSNG 11 5/M 21 36.2 36.1 36.5 22 39.0 39.6 39.7 23 31.0 30.8 31.2 24 34.5 35.2 35.0 25 31.0 32.3 32.1

Table 12-33

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 9 DSNG 10 DSNG 11 6/M 26 31.1 30.6 31.5 27 31.6 32.4 32.7 28 29.8 30.8 32.0 29 37.5 38.0 38.9 thirty 35.7 35.8 36.8

Table 12-34

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 9 DSNG 10 DSNG 11 7/M 31 32.1 31.9 31.9 32 31.5 31.7 31.3 33 33.4 33.4 33.3 34 37.0 37.7 38.1 35 33.4 34.1 34.7

Table 12-35

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 9 DSNG 10 DSNG 11 8/M 36 30.0 30.0 30.1 37 34.6 35.2 35.4 38 34.3 34.5 34.7 39 29.6 28.8 30.5 40 32.7 33.6 33.0

Table 12-36

Individual body weight

Tested product (dose) one 2 3 four 5 6 7 eight 9 ten eleven 12 Carrier mg/kg 0 - - - - - - - - - - - ERX1000-4 mg/kg - 2 - - - - - - - - - - ERX1006 mg/kg - - 2 - - - - - - - - - ERX1007 mg/kg - - - 2 - - - - - - - - ERX1037 mg/kg - - - - 2 - - - - - - - ERX1060 mg/kg - - - - - 2 - - - - - - ERX1077 mg/kg - - - - - - 2 - - - - - ERX1107 mg/kg - - - - - - - 2 - - - - ERX1149 mg/kg - - - - - - - - 2 - - - ERX1168 mg/kg - - - - - - - - - 2 - - ERX1177 mg/kg - - - - - - - - - - 2 - Pristimerin mg/kg - - - - - - - - - - - 2

Group / gender Animal number Phase Data are presented in "d" Day DSNG 9 DSNG 10 DSNG 11 9/M 41 31.1 31.8 30.7 42 34.4 34.3 36.1 43 38.9 38.6 39.1 44 28.9 29.9 29.3 45 32.0 32.8 32.6

6. PROTOCOL

6.1 Test products and study design

Table 13

Study plan

Group Tested product Dose (mg/kg) Animal numbers one Carrier 0 0-5 2 ERX1000 2 6-10 3 ERX1006 2 11-15 four ERX1007 2 16-20 5 ERX1037 2 21-25 6 ERX1060 2 26-30 7 ERX1077 2 31-35 eight ERX1107 2 36-40 9 ERX1149 2 41-45 ten ERX1168 2 46-50 eleven ERX1177 2 51-55 12 Pristimerin 2 56-60

Vehicle contains 1% methylcellulose (400 cps) in citric acid/phosphate buffer

6.2. Dose formulation details

Table 14

Acclimatization Introduction All 75 animals are given 0.9% saline (approximately 0.1 ml per animal) daily on days 11-14 of the pre-dose phase. The 0.9% saline solution is distributed as a batch. Dose Administration Daily (1-10 days) Dose volume 2 ml/kg Dose route oral probe Cooking frequency Preparations are prepared twice during the study (each for 5 days) and divided into daily aliquots.
Preparations can be prepared up to 24 hours in advance. Media mixing instructions Ingredients: citric acid monohydrate (4.204 mg/ml), sodium phosphate dibasic heptahydrate (4.124 mg/ml), purified water, methylcellulose (400 cps) (1%)
The purified water is heated to about 75°C. Dissolve citric acid and sodium phosphate dibasic heptahydrate in purified water. Add methylcellulose. Stir. Cool down. Bring to final volume with cold purified water, mix. Measure and record the pH. pH should be 4±0.2. If the pH does not fall within the appropriate range, adjust if necessary with HCl or NaOH. Media Storage Conditions Store frozen for up to 7 days. Storage conditions of the tested product Store at ≤-60°C, protect from light. Verification tube Verification tube is not made. Dose Mixing Instructions Groups 2-12
Weigh a suitable amount of the test product into a mixing container. Add a small amount of carrier and mix with a spatula to obtain a homogeneous paste. Add the remaining media to the final volume. Mix with a homogenizer, if necessary, to obtain a homogeneous suspension or solution.
Divide the drug batch into daily aliquots. Storage conditions of the drug Frozen and protected from light. Elimination of the drug After completion of in vivo testing, the remaining doses of the drug are disposed of. Return shipment of test product Covance is holding the remaining test product for possible use in future research.

6.3 Detailed description of the test animals and their conditions of detention

Table 15

Covance ACUA Protocol 04811-B View and group Mouse, C57BL/6 diet-induced obesity (AO) Floor Male Source Taconic Vendor nomenclature C57BL/6NTac (AO) Approximate age 21-22 weeks before the start of the study Order Quantity 75 Number of participants in the study 60 Supplier / type of feed TD95217 Feeding details Feed ad libitum, see Lifetime Parameters for fasting details.
Give sufficient food throughout the study on Day 1 of the administration phase. Food fortification Animals do not receive food with special enrichment. Water Greenfield - tap water
Gel plates are not placed with these animals; animals are observed and if they do not take water from the automatic drinker, they are placed on water bottles. Accommodation Individual placement in shoe box type cages with a layer of sawdust and nests. Acclimatization Animals are acclimatized to housing conditions for at least 1 week prior to the introduction of the acclimatizing vehicle. Environmental conditions Light day: light 05:00-17:00
12 hours light, 12 hours dark (may be interrupted for research activities)
Temperature: 68-79°F
Relative Humidity: 30%-70%

6.4 Lifetime parameters

6.4.1 Pre-administration phase

Table 16

Acclimatization Introduction Days 11-14 (approximately at 15:00)
Note: all 75 animals are administered an autonomous acclimatization dose Clinical Observations Check for dead or dying animals daily. Record abnormal changes. Randomization On day 14, animals are manually excluded and randomized based on body weight. Body mass Body weights are determined daily on days 11-14 for all animals at approximately 15:00 food intake Food intake is determined daily on days 11-14 of the pre-dose phase at approximately 15:00
If consumption is not 0-6 grams in 24 hours, reweigh once and record. Glucose measurements Day 14
Approximately 08:00: Fasting mice in clean shoe box type cages.
Approximately 2:00 pm: Approximately 5 µl of blood is collected through a tail clamp and blood glucose is measured using Aviva blood glucose meters. Measurements with glucometers are carried out twice. If the values differ by more than 20 mg/dl (the calculated value of the meter), then record the triple value. Animals are fed after the last scheduled blood collection with a glucometer.

6.4.2. Insertion phase

Table 17

Clinical Observations Check for dead or dying animals daily. Record abnormal changes. Product Test Introduction Daily at 15:00 (± 30 minutes)
The dose volume is calculated based on the most typical body weight.
Animals are administered in order. Body mass Daily at 15:00 (± 30 minutes) (before administration) food intake Daily at 15:00 (± 30 minutes) (before administration)
If the intake value is not 0-6 grams within 24 hours, reweigh once and document. Glucose measurements Day 11
Approximately 08:00: Fasting mice in clean shoe box type cages.
Approximately 2:00 pm: Approximately 5 µl of blood is collected through a tail clamp and blood glucose is measured using Aviva blood glucose meters. Measurements with glucometers are carried out twice. If the values differ by more than 20 mg/dl (the calculated value of the meter), then record the triple value.

Claims (14)

1. A compound having a structure

2. A pharmaceutical composition for the treatment of obesity, containing an effective amount of a compound according to claim 1 and a pharmaceutically acceptable excipient. 3. The pharmaceutical composition according to claim 2, wherein said composition is administered intraperitoneally. 4. Pharmaceutical composition according to claim 2, wherein said composition is administered orally. 5. A method of treating obesity in a subject in need thereof, comprising administering to the subject an effective amount of a compound of claim 1. 6. The method of claim 5, wherein the administration comprises intraperitoneal administration. 7. The method of claim 6, wherein the administration comprises oral administration. 8. Composition according to claim 2, containing a compound of the following structure:

9. A kit for the treatment of obesity, containing the composition according to p. 2 and instructions for use for the treatment of obesity. 10. A kit for the treatment of obesity, containing the composition according to p. 8 and instructions for use for the treatment of obesity. 11. A compound having a structure

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