Patent Grant
10316056
The contents of the electronic sequence listing (seq.txt; Size: 1.41 kilobytes; and Date of Creation: Apr. 9, 2018) is herein incorporate by reference in its entirety.
The present invention relates to chemical compounds and to the therapeutic use thereof, in particular for improving muscle quality in mammals.
More particularly, the invention makes it possible to improve muscle quality in obese mammals.
The invention also makes it possible to improve the muscle quality of sarcopenic mammals.
The invention also relates to the use of these chemical compounds in the treatment and/or prevention of obesity in mammals.
Muscle atrophy can result from several different causes: under nutrition, non-use of the muscles (for example immobilization following a fracture), cancer or other serious disease (heart or kidney failure) inducing cachexia, or resulting naturally from the aging of individuals (sarcopenia). This atrophy can result from a reduction in protein synthesis and/or from an increase in proteolysis and, as appropriate, is accompanied by fibrosis and/or by infiltration by adipose tissue. The identification of the factors and mechanisms controlling muscle protein synthesis and muscle proteolysis thus represents a prerequisite for designing appropriate treatments for these pathological conditions.
Muscle protein synthesis is essential, and is essentially controlled at the translational level. It requires of course an adequate nutritional intake of amino acids. It is stimulated by physical activity and regulated by numerous factors, at the forefront of which are IGF-1 and androgens (Little et al., 2009).
Myofibril proteolysis is performed via the proteasome, while the mitochondria are destroyed by autophagy (Zhao et al., 2008). Satellite cell apoptosis mechanisms are also described (Murphy et al., 2010).
Myostatin, produced in an autocrine manner by the muscles themselves, represents a particularly important factor, since it acts both by stimulating proteolysis and by inhibiting protein synthesis. It also stimulates fibrosis (Li et al., 2008).
Aging is accompanied by a modification of the various regulatory factors (Walston et al., 2012): physical activity is often reduced, protein/vitamin nutrition may be insufficient and, following meals, the contents of circulating amino acids, an increase of which is required to stimulate protein synthesis, show a reduced increase that may be due to splanchnic sequestration (Boirie et al., 1997). Moreover, aging is accompanied by considerable hormonal modifications: an increase in myostatin (Leger et al., 2008), a reduction in androgens (Seidman, 2007) and in growth hormone (Macell et al., 2001; Sattler, 2013), and also an increase in inflammation markers (IL-6, TNF-α etc., Schaap et al., 2009; Verghese et al., 2011), will in particular be noted. These various modifications are unfavorable for protein synthesis, whereas these promote proteolysis, hence the gradual reduction in muscle size (sarcopenia). They also cause a modification in the distribution of muscle fiber types to the detriment of the fast fibers, which is reflected by a decrease in muscle strength (dynapenia). Finally, the development of connective tissue within the muscles (fibrosis) is witnessed.
In an obesity context, the situation is worsened for several additional reasons: fat infiltration of the muscles worsens the inflammatory context, insulin resistance reduces the effect of IGF-1 on protein synthesis, without considering that mobility is reduced by the excess weight (Stenholm et al., 2009).
In any event, in the absence of treatment, sarcopenia is a process which can only get worse, until total loss of mobility. However, sarcopenia is not the only process which results in skeletal muscle atrophy. Atrophy also occurs during immobilization (for example following a fracture), during prolonged fasting (or a slimming diet), or during serious pathological conditions (for example cancers, AIDS) which cause cachexia.
Mention may also be made of various muscle dystrophies of genetic origin. These various situations have a certain number of characteristics in common with sarcopenia, but with a respective weight different than the triggering factors (Tisdale, 2007; Saini et al., 2009).
Known Possible Treatments
Various methods for preventing/treating sarcopenia have thus been envisaged and tested. They are first and foremost physical exercise, the effectiveness of which is established (Bonnefoy et al, 2000; Bonnefoy, 2008; Ryan et al., 2013). Thus, following exercise carried out over a period of 8 weeks, increases in muscle strength of 180% and in muscle mass of 11% have been observed (Fiatarone et al., 1990). However, optimal effectiveness would require several hours of physical exercise per day, which is difficult to envision over long periods of time.
An increased intake of protein synthesis substrates, whether by giving rapidly digestive proteins according to an optimized timing (Coëffier et al., 2009; Aussel et al., 2013), and also a supplement of certain amino acids or their metabolites (leucine, HMB [β-hydroxy-β-methylbutyrate], citrulline, ornithine), can increase muscle protein synthesis (Li & Heber, 2011).
Various pharmaceutical treatments aim to correct the modifications of the hormonal context associated with aging (Crenn, 2013). They comprise:
The great diversity of these treatments attests to the difficulty of treating a multifactorial pathological condition, the triggering factors of which have not been totally identified. Furthermore, several candidate molecules have side effects (in the case of sex hormones, SARMs or β-agonists, for example), or have as yet been studied only on animal models. All these elements explain the lack of available medicaments on the market.
To date, research studies target more particularly myostatin, by inhibiting its action with, for example, anti-myostatin antibodies or anti-receptor antibodies (Dumonceaux et al., 2010; Greenberg, 2012; Sakuma & Yamaguchi, 2012; Arounleut et al., 2013; Buehring & Binkley, 2013; Collins-Hooper et al., 2014; White & Le Brasseur, 2014).
Phytoecdysones, and more particularly the 20-hydroxyecdysone (20E), have been the subject of numerous pharmacological studies, which began in Japan and then in Uzbekistan, and have subsequently developed in various other countries.
These studies have revealed the antidiabetic and anabolic properties of this molecule. Its stimulating effects on protein syntheses in muscles are observed in rats in vivo (Syrov, 2000; Tóth et al., 2008; Lawrence, 2012) and on C2C12 murine myotubes in vitro (Gorelick-Feldman et al., 2008). It is an effect at the level of translation, which involves the phosphorylation of the p70S6K ribosomal protein, at the end of a cascade involving the Akt/PkB protein kinase, a pathway also used by IGF-1 to stimulate protein synthesis.
Using the same C2C12 cells, Zubeldia et al. (2012) have moreover shown that an Ajuga turkestanica extract enriched with phytoecdysones (20-hydroxyecdysone and turkesterone) inhibits the transcription of myostatin and of caspase 3 (a protein involved in apoptosis processes).
Moreover, 20-hydroxyecdysone has antifibrotic properties, which have not been demonstrated on muscles, but in the kidneys, where the fibrosis mechanisms take place very similarly (Hung et al., 2012). It thus opposes the effects of TGFβ, a protein similar to myostatin, and in particular the stimulation of Smad 2,3 caused by this substance. It can thus be considered that 20-hydroxyecdysone could have similar effects on muscles (or the heart).
20-Hydroxyecdysone reduces body fat in mice fed with a fat-enriched diet (Kizelsztein et al., 2009; Foucault et al., 2012) or in ovariectomized female rats, a model of menopause (Seidlova-Wuttke et al., 2010).
Some of the effects described above in animal models have been found in clinical studies, which are even fewer in number. Thus, 20-hydroxyecdysone increases physical capacity (Azizov et al., 1995; Gadhzieva et al., 1995) and muscle mass (Simakin et al., 1988) and causes a loss of abdominal fat mass in obese and overweight volunteers (Wuttke et al., 2013; Foucault et al., 2014; PCT patent application WO 2013/068704).
However, 20E and the metabolites thereof have poor bioavailability in mice (Dzhukharova et al., 1987; Hikino et al., 1972), in rats (Kapur et al., 2010 and Seidlova-Wuttke et al., 2010) and in humans (Brandt 2003; Bolduc, 2006). Their overall performance is among other things not entirely satisfactory in relation to muscle quality improvement applications.
Several studies have shown that turkesterone (11α,20-dihydroxyecdysone), a metabolite derived from 20E, shows a greater activity than that of 20E in vivo (Syrov et al., 2001: Bathori et al., 2008). There is still today, for therapeutic applications targeting an improvement in muscle quality both in obese mammals and in sarcopenic mammals, a need for novel compounds which have good bioavailability, expressed more particularly in terms of high plasma exposure coefficient, while at the same time having an overall activity greater than that of 20E on muscle quality improvement, this overall activity being expressed in terms of performance relating to inhibition of myostatin gene expression combined with increased protein synthesis in the mammal.
The inventors have now discovered that, entirely unexpectedly, certain compounds of the steroid family corresponding to a particular general formula, the structure of which differs from that of 20E and metabolites thereof, have a plasma exposure coefficient that is higher than that of said 20E and effects that are greater than or equal to those of 20-hydroxyecdysone (20E) with respect to the inhibition of myostatin and the stimulation of protein synthesis via phosphorylation of the S6K1 protein. These effects make it possible to improve the muscle quality and/or strength in sarcopenic mammals and sarcopenic obese mammals.
The compounds of the invention do not interact with steroid nuclear receptors of the sex sphere (androgen receptors and estrogen receptors). They show good chemical stability in plasma and in microsomes. Finally, several of them have a pharmacokinetic profile that is much improved compared with 20-hydroxyecdysone. They also induce better inhibition of myostatin gene expression and a better improvement of protein synthesis.
The invention thus provides a compound of general formula (I) below:
wherein:
Another particular form of the invention uses the compound of general formula (I) mentioned above wherein Q represents a carbonyl group.
One particular form of the invention uses the compound of general formula (I), wherein:
Another particular form of the invention uses the compound of general formula (I), wherein Q represents a CHNR2R3 group, with R2 and R3 being chosen from: a hydrogen atom; a (C1-C6) alkyl group; a (C1-C6)W(C1-C6) group; a cycloalkyl group; a (C1-C6)CHF2 group; a (C1-C6)A group, with A representing a heterocycle defined as above; a group of COR4 type,
Another particular form of the invention uses the compound of general formula (I), wherein:
Another particular form of the invention uses the compound of general formula (I), wherein Q represents a C═NOR5 group, R5 being defined as above.
Another particular form of the invention uses the compound of general formula (I), wherein:
Another particular form of the invention uses the compound of general formula (I), wherein V—U is a C═C ethylenic bond.
Another particular form of the invention uses the compound of general formula (I), wherein X is an N—OR5 group, R5 being defined as above.
Another particular form of the invention uses the compound of general formula (I), chosen from the following compounds:
Another subject of the invention relates to the use of a compound of general formula (I) as a medicament, in particular in a pharmaceutically acceptable carrier.
Another subject of the invention uses the compound of general formula (I), for use in the treatment and/or prevention of sarcopenia and of sarcopenic obesity, and the associated complications and/or pathological conditions thereof, such as loss of strength, of muscle mass, of physical performance and capacity and of mobility in mammals. The physical performance and capacity can be characterized by means of walking tests and physical effort tests.
Another subject of the invention uses the compound of general formula (I), for use in the treatment and/or prevention of obesity and the complications thereof and/or of the associated pathological conditions, advantageously type 2 diabetes or metabolic syndrome in mammals.
The object of the invention is to develop novel chemical compounds which meet in particular the objectives set above, in relation to therapeutic applications for the treatment and/or prevention of obesity and/or of sarcopenia in mammals. The latter compounds are novel since they do not exist in the chemical databases. They can advantageously be synthesized according to industrializable processes, that is to say processes with a minimum number of synthesis steps and an optimal yield. They have effects greater than those of 20E in terms of the inhibition of myostatin and the stimulation of protein synthesis via the phosphorylation of the S6K1 protein. They show good chemical stability in plasma and in microsomes. They have an improved pharmacokinetic profile and a defined dosage regimen. They stimulate muscle anabolism in C2C12 cells and show an anti-hyperglycemic effect.
In the context of the present invention, the term “aryl group” is intended to mean an aromatic ring having 5 to 8 carbon atoms or several fused aromatic rings having 5 to 14 carbon atoms. In particular, the aryl groups can be monocyclic or bicyclic groups, preferably phenyl or naphthyl. Advantageously, it is a phenyl group (Ph).
In the context of the present invention, the term “heteroaryl group” is intended to mean any hydrocarbon-based aromatic group of 3 to 9 atoms containing one or more heteroatoms, such as for example sulfur, nitrogen or oxygen atoms. The heteroaryl according to the present invention may consist of one or more fused rings. Examples of a heteroaryl group are furyl, isoxazyl, pyridyl, thiazolyl, pyrimidyl, benzimidazol, benzoxazole and benzothiazole groups. Advantageously, the heteroaryl group is chosen from furyl, pyridyl and thiazolyl groups. Advantageously, it is the furyl group.
In the context of the present invention, the term “halogen atom” is intended to mean any halogen atom, advantageously chosen from Cl, Br, I or F, in particular chosen from F, Cl or Br, in particular F or Cl.
In the context of the present invention, the term “C1-C6 alkyl group” is intended to mean any linear or branched alkyl group having from 1 to 6 carbon atoms, in particular methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl or n-hexyl groups. Advantageously, it is a methyl, ethyl, isopropyl or t-butyl group, in particular a methyl or ethyl group, more particularly a methyl group.
In the context of the present invention, the term “C3-C6 cycloalkyl group” is intended to mean any saturated and hydrocarbon-based ring comprising from 3 to 6 carbon atoms, in particular the cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl group. Advantageously, it is a cyclopropyl or cyclohexyl group.
In the context of the present invention, the term “(C1-C6 alkyl group) aryl” is intended to mean any aryl group as defined above, bonded by means of a C1-C6 alkyl group as defined above. In particular, an example of a (C1-C6 alkyl group) aryl is a benzyl or —(CH2)2 phenyl group.
In the context of the present invention, the term “pharmaceutically acceptable” is intended to mean what is of use in the preparation of a pharmaceutical composition which is generally safe, nontoxic and neither biologically undesirable or undesirable in another way, and which is acceptable for both veterinary use and human pharmaceutical use.
In the context of the present invention, the term “pharmaceutically acceptable salts of a compound” is intended to mean salts which are pharmaceutically acceptable, as defined herein, and which have the desired pharmacological activity of the parent compound. Such salts comprise:
In the context of the present invention, the term “solvate of a compound” is intended to mean any compound obtained by addition of an inert solvent molecule to the compound according to the invention, the solvate forming because of their mutual attraction force. The solvates are, for example, alkoxides of the compound. A hydrate is a solvate in which the inert solvent used is water. It may be mono-, di- or trihydrated.
In the context of the present invention, the term “tautomer” is intended to mean any constitutional isomer of the compounds according to the present invention which are interconvertible by means of the reversible chemical reaction known as tautomerization. In most cases, the reaction occurs by migration of a hydrogen atom accompanied by a change in location of a double bond. In a solution of a compound capable of tautomerization, an equilibrium between the two tautomers is created. The ratio between tautomers then depends on the solvent, on the temperature and on the pH. Tautomerism is thus the conversion of one functional group into another, usually by concomitant shift of a hydrogen atom and of a π bond (double or triple bond). Common tautomers are, for example, the following pairs: aldehydes/ketones—alcohols or more specifically enol; amides—imidic acids; lactams—lactims; imines—enamines; enamines—enamines. In particular, it may include a ring-chain tautomerism which takes place when the movement of the proton is accompanied by the conversion of an open structure to a ring.
Description of the General Syntheses and Schemes
The compounds of general formula (I) can be prepared by applying or adapting any method known per se to those skilled in the art and/or within the scope of the latter, in particular those described by Larock (1989), or by applying or adapting the processes described in the procedures which follow.
The various groups refer to the definitions given above.
Scheme A: The 20-hydroxyecdysone A1 can be reduced to the compound A2 by the action of zinc in acetic acid as described in Zhu et al. (2002). This compound A2 can undergo oxidative cleavage at C20-C22 of the chain by reaction of PCC in pyridine to give the compound A3. The alkyloximes of R5ONH2 type react with the carbonyl at C20 to give the corresponding imines A4 and also the compound A5 from double reaction at C20 and C6.
Scheme B: The alkyloximes of R5ONH2 type react with the carbonyl at C6 of the compound A1 to give the oxime B1 and also, optionally, the compounds B2 (Z conformer) and B′2 (E conformer) from elimination of the hydroxyl at C14-C15. These 3 compounds can independently undergo a chain cleavage as described in scheme A so as to give the compounds B3 and B4, with the (Z)-oxime compound B′3 as by-product. Alkyloximes of R5ONH2 type react with the carbonyl at C6 of the compounds B3 or B4 to give the compounds B5 and B6.
Scheme C: The compound A1 can undergo an oxidative cleavage as described on scheme A to give the compound C1. This compound, called poststerone in the literature can be subjected to the action of an alkoxime of R5ONH2 type on the carbonyl at C20, thereby making it possible to obtain the compound C2, the compound C3 from double reaction at C6 and C20 and the compound C4 from elimination of the hydroxyl at C14-C15.
Scheme D: The mixture of (E) and (Z) conformers B3 and B′3 derived from scheme B is reacted with titanium chloride, the action of which is to dehydrate the (Z) compound B′3 so as to obtain D1. The carbonyl at C17 of the compound B3 isolated in the previous step undergoes a reductive amination with R3NH2 in the presence of cyanoborohydride so as to give the compound D2 which can be acylated with an acid chloride R4COCl, making it possible to obtain the compound D3.
Scheme E: The poststerone C1 undergoes a reductive amination and then an acylation of the same type as those described in scheme D and makes it possible to obtain the compounds E1 and then E2.
Scheme F: The secondary amine of the compound E1 derived from scheme F is alkylated with a bromoalkyl compound so as to give the tertiary amine F2.
Scheme G: The poststerone C1 can be brominated at C21 using bromine so as to give the brominated compound G1 which can be alkylated with a nucleophile WR, W possibly being an amine or a thiol, and giving the compound G2.
Scheme H: The brominated compound G1 obtained in scheme G can react with alkoxide compounds of OR type in order to obtain the ethereal compounds H1.
Scheme I: The compounds G2 derived from scheme G can undergo a reduction of the carbonyl at C20 using sodium borohydride so as to give the alcohols I2.
Scheme J: The compounds G2 derived from scheme G can undergo the reaction at C20 of an alkoxamine of R5ONH2 type as described in scheme C and makes it possible to obtain the compound J1.
Materials and Methods
The proton (1H) nuclear magnetic resonance (NMR) spectra are performed on a Bruker Avance DPX300 apparatus (300.16 MHz). The chemical shifts (δ) are measured in parts per million (ppm). The spectra are calibrated on the chemical shift of the deuterated solvent used. The coupling constants (J) are expressed in Hertz (Hz) and the multiplicity is represented in the following way: singulet (s), doublet (d), doublet of doublets (dd), triplet (t), triplet of doublets (td), quadruplet (q), multiplet (m). The mass spectra (MS) are carried out by an Agilent Technologies MSD, type G1946A, spectrometer, and the samples are ionized by an “atmospheric pressure chemical ionization” (APCI) source.
Abbreviations
By way of illustrative examples of the invention, the compounds represented in table 2 were synthesized.
20 g (41.6 mmol) of 20-hydroxyecdysone (commercially available) are dissolved in 280 ml of acetic acid and the solution is heated to 67° C. 27.2 g (416 mmol) of zinc powder are added portionwise and the reaction medium is heated at 67° C. for 18 h. The solution is then filtered at 20° C. through a celite cake which is washed with 50 ml of methanol. The filtrate is evaporated off to give 33.7 g of brown oil which is purified by flash chromatography on a silica gel cartridge (90/10 dichloromethane/methanol) to give 9.52 g of yellow powder (yield: 49%) of (2S,3R,5R,10R,13S,17S)-2,3-dihydroxy-10,13-dimethyl-17-[(1R,2R)-1,2,5-trihydroxy-1,5-dimethylhexyl]-1,2,3,4,5,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-6-one.
LC-MS: m/z=465.3 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6)δ 5.72-5.43 (m, 1H(C7)), 4.42-4.32 (m, 2H), 4.13 (s, 1H), 3.76-2.62 (m, 2H), 3.2-3.1 (m, 2H), 2.21-2.14 (m, 2H), 1.90-1.02 (m, 28H), 1.03-0.77 (m, 6H).
9.52 g (20.28 mmol) of (2S,3R,5R,10R,13S,17S)-2,3-dihydroxy-10,13-dimethyl-17-[(1R,2R)-1,2,5-trihydroxy-1,5-dimethylhexyl]-1,2,3,4,5,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-6-one are dissolved in 46 ml of pyridine and 276 ml of dichloromethane. 6.69 g (30.4 mmol) of pyridinium chlorochromate are added portionwise over the course of 10 min and the reaction medium is stirred at 20° C. for 2 h 30. The pyridine and the dichloromethane are then evaporated off under vacuum and the residue is purified by flash chromatography on a silica gel cartridge (95/5 dichloromethane/methanol) to give 4 g of beige powder (yield: 56%) of (2S,3R,5R,10R,13S,17S)-17-acetyl-2,3-dihydroxy-10,13-dimethyl-1,2,3,4,5,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-6-one.
LC-MS: m/z=347.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6) δ 5.68-5.46 (m, 1H(C7)), 4.41-4.37 (m, 2H), 3.76-3.55 (m, 2H), 2.83-2.54 (m, 2H), 2.33-1.95 (m, 6H), 1.90-1.30 (m, 10H), 1.28-1.18 (m, 1H), 0.88-0.42 (m, 6H).
328 mg (0.947 mmol) of (2S,3R,5R,10R,13S,17S)-17-acetyl-2,3-dihydroxy-10,13-dimethyl-1,2,3,4,5,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-6-one (14-deoxypoststerone) prepared in step 2 are dissolved in 1.2 ml of ethanol and 200 mg (0.994 mmol) of but-3-enoxyammonium 2,2,2-trifluoroacetate are added portionwise. The reaction medium is brought to reflux for 20 h. The solvent is evaporated off and the residue is purified by preparative chromatography on a C18 column (60/40 acetonitrile/water) to give 24 mg of beige powder (yield: 6%) of compound No. 1 (2S,3R,5R,10R,13S,14S,17S)-17-(N-but-3-enoxy-C-methylcarbonimidoyl)-2,3-dihydroxy-10,13-dimethyl-1,2,3,4,5,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-6-one and 57 mg of beige powder (yield: 14%) of compound No. 2 (2S,3R,5R,10R,13S,14R,17S)-17-(N-but-3-enoxy-C-methylcarbonimidoyl)-2,3-dihydroxy-10,13-dimethyl-1,2,3,4,5,9,11,12,14,15,16,17-dodecahydrocyclopenta[a]phenanthren-6-one.
Compound No. 1:
LC-MS: m/z=416.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6)—C14 beta epimer—δ 5.83-5.72 (m, 1H), 5.70 (s, 1H(C7)), 5.1-5 (m, 2H), 4.40-4.36 (m, 2H), 4 (t, 2H), 3.77-3.71 (m, 2H), 2.80-2.60 (m, 1H), 2.40-1.20 (m, 20H), 0.82-0.74 (m, 6H).
Compound No. 2:
LC-MS: m/z=416.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-D6)—C14 alpha epimer—δ 5.87-5.72 (m, 1H), 5.48 (s, 1H(C7)), 5.1-4.9 (m, 2H), 4.40-4.36 (m, 2H), 4 (t, 2H), 3.77-3.71 (m, 2H), 2.80-2.60 (m, 1H), 2.44-1.23 (m, 20H), 0.83 (s, 3H), 0.47 (s, 3H).
Compounds Nos. 3 to 6 were prepared according to the same scheme, in the form of C14 alpha and C14 beta epimers.
1H NMR (300 MHz, DMSO-d6) δ
1LCMS purity, UV at 254 nm
According to the same procedure as that described in step 3 of scheme A, 788 mg of beige powder (yield: 37%) of compound (a) [(2S,3R,5R,6E,10R,13R,14S,17S)-6-methoxyimino-10,13-dimethyl-17-[(1R,2R)-1,2,5-trihydroxy-1,5-dimethylhexyl]-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthrene-2,3,14-triol] were prepared from 20-hydroxyecdysone and from O-methylhydroxylamine hydrochloride. 667 mg (yield: 32%) of elimination compound (b) [(2R,3R)-2-[(2S,3R,5R,6Z,10R,13R,17S)-2,3-dihydroxy-6-methoxyimino-10,13-dimethyl-1,2,3,4,5,9,11,12,16,17-decahydrocyclopenta[a]phenanthren-17-yl]-6-methylheptane-2,3,6-triol] could also be isolated, and also 34 mg (yield: 2%) of elimination compound (c) [(2R,3R)-2-[(2S,3R,5R,6E,10R,13R,17S)-2,3-dihydroxy-6-methoxyimino-10,13-dimethyl-1,2,3,4,5,9,11,12,16,17-decahydrocyclopenta[a]phenanthren-17-yl]-6-methylheptane-2,3,6-triol] could also be likewise isolated.
Compound (a):
LC-MS: m/z=510.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6)δ 6.25 (s, 1H(C7)), 4.45-4.35 (m, 3H), 4.31-4.29 (m, 1H), 4.14 (s, 1H), 3.74-3.69 (m, 4H), 3.6-3.5 (m, 1H), 3.17-3.08 (m, 1H), 2.87-2.75 (m, 1H), 2.26-2.20 (m, 2H), 2.05-1.1 (m, 15H), 1.1-0.98 (m, 11H), 0.73 (s, 6H).
Compound (b):
LC-MS: m/z=492.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6) δ 6.04 (s, 1H), 5.77 (s, 1H), 4.45-4.30 (m, 2H), 4.25 (s, 1H), 4.11 (s, 1H), 3.75-3.65 (m, 5H), 3.63-3.55 (m, 1H), 3.20-3.08 (m, 2H), 2.17-1.90 (m, 3H), 1.70-1.20 (m, 11H), 1.15-0.93 (m, 14H), 0.74 (s, 3H).
Compound (c):
LC-MS: m/z=492.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6)δ 6.55 (s, 1H), 5.81 (s, 1H), 4.44-4.26 (m, 3H), 4.09 (s, 1H), 3.79-3.67 (m, 5H), 3.62-3.54 (m, 1H), 3.16-3.08 (m, 1H), 2.30-1.90 (m, 4H), 1.70-1.20 (m, 11H), 1.15-0.92 (m, 14H), 0.73 (s, 3H).
Starting from the Isolated Compound (b):
According to the same procedure as that described in step 2 of scheme A, 267 mg of beige powder (yield: 55%) of compound (d) [1-[(2S,3R,5R,6Z,10R,13R,17S)-2,3-dihydroxy-6-methoxyimino-10,13-dimethyl-1,2,3,4,5,9,11,12,16,17-decahydrocyclopenta[a]phenanthren-17-yl]ethanone] were prepared from compound (b).
Compound (d):
LC-MS: m/z=374.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6) δ 6.09 (s, 1H), 5.81-5.75 (m, 1H), 4.39-4.37 (m, 1H), 4.30-4.26 (m, 1H), 3.76 (s, 3H), 3.72-3.68 (m, 1H), 3.65-3.55 (m, 1H), 3.2-3 (m, 2H), 2.75-2.60 (m, 1H), 2.29-2.10 (m, 5H), 1.74-1.23 (m, 8H), 0.74-0.70 (m, 6H).
According to the same procedure as that described in step 3 of scheme A, 81 mg of white powder (yield: 71%) of 1-[(2S,3R,5R,6Z,10R,13R,17S)-2,3-dihydroxy-6-methoxyimino-10,13-dimethyl-1,2,3,4,5,9,11,12,16,17-decahydrocyclopenta[a]phenanthren-17-yl]ethanone oxime were prepared from compound (d).
Compound No. 7:
LC-MS: m/z=389.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6) δ 10.53 (s, 1H), 6.09 (s, 1H), 5.04 (s, 1H), 4.37 (d, 1H), 4.30-4.26 (m, 1H), 3.77-3.67 (m, 4H), 3.65-3.55 (m, 1H), 3.15-3.03 (m, 1H), 2.80-2.65 (m, 2H), 2.25-2.12 (m, 1H), 2.05-1.99 (m, 1H), 1.79 (s, 3H), 1.74-1.20 (m, 8H), 0.76-0.66 (m, 6H).
According to the same procedure as that described in step 2 of scheme A, 891 mg of beige powder (yield: 36%) of compound (e) [1-[(2S,3R,5R,6E,10R,13R,14S,17S)-2,3,14-trihydroxy-6-methoxyimino-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-17-yl]ethanone] were isolated and also 23 mg (yield: 0.9%) of compound (f): [1-[(2S,3R,5R,6Z,10R,13R,14S,17S)-2,3,14-trihydroxy-6-methoxyimino-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-17-yl]ethanone] were isolated from 3.5 g of the isolated compound (a) [2S,3R,5R,6E,10R,13R,14S,17S)-6-methoxyimino-10,13-dimethyl-17-[(1R,2R)-1,2,5-trihydroxy-1,5-dimethylhexyl]-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthrene-2,3,14-triol].
Compound (e):
LC-MS: m/z=392.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6) 67 6.28 (s, 1H(C7)), 4.74 (s, 1H), 4.42-4.36 (m, 1H), 4.32-4.28 (m, 1H), 3.76-3.70 (m, 4H), 3.68-3.52 (m, 1H), 3.20-3.12 (m, 1H), 2.90-2.76 (m, 1H), 2.30-2.00 (m, 5H), 1.90-1.50 (m, 8H), 1.49-1.24 (m, 3H), 0.72 (s, 3H), 0.45 (s, 3H).
Compound (f):
LC-MS: m/z=392.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6) δ 5.71 (s, 1H(C7)), 4.45 (s, 1H), 4.45-4.41 (m, 1H), 4.26-4.23 (m, 1H), 3.76-3.70 (m, 4H), 3.65-3.55 (m, 1H), 3.18-3.09 (m, 1H), 2.90-2.80 (m, 1H), 2.22-2.00 (m, 5H), 1.88-1.22 (m, 11H), 0.73 (s, 3H), 0.47 (s, 3H).
According to the same procedure as that described in step 3 of scheme A, 46 mg of white powder (yield: 48%) of compound No. 19 [(2S,3R,5R,6E,10R,13R,14S,17S)-17-(N-(2-methoxyethoxy)-C-methylcarbonimidoyl)-6-methoxyimino-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthrene-2,3,14-triol] were prepared from 233 mg of compound (e).
Compound No. 19:
LC-MS: m/z=465.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6) δ 6.28 (s, 1H(C7)), 4.66 (s, 1H), 4.44-4.38 (m, 1H), 4.34-4.28 (m, 1H), 4.10-4.01 (m, 2H), 3.75-3.70 (m, 4H), 3.65-3.45 (m, 3H), 3.24 (s, 3H), 2.98-2.76 (m, 2H), 2.30-1.90 (m, 4H), 1.80-1.24 (m, 12H), 0.73 (s, 3H), 0.49 (s, 3H).
Compound No. 21 was prepared according to the same scheme.
1H NMR (300 MHz, DMSO-d6) δ
1LCMS purity, UV at 254 nm
According to the same procedure as that described in step 3 of scheme A, 64 mg of white powder (yield: 22%) of (2S,3R,5R,10R,13R,14S,17S)-17-(N-ethoxy-C-methyl-carbonimidoyl)-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one were prepared from poststerone (obtained by oxidative cleavage of the chain of 20-hydroxyecdysone according to the same procedure as that described in step 2 of scheme B).
Compound No. 23:
LC-MS: m/z=406.2 (MH+) UV purity at 254 nm=93%.
1H NMR (300 MHz, CD3OD) δ 5.82 (s, 1H(C7)), 4.04 (q, 2H), 3.97-3.92 (m, 1H), 3.89-3.80 (m, 1H), 3.22-3.10 (m, 1H), 3.04 (t, 1H), 2.43-1.55 (m, 15H), 1.45-1.37 (m, 1H), 1.21 (t, 3H), 0.96 (s, 3H), 0.64 (s, 3H).
Compounds Nos. 24 to 36 were prepared according to the same scheme.
1H NMR (300 MHz, DMSO-d6) δ
1LCMS purity, UV at 254 nm
180 mg (0.46 mmol) of compound (e) [1-[(2S,3R,5R,6E,10R,13R,14S,17S)-2,3,14-trihydroxy-6-methoxyimino-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-17-yl]ethanone] obtained in step 2b of method B are dissolved in 5 ml of methanol and 0.21 ml (2.76 mmol) of 2,2-difluoroethanamine is added to the reaction medium. The pH of the solution is adjusted to 6 using the sufficient amount of concentrated acetic acid. 31.8 mg (0.506 mmol) of sodium cyanoborohydride are then added portionwise and the suspension obtained is refluxed for 20 h. The solvent is evaporated off and the residue obtained is taken up in 20 ml of water and the pH is adjusted to 8 using a saturated sodium bicarbonate solution. This aqueous phase is extracted with two times 15 ml of butanol and the butanol phase is dried over solvate, filtered and evaporated to give a yellow solid, which, taken up in 30 ml of isopropyl ether and filtered, gives, after drying, 134 mg (yield: 62%) of compound No. 39 (2S,3R,5R,6E,10R,13R,14S,17S)-17-[1-(2,2-difluoroethylamino)ethyl]-6-methoxyimino-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthrene-2,3,14-triol in the form of a yellow powder.
Compound No. 39:
LC-MS: m/z=457.4 (MH+) UV purity at 254 nm=97%.
1H NMR (300 MHz, DMSO-d6) δ 6.30-6.23 (m, 1H), 5.95-5.70 (m, 1H), 4.43-4.25 (m, 3H), 3.72 (s, 3H), 3.65-3.55 (m, 1H), 3.42-3.32 (m, 1H), 2.88-2.76 (m, 2H), 2.29-2.23 (m, 1H), 1.99-1.15 (m, 16H), 1.05-0.82 (m, 3H), 0.73 (s, 3H), 0.61-0.53 (m, 3H).
134 mg (0.285 mmol) of compound No. 39 [(2S,3R,5R,6E,10R,13R,14S,17S)-17-[1-(2,2-difluoroethylamino)ethyl]-6-methoxyimino-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthrene-2,3,14-triol] are dissolved in 2 ml of THF and 52 mg (0.854 mmol) of sodium bicarbonate are added to the reaction medium under an argon atmosphere. 30 μL (0.299 mmol) of furoyl chloride are added and the reaction medium is stirred for 20 h at 20° C. The solution is then poured onto 5 ml of water and extracted two times with 10 ml of butanol. The butanol phase is evaporated off to give 118 mg of solid purified by flash chromatography on a silica gel cartridge (95/5 dichloromethane/MeOH) to give 100 mg of white powder (yield: 60%) of compound No. 37: N-(2,2-difluoroethyl)-N-[1-[(2S,3R,5R,6E,10R,13R,14S,17S)-2,3,14-trihydroxy-6-methoxyimino-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-17-yl]ethyl]furane-2-carboxamide.
Compound No. 37:
LC-MS: m/z=551.3 (MH+) UV purity at 254 nm=93%.
1H NMR (300 MHz, DMSO-d6) δ 7.87 (s, 1H), 7.03 (s, 1H), 6.64 (s, 1H), 6.25 (s, 1H), 4.58 (d, 1H), 4.43-4.27 (m, 3H), 3.95-3.83 (m, 1H), 3.75-3.65 (m, 4H), 3.63-3.49 (m, 2H), 2.85-2.68 (m, 1H), 2.31-2.18 (m, 1H), 2.01-1 (m, 17H), 0.73-0.15 (m, 6H).
Compounds Nos. 38 and 40 were prepared according to the same scheme.
1H NMR (300 MHz, DMSO-d6) δ
1LCMS purity, UV at 254 nm
5 g (13.8 mmol) of poststerone (obtained by oxidative cleavage of the chain of 20-hydroxyecdysone according to the same procedure as that described in step 2 of scheme B) are dissolved in 250 ml of methanol and 7.2 ml (83 mmol) of 2-methoxyethylamine are added dropwise. The pH of the solution is then brought to pH 6 by adding concentrated acetic acid, and 250 ml of THF are added. 0.954 g of sodium cyanoborohydrure are added portionwise and the reaction medium is brought to reflux for 20 h. The solvents are evaporated off and the crude product obtained is taken up in 100 ml of water and the pH is adjusted to 8 by adding a saturated sodium bicarbonate solution. The medium is extracted three times with 80 ml of butanol and the butanol phase is evaporated off to give a brown foam which, taken up with 5 ml of ethyl acetate, gives, after filtration and drying, 3.32 g (yield: 57%) of compound No. 42: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[1-(2-methoxyethylamino)ethyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one in the form of a gray powder.
Compound No. 42:
LC-MS: m/z=422.2 (MH+) UV purity at 254 nm=95%.
1H NMR (300 MHz, DMSO-d6) δ 5.70-5.60 (m, 1H(C7)), 4.80-4.62 (m, 1H), 4.55-4.47 (m, 1H), 4.43-4.35 (m, 1H), 3.78-3.70 (m, 2H), 3.68-3.50 (m, 3H), 3.30-3.18 (m, 5H), 3.10-2.91 (m, 1H), 2.30-0.9 (m, 18H), 0.82 (s, 3H), 0.59 (s, 3H).
13C NMR (75 MHz, DMSO-d6) δ 202.9 (C6), 120.5, 82.9, 66.7, 58.1, 46.2, 37.8, 30.5, 23.9, 6.2.
According to the same procedure as step 2 of example 5, 89 mg (yield: 58%) of compound No. 41 [2-methoxy-N-(2-methoxyethyl)-N-[1-[(2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-6-oxo-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-17-yl]ethyl]acetamide] were obtained in the form of an orange powder from compound No. 42.
Compound No. 41:
LC-MS: m/z=494.4 (MH+) UV purity at 254 nm=94%.
1H NMR (300 MHz, DMSO-d6) δ 5.63 (s, 1H(C7)), 4.88-4.7 (m, 1H), 4.5-4.35 (m, 2H), 4.2-3.9 (m, 2H), 3.76 (s, 1H), 3.68-3.52 (m, 1H), 3.5-3.3 (m, 4H), 3.28-3.18 (m, 6H), 3.08-2.9 (m, 1H), 2.3-0.95 (m, 18H), 0.88-0.75 (m, 3H), 0.7-0.42 (m, 3H).
Compounds Nos. 43 to 75 were prepared according to the same scheme:
1H NMR (300 MHz, DMSO-d6) δ
1LCMS purity, UV at 254 nm
155 mg (0.368 mmol) of compound No. 42 [(2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[1-(2-methoxyethylamino)ethyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one] prepared according to the technique described in step 1 of example 5 are dissolved in 2.5 ml of DMF and 61.8 mg (0.735 mmol) of sodium bicarbonate are added to the reaction medium, along with 0.034 ml (0.552 mmol) of iodomethane. The suspension obtained is stirred at 20° C. for 20 h. The solution is then poured onto 15 ml of water and extracted three times with 15 ml of butanol. The butanol phase is evaporated off to give 220 mg of powder purified by flash chromatography on a silica gel cartridge (95/5 dichloromethane/MeOH) to give 40 mg of white powder (yield: 25%) of compound No. 76: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[1-(2-methoxyethyl(methyl)amino)ethyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one in the form of a white powder.
Compound No. 76:
LC-MS: m/z=436.3 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6)δ 5.61 (s, 1H(C7)), 4.64 (s, 1H), 4.47-4.34 (m, 2H), 3.75 (s, 1H), 3.67-3.50 (m, 1H), 3.25-3.16 (m, 5H), 3.05-2.85 (m, 1H), 2.27-1.15 (m, 20H), 0.90-0.70 (m, 6H), 0.59 (s, 3H).
Compounds Nos. 77 to 80 were prepared according to the same scheme.
1H NMR(300 MHz, DMSO-d6) δ
1LCMS purity, UV at 254 nm
1 g (2.76 mmol) of poststerone (obtained by oxidative cleavage of the chain of 20-hydroxyecdysone according to the same procedure as that described in step 2 of scheme B) is dissolved in 20 ml of methanol. The solution is cooled to 0° C. and 0.284 ml (5.52 mmol) of bromine is added dropwise and the reaction medium is stirred for 1 h at this temperature, then left at ambient temperature for 16 h. The reaction medium is poured onto 50 ml of a saturated sodium bicarbonate solution and extracted three times with 100 ml of ethyl acetate. The organic phases are washed with 50 ml of saturated sodium bicarbonate solution, then salt water, dried over sodium sulfate, and filtered, and the solvent is evaporated off to give 833 mg of powder, which, taken up in 30 ml of dichloromethane, gives, after filtration and desiccation, 412 mg (yield: 31%) of compound No. 102: 2S,3R,5R,10R,13R,14S,17S)-17-(2-bromoacetyl)-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one in the form of a yellow powder.
Compound No. 102:
LC-MS: m/z=443.1 (MH+) UV purity at 254 nm=91%.
1H NMR (300 MHz, DMSO-d6) δ 5.69-5.63 (m, 1H(C7)), 5.08 (s, 1H), 4.42-4.35 (m, 3H), 4.33-4.22 (m, 1H), 3.77 (s, 1H), 3.66-3.58 (m, 1H), 3.39 (t, 1H), 3.10-2.95 (m, 1H), 2.25-1.20 (m, 13H), 0.83 (s, 3H), 0.51 (s, 3H).
50 mg (0.103 mmol) of compound No. 102 [(2S,3R,5R,10R,13R,14S,17S)-17-(2-bromoacetyl)-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one] are dissolved in 1 ml of DMF and 42.7 mg of potassium carbonate are added, along with 10.78 μl (0.124 mmol) of morpholine. After stirring for 18 h at 20° C., the reaction medium is poured onto 10 ml of water and this aqueous phase is extracted two times with 15 ml of butanol. The organic phase is evaporated off to give 71 mg of powder purified by flash chromatography on a silica gel cartridge (90/10 dichloromethane/MeOH) to give 28 mg (yield: 60%) of compound No. 81: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-17-(2-morpholinoacetyl)-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one in the form of a white powder.
Compound No. 81:
LC-MS: m/z=448.4 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6) δ 5.65 (s, 1H(C7)), 5.02 (s, 1H), 4.45 (d, 1H), 4.40-4.37 (m, 1H), 3.77 (s, 1H), 3.68-3.53 (m, 5H), 3.34-3.24 (m, 4H), 3.08-2.95 (m, 1H), 2.45-1.17 (m, 16H), 0.82 (s, 3H), 0.48 (s, 3H).
Compounds No. 82 to 94 were prepared according to the same scheme.
1H NMR (300 MHz, DMSO-d6) δ
1LCMS purity, UV at 254 nm
100 mg (0.227 mmol) of compound No. 102 [2S,3R,5R,10R,13R,14S,17S)-17-(2-bromoacetyl)-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one] prepared in step 1 of example 7 are dissolved in 2 ml of ethanol and 0.102 ml (0.272 mmol) of a solution of sodium ethoxide at 21% in ethanol, diluted in 1 ml of ethanol, is added dropwise and the solution obtained is brought to reflux for 30 min. The reaction medium cooled to 20° C. is poured onto 25 ml of water and extracted with two times 20 ml of butanol. The organic phase is evaporated off to give 30 mg of an oil purified by flash chromatography on a silica gel cartridge (95/5 dichloromethane/MeOH) to give 13.5 mg (yield: 14%) of compound No. 95: (2S,3R,5R,10R,13R,14S,17S)-17-(2-ethoxyacetyl)-2,3,14-trihydroxy-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one in the form of a yellow oil.
Compound No. 95:
LC-MS: m/z=407.2 (MH+) UV purity at 254 nm=93%.
1H NMR (300 MHz, DMSO-d6)δ 5.65-5.59 (m, 1H(C7)), 4.96 (s, 1H), 4.46 (d, 1H), 4.41-4.36 (m, 1H), 4.03 (q, 2H), 3.77 (s, 1H), 3.68-3.55 (m, 1H), 3.08-2.90 (m, 1H), 2.75-2.62 (m, 1H), 2.3-2.15 (m, 2H), 1.92-1.42 (m, 13H), 1.18 (t, 3H), 0.83 (s, 3H), 0.58-0.49 (m, 3H).
Compound No. 96 was prepared according to the same scheme
1H NMR(300 MHz, DMSO-d6) δ
1LCMS purity, UV at 254 nm
157 mg (0.360 mmol) of compound No. 87 [(2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[2-(2-hydroxyethyl(methyl)amino)acetyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one] obtained according to the method of step 2 of example 7 are dissolved in 7.5 ml of ethanol and 21.14 mg (0.559 mmol) of sodium borohydride are added portionwise. After stirring for 16 h at 20° C., the reaction medium is poured onto 20 ml of water and extracted with three times 15 ml of butanol. The organic phase is evaporated off to give a powder purified by flash chromatography on a silica gel cartridge (85/14/1 dichloromethane/MeOH/NH4OH) to give 96 mg (yield: 60%) of compound No. 97: (2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-17-[1-hydroxy-2-(2-hydroxyethyl(methyl)amino)ethyl]-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one in the form of a white powder.
Compound No. 97:
LC-MS: m/z=438.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6) δ 5.6 (s, 1H(C7)), 5.32-5.2 (m, 2H), 4.77 (s, 1H), 4.47 (d, 1H), 4.42-4.38 (m, 1H), 3.92-3.57 (m, 4H), 3.3-2.95 (m, 4H), 2.82 (s, 3H), 2.31-1.18 (m, 16H), 0.85 (s, 3H), 0.69 (s, 3H).
13C NMR (75 MHz, DMSO-d6)δ 203.2, 164.9, 121.0, 82.8, 66.9, 59.0, 55.6, 50.6, 46.9, 40.7, 37, 34, 31.9, 31.1, 30.3, 24.5, 23.2, 20.4, 16.3.
Compounds No. 98 to 100 were prepared according to the same scheme.
1H NMR (300 MHz, DMSO-d6) δ
1LCMS purity, UV at 254 nm
30 μl (0.301 mmol) of methoxylamine hydrochloride are dissolved in 0.6 ml of pyridine and 136 mg (0.301 mmol) of compound No. 81 [(2S,3R,5R,10R,13R,14S,17S)-2,3,14-trihydroxy-10,13-dimethyl-17-(2-morpholinoacetyl)-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-6-one] prepared in step 2 of example 7 are added portionwise. After stirring for 36 h at 20° C., the reaction medium is taken up in 10 ml of dichloromethane and this solution is washed two times with salt water, dried over sodium sulfate, filtered and evaporated to give a powder purified by flash chromatography on a silica gel cartridge (90/10 dichloromethane/MeOH) to give 81 mg (yield: 53%) of compound No. 101: (2S,3R,5R,6E,10R,13R,14S,17S)-6-methoxyimino-17-(N-methoxy-C-(morpholinomethyl)carbonimidoyl)-10,13-dimethyl-2,3,4,5,9,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthrene-2,3,14-triol in the form of a yellow powder.
Compound No. 101:
LC-MS: m/z=506.2 (MH+) UV purity at 254 nm=99%.
1H NMR (300 MHz, DMSO-d6) δ mixture of C6 (Z) and (E) conformers: 6.28 (s,0.45H (C7-conformer E), 5.72 (s,0.55H (C7-conformer Z), 4.62 (s,0.45H-conformer E), 4.53 (s,0.55H-conformer Z), 4.47-4.35 (m,1H), 4.33-4.21(m,1H), 3.77-3.70 (m,7H), 3.60-3.48 (m,5H), 3.16-3.06 (m,1H), 2.90-2.70 (m,2H), 2.45-1.20 (m,18H), 0.72 (s,3H), 0.64-0.57 (m, 3H).
Cascade for Screening and Characterization of the Biological Effects of the 20-Hydroxyecdysone Derivatives
The development of the screening test was initiated on the basis of the studies in the literature and was based on the characteristics of the pathology of sarcopenia. At the physiopathological level, this disease is characterized by a decrease in protein synthesis and an increase in proteolysis. The development of future medicaments should thus be screened on molecular factors in relation to these two phenomena.
At the cellular level, on cultures of muscle cells derived from the C2C12 murine line, Gorelick-Feldman et al. (2008) showed that treatment with phytoecdysones increases protein synthesis by +20% on average. The first development studies were based on the culture and treatment conditions described by Gorelick-Feldman in the presence of reference products (IGF-1 and 20-hydroxyecdysone or 20E). Measurements of tritiated leucine incorporation into these cells were carried out in order to evaluate the de novo protein synthesis. These first results made it possible to determine that the optimal sequence for observing the effects of phytoecdysones on protein synthesis was to differentiate the cells for 5 days, then to add the tritiated leucine for 2 h 30 in the presence of IGF-1 or of 20E.
The analysis of the literature showed that molecules such as IGF-1 increased protein synthesis only by 20%, while at the same time activating targets of this signaling pathway in a more sustained manner that could reach stimulations of about 200% [Kazi et al., 2010]. These targets comprise phosphorylations that activate proteins such as Akt or S6 kinase. Furthermore, in the same C2C12 cell system, Zubeldia et al. (2012) analyzed the phenomena of apoptosis and proteolysis. In their study, they in particular reported that plant extracts containing phytoecdysones such as turkesterone or 20E were capable, after 24 h of treatment of differentiated C2C12 cells, of inhibiting myostatin and caspase 3 gene expression by a factor of 4 and 2, respectively [Zubeldia et al., 2012].
After several experiments in which the C2C12 cells differentiated into myotubes were incubated in the presence of IGF-1 or 20E for 2 h 30 or 6 h, two screening tests were developed. Thus, the phosphorylation of the S6 protein kinase and the expression of the myostatin gene were studied in order to determine their modulation by a growth hormone or an ecdysone and to characterize these modulations from a statistical point of view.
Protocols
Inhibition of Myostatin Expression in C2C12 Cells:
The C2C12 myoblast cells (ATCC CRL-1772) are seeded into 24-well plates at a density of 30 000 cells per well and cultured in a DMEM medium containing glucose in a proportion of 4.5 g/l and supplemented with fetal calf serum (10%) and with antibiotics (penicillin and streptomycin). Forty-eight hours later, the myoblasts are induced to differentiate by partial serum depletion (2% instead of 10%) for 5 days. The cells are then placed in a medium which is glucose-depleted (DMEM containing 1 g/l of glucose) and serum-free in the presence of the test molecules or of the references (100 ng/ml IGF-1 or 10 μM 20E) for 6 h. At the end of the experiment, the messenger RNAs (mRNAs) are extracted using the conventional methodology based on phenol and chloroform. Briefly, the cells are lysed in a Trizol solution (Sigma T9424) containing a strong acid and phenol. The mRNAs are separated from the proteins by addition of chloroform and then centrifugation. They are then precipitated from isopropanol and then suspended at a concentration of 1 μg/μl in an RNAses-free and DNAses-free ultrapure water. 1 μg of mRNA is then converted by reverse transcription into complementary DNA by the AMV enzyme in the presence of a primer and of a mixture of nucleotides according to the protocol given by the supplier (Applied Biosystems 4368814). The gene expression is studied by chain reaction initiated by a polymerase enzyme and commonly referred to as PCR under quantitative conditions, hence the specific name qPCR. The qPCRs are carried out on a 7900HT Fast real-Time PCR detection system (Applied Biosystems). The programming conditions are standard and consist of 1 cycle at 95° C. for 15 min, followed by 40 cycles at 95° C. for 15 s and at 60° C. for 1 min and the program ends with a melt curve step between 60° C. and 95° C. The samples analyzed all contain 100 ng of cDNA, a qPCR buffer including the enzyme, the mixture of oligonucleotides and the intercalating agent (sybergreen or SYBRgreen), and the pair of primers specific for the gene studied, strategically chosen between two exon sequences and at a final concentration of 200 nM. Fluorescent probes bind to the double-stranded DNA and are fluorescent only once bound to the DNA. A fluorescence threshold is established by the machine's program. When the amount of DNA allows the fluorescent probe to exceed this threshold, a PCR cycle number, called “Ct” for “Cycle Threshold”, is obtained. It is this value which forms the basis of the calculations to quantify the DNA relatively. A ratio R is established between the amount of starting DNA of a sample and that of a control, which has not undergone treatment (i.e. R=2−(Ct sample−Ct control)) and this measurement will be related to that of a housekeeping gene known not to be modulated by the treatment (i.e. R=2−ΔΔCt).
The primers used are reported in the following table:
Phosphorylation of the S6 Kinase:
The C2C12 myoblast cells (ATCC CRL-1772) are seeded into 6-well plates at a density of 170 000 cells per well and cultured in a DMEM medium containing glucose in a proportion of 4.5 g/l and supplemented with fetal calf serum (10%) and with antibiotics (penicillin and streptomycin). Forty-eight hours later, the myoblasts are induced to differentiate by partial serum depletion (2% instead of 10%) for 5 days. The cells are then placed in a medium which is glucose-depleted (DMEM containing 1 g/l of glucose) and serum-free in the presence of the test molecules or of the references (100 ng/ml IGF-1 or 10 μM 20E) for 2 h. At the end of the experiment, the cells are lysed in a commercial lysis buffer (Invitrogen FNN0011) supplemented with a commercial mixture of anti-proteases (Roche 05056489001). After centrifugation, the cytoplasmic fraction containing the soluble proteins is kept and the protein concentration is determined using a commercial kit (Biorad 500-0114), the principle of which is composed of assaying by the Lowry method. The assaying of the S6 kinase phosphorylation is carried out using a commercial ELISA (Enzyme Linked ImmunoSorbent Assay) kit (Cell signaling 7063). Briefly, 50 μg of protein lysate are deposited in the wells of a 96-well microplate and incubated overnight at 4° C. with the solution of antigen specific for the pS6 kinase threonine 389 antibody. The binding of the antigen to the bottom of the wells is done electrostatically. The solution of antibody to be assayed (pS6K T389) is then incubated at 37° C. in the wells for 2 hours. The antibodies bind specifically to the antigen. The wells are then washed with washing buffer in order to remove the antigen-specific primary antibodies to be assayed which are in excess. The third step consists in binding the detection antibody. The solution of detection antibodies is incubated at 37° C. in the wells for 1 hour. The wells are then washed in order to remove the excess detection antibodies. It should be noted that the detection antibodies are coupled to an enzyme which, in the presence of its substrate, converts it into a reaction product that can be detected and measured by virtue of the appearance of a coloration. The final step consists in revealing the bound antibodies. A revealing solution containing the substrate for the enzyme, in this case TMB (3,3′,5,5′-tetramethylbenzidine), is incubated at 37° C. in the dark for 30 min. The appearance of a blue coloration in the substrate indicates the presence of the antibody to be assayed. In order to prevent any saturation phenomenon, a stop solution (generally containing sodium hydroxide) is added and brings about a change in coloration, which goes from blue to yellow. The strength thereof is proportional to the amount of enzyme present and thus to the concentration of antibody sought. The strength of the signal is measured using spectrophotometry at a wavelength of 450 nm.
Evaluation of the Effect of the Molecules in a Model of Mice Subjected to a High-fat Diet
The 20E as comparative compound and the compounds according to the invention (Nos. 51 and 93) were administered orally, at a dose of 5 mg/kg of body weight, to 12-week-old C57BL/6J mice subjected to a high-fat diet for 6 weeks. The effect of the compounds on the weight and the amount of proteins of the Soleus muscle and also the transcripts of genes involved in myogenesis were evaluated.
Myogenesis, which is the process for forming muscle tissues, is controlled by several myogenic transcription factors which act as end effectors of the signaling cascade and produce transcripts involved in the various stages of development. The roles of the transcription factors have been described in various journals (Sabourin and Rudnicki 2000 and Le Grand and Rudnicki 2007). The Pax7 protein (Paired-box protein 7) maintains a population of satellite cells in quiescence and, with Myf5 (Myogenic factor 5), plays a role in the expansion of activated myoblasts. The MyoD protein (Myoblast Determination protein) appears to determine the differentiation potential of activated myoblasts, and cooperates with myogenin and the MEF2 (Myocyte Enhancer Factor 2) protein to control and bring about differentiation. Finally, MRF4 (Muscle-specific Regulatory Factor 4) is required for hypertrophy, even though it probably plays other roles. Quite obviously, these transcription factors do not act alone, but exist in the context of complex signaling cascades which control each step of myogenesis (Knight and Kothary, 2011).
The amount of proteins is determined by first lysing the muscles sampled in a 0.1N NaOH solution with the FastPrep technique. The proteins are quantified by means of a colorimetric assay derived from the Lowry method.
In order to carry out the gene expression analysis, the muscle tissues were homogenized in a Trizol solution (500 μl), and the RNAs were extracted and purified using the phenol/chloroform method. An amount of 1 pg of RNA was used as template for the synthesis of the first cDNA strand using oligo (dT)s as primers and the AMV reverse transcriptase enzyme as described by the supplier (Applied Biosystems 4368814). The q-PCRs were then carried out using a 7900HT machine equipped with a rapid system for real-time detection by PCR (Applied Biosystems) and the standard qPCR program (1 cycle of 95° C. for 15 min, 40 cycles of 95° C. for 15 s and 60° C. for 1 min, a 60-95° C. melt curve for the Sybergreen probes). The experiments are carried out in a Sybergreen SYBR master mix (Applied Biosystems) containing the 100 ng cDNA samples and a set of primers which bind to the two different exons and at a final concentration of 200 nM.
The relative differences in gene expression between treatments are expressed as increase or decrease in the number of cycle times [Ct] compared with the control group, the [Ct] value of each gene having been standardized with the beta-actin gene.
Oral Pharmacokinetic Study of the Molecules in Rats
The pharmacokinetics of the compounds were evaluated orally using male Wistar rats (Charles River). The 20E as comparative compound was administered at a dose of 50 mg/kg of body weight. The novel compounds according to the invention were administered at a dose of 10 mg/kg of body weight in the form of a mixture of 4 to 6 products. After administration, the blood was sampled from the tail at t=0.25 h, 0.5 h, 1 h, 3 h, 6 h and 8 h. The blood samples were centrifuged and the plasmas removed. The assaying of the plasma samples made it possible to determine the pharmacokinetic parameters, namely the Cmax, which corresponds to the maximum concentration observed after the administration of the molecule, the Tmax, which is the time required to reach the maximum concentration after administration of the molecule, and the AUC: area under the curve composed of the various concentrations of compounds at the various sampling times.
Results
The Effects on Myostatin Expression
The following 38 compounds: 4, 5, 7, 21, 25, 27 to 29, 31 to 33, 38, 41, 43, 46, 47, 51 to 54, 62 to 65, 67, 68, 71, 75, 76, 79, 81, 86, 89, 92 to 94, 99 and 101 very significantly inhibit myostatin expression in muscle cells.
The following 15 compounds: 19, 23, 30, 35 to 37, 48, 56, 57, 60, 73, 83, 85, 88 and 91 significantly inhibit myostatin expression in muscle cells.
The Effects on Protein Synthesis Via S6K1 Phosphorylation
The following 8 compounds: 28, 42, 62, 67, 86, 89, 93 and 94 very significantly stimulate S6Ka phosphorylation at levels equivalent to IGF-1 (130-140%).
The following 12 compounds: 32, 41, 43, 46, 51, 52, 63, 76, 81, 88, 91 and 92 significantly stimulate S6K1 phosphorylation at levels equivalent to that of 20E (120%).
Study of the Molecules in a Model of Mice Subjected to a High-fat Diet
The in vivo study is carried out by evaluating the effect of 20E as comparative compound and of the molecules according to the invention (Nos. 51 and 93) administered orally, at a dose of 5 mg/kg of body weight, to C57BL/6 mice subjected to a high-fat diet for 6 weeks. The effect of the molecules on the weight and the amount of proteins of the Soleus muscle and also the transcripts of genes involved in myogenesis were evaluated.
The effects of 20E as comparative compound and of the compounds according to the invention Nos. 51 and 93 on the weight of the muscle are illustrated in
All three of the 20E and the compounds administered at 5 mg/kg induce increases in the weight and in the amount of proteins of the Soleus muscle compared with the control group. The compounds in accordance with the invention show just as high an effectiveness as that of 20E. A significant increase in the protein content is even noted with compound No. 93.
The effects of 20E as comparative compound and of the administered compounds according to the invention No. 51 and No. 93, on the myostatin transcript of the Soleus muscle, are illustrated in
The 20E and the compounds Nos. 51 and 93 comparably inhibit myostatin expression in the Soleus muscle. These molecules also inhibited the myostatin transcript in the in vitro studies in the C2C12 cell lines as presented in table 4 above.
The effects of the 20E as comparative compound and of the compounds according to the invention Nos. 51 and 93 on the transcripts of MyoD and of myogenin, which are genes involved in Soleus muscle myogenesis, are respectively illustrated in
The 20E and compounds Nos. 51 and 93 induce an increase in the transcripts of the MyoD gene which determines the differentiation potential and the Myf5 gene involved in the proliferation of myocytes. They also induce an increase in the transcript of the myogenin gene involved in the early differentiation of the myocytes.
Pharmacokinetic Study of the Molecules in Rats
The pharmacokinetics of 20E and of compounds according to the invention were evaluated in rats by oral administration at a dose of 10 mg/kg in the case of the compounds and 50 mg/kg in the case of 20E.
Taking into account the 20E dose which is 5 times higher (50 mg/kg) compared with that of the compounds according to the invention (10 mg/kg), the coefficient of exposure Cexp [Cexp=(Dose20E×AUCcompound): (Dosecompound×AUC20E)] demonstrates the improvement in the pharmacokinetic profile of all compounds tested compared with 20E. Thus, this study in rats shows that compounds Nos. 31; 46; 51 and 93 have a better plasma exposure compared with 20E.
Overview
The table illustrated in
With regard to the myostatin gene expression, the results were expressed as percentage myostatin gene expression in the cells in contact with the compounds related to the expression in the control cells. A represents a percentage of less than 70%, B represents a percentage of between 71% and 85%.
With regard to the protein synthesis analysis, the results are expressed as percentage increase in S6K phosphorylation in the muscle cells. A represents values greater than 130%, B represents values of between 110% and 129%.
The most attractive products are of category AA or AB, namely of category A in terms of their gene expression on myostatin combined with a category A or B in terms of protein synthesis.
| Number | Date | Country | Kind |
|---|---|---|---|
| 14 54538 | May 2014 | FR | national |
This application is a divisional of application Ser. No. 15/311,967 filed Jan. 3, 2017, which is a § 371 application from PCT/FR2015/051332 filed May 20, 2015, which claims priority from French Patent Application No. 14 54538 filed May 20, 2014, each of which is herein incorporated by reference in its entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 20070259837 | Meier et al. | Nov 2007 | A1 |
| 20090298175 | Hormann et al. | Dec 2009 | A1 |
| 20140309203 | Lafont et al. | Oct 2014 | A1 |
| Number | Date | Country |
|---|---|---|
| 2006007910 | Jan 2006 | WO |
| 2009114201 | Sep 2009 | WO |
| 2013068704 | May 2013 | WO |
| Entry |
|---|
| Kumpun et al., “The metabolism of 20-hydroxyecdysone in mice: Relevance to pharmacological effects and gene switch applications of ecdysteroids,” The J. Steroid Biochem. and Molecular Biol., Aug. 1, 2011, pp. 1-9, vol. 126, No. 1-2. |
| Syrov, “Comparative experimental investigation of the anabolic activity of phytoecdysteroids and steranabols,” Pharm. Chem. J., Apr. 1, 2000, pp. 193-197, vol. 34, No. 4. |
| Toth et al., “20-Hydroxyecdysone increases fiber size in a muscle-specific fashion in rat,” Phytomedicine, Sep. 3, 2008, pp. 691-698, vol. 15, No. 9, Gustav Fischer Verlag, Stuttgart, DE. |
| Kizelsztein et al., 20-Hydroxyecdysone decreases weight and hyperglycemia in a diet-induced obesity mice model, American J. of Physiol.: Endocrinology and Metab., Mar. 1, 2009, pp. E433-E439, vol. 296, No. 3, American Physiological Society, Bethesda, MD, US. |
| Zhao et al., “Coordinate activation of autophagy and the proteasome pathway by FoxO transcription factor,” Autophagy, 2008, pp. 378-380, vol. 4, No. 3. |
| Little et al. “Resistance exercise and nutrition to counteract muscle wasting,” Appl. Physiol. Nutr. Metab., Oct. 2009, pp. 817-828, vol. 34. |
| Bathori et al., “Complex Phytoecdysteroid Cocktail of Silene otites (Caryophyllaceae),” Archives of Insect Biochem. and Physiol., 1999, pp. 1-8, vol. 41. |
| Communications to the Editor, “Synthesis of Ecdysterone,” Chem. Pharm. Bull., 1969, pp. 1970-1973, vol. 17. |
| Galbraith et al., “The Structure of Podecdysone B, a New Phytoecdysone,” Chemical Communications, 1969, pp. 402-403. |
| Number | Date | Country | |
|---|---|---|---|
| 20180327444 A1 | Nov 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15311967 | US | |
| Child | 15949010 | US |
