Patent Application
20160083355
The subject of the present invention is novel families of compounds which are aromatic amine, imine, aminoadamantane and benzodiazepine derivatives, medicaments comprising same and the use thereof as inhibitors of the toxic effects of toxins with intracellular activity, such as, for example, ricin, and of viruses that use the internalization pathway for infecting cells.
Toxins with an intracellular mode of action, or AB toxins, are enzymes that are organized into several domains: a catalytic domain A which carries the toxic activity and one or more B domains which provide cell recognition and enable transmembrane translocation of the A fragment into the cytoplasm (Falnes, P. O.; Sandvig, K. Curr. Opin. Cell. Biol. 2000, 12, 407). There are many bacterial toxins with intracellular activity: for example, and nonexhaustively, diphtheria toxin and cholera toxin have an A domain which carries an ADP-ribosyltransferase enzyme activity; the major clostridial toxins have a glucosyltransferase activity; botulinum toxin, tetanus toxin and anthrax lethal toxin are metalloproteases; Shiga toxins and ricin have an N-glucosidase activity.
More particularly, ricin is a toxalbumin produced by a shrub of the family Euphorbiaceae, the castor oil plant (Ricinus communis). It is present at a concentration ranging from 1% to 10% in the castor oil plant seed.
It is more specifically a 66 kDa glycoprotein composed of two chains linked by a disulfide bridge. The A chain (RTA, 267 amino acids) performs the catalytic function of ricin (N-glucosidase), while the B chain (RTB, lectin of 262 residues) plays the role of transporter allowing ricin to enter the cell.
The ribosomal-RNA depurination enzymatic activity, located on the A chain, causes the arrest of protein synthesis in poisoned cells and results in cell death.
The B chain has two galactose-binding sites and enables binding of the toxin to glycoreceptors present at the cell surface. The ricin can then penetrate into these cells via multiple endocytosis pathways, so as to reach the trans-Golgi network, where it is conveyed to the endoplasmic reticulum (ER) by retrograde transport.
The toxin is then partially unfolded and the A chain is translocated into the cytosol by the Sec61p translocon which is normally used to translocate newly formed proteins into the ER or to transport incorrectly folded proteins out of the ER and to the cytoplasm so as to be degraded therein. Ricin is capable of escaping this proteolysis, thereby allowing it to bind to the ribosome with great efficiency and to cleave the adenine at position 4324 (hereinafter A4324) of the 28S RNA of the 60S ribosomal subunit. It can thus inactive up to 2000 ribosomes per minute.
Ricin is a cytotoxin which can be easily extracted in large amounts. Its toxicity differs according to the routes of introduction: via the digestive route, because it is absorbed little or inactivated by the digestive enzymes, it is approximately 1000 times less toxic than via the pulmonary route (inhalation) or the parenteral route. There are many symptoms of poisoning, which depend on the route of introduction, and they appear in a few hours and can result in death in 2 to 3 days. There is no antidote in the event of poisoning, treatment being essentially symptomatic. Since ricin is also very soluble in water and can disperse in aerosol form, this toxin is considered to be a major bioterrorist agent (category B agent on the list of the US centers for disease control (CDC, Atlanta)).
In view of the potential for the use of ricin, of other intracellular toxins or of viruses as a biological weapon, it is therefore essential to have medical countermeasures which inhibit the action of a toxin or of a virus.
In order to counter the threat posed by ricin, several types of antitoxins have been developed: neutralizing antibodies, enzymatic activity inhibitors (small molecules and substrate analogs, soluble receptor mimics).
The enzymatic activity inhibitors were described following the elucidation of the mechanism of action of ricin studied firstly by Robertus et al. (Lord, J. M.; Robertus, L. M.; Robertus, J. D. FASEB J. 1994, 8, 201). This author described the depurination mechanism of ricin, the N-glycosylase attacking the 28S RNA of the ribosome. After cleavage of an adenine base (A4324), the rRNA obtained will no longer be able to bind the elongation factors necessary for the movement of the ribosome along the mRNA, which stops protein synthesis and causes death of the cell.
The adenosine targeted is stabilized at the active site by formation of H bonds between the purine ring of the adenosine and certain residues of RTA: valine at position 81 (V81), glutamic acid at position 177 (E177) and arginine at position 180 (R180).
R180 will subsequently allow partial protonation of the adenine base, weakening the link between the base and the ribose. The adenine is then released and the oxonium ion formed, stabilized by E177, is trapped by the water activated by R180, thus forming the ribose.
Thus, there are among the enzyme activity inhibitors:
However, pteroic acid, and also its derivative, are mediocre inhibitors of the N-glycosylase activity, with an inhibition constant of about 0.6 mM (Miller, D.; Ravikumar, K.; Shen, H.; Suh, J.; Kerwin, S.; Robertus, J. D. J. Med. Chem. 2002, 45, 90. Yan, X. et al. J. Mol. Biol. 1997, 266, 1043).
Since ricin recognizes a very precise sequence in the 28S RNA, the SRL loop (Sarcin-Ricin Loop), inhibitors were designed on the basis of this nucleotide sequence. They are transition state analogs of the natural substrate for ricin which have noncleavable groups at the level of the target adenine. The studies by Schramm (Schramm, V. L. et al. Biochemistry 2001, 40 (23), 6845; Roday, S. et al. Biochemistry 2004, 43, 4923) thus made it possible to identify various inhibitors, one of the best compounds of which (P14) is represented below. This modified RNA is capable of inhibiting the enzymatic activity in vitro, with a Ki of 0.18 μM.
In addition, the development of circular oligonucleotides derived from the sequence of the SRL loop is materialized through the obtaining of molecules which inhibit RTA at micromolar and nanomolar concentrations (Sturm, M. B.; Roday S.; Schramm, V. L. J. Am. Chem. Soc., 2007, 129, 5544-5550).
In vitro selection methods were also used to generate RNA ligands, or aptamers, specific for the RTA catalytic chain. These aptamers bear no resemblance to the natural RTA substrate (SRL loop) and are not depurinated by ricin. This study made it possible to identify an aptamer of 31 nucleotides (31RA) capable of interacting with RTA (high-affinity complex, Kd=7.3 nM) and of competitively inhibiting ribosome depurination (IC50=100 nM) (Hesselberth, J. R.; Miller, D.; Robertus, J. D.; Ellington, A. D. J. Biol. Chem. 2000, 275, 4937).
The therapeutic potential of all these enzymatic inhibitors nevertheless appears to be weak. This is because they are compounds that are active only on enzymatic tests; no cell protection or protection on animals has been reported for these molecules. Furthermore, these molecules exhibit deficiencies such as a weak efficacy as regards pteroic acid and its derivatives, or instability in biological media and poor effectiveness in penetrating cells, as regards the RNA derivatives.
Neutralizing monoclonal antibodies have also been developed. Thus, antibodies directed against the RTB chain are capable of protecting mice poisoned with 10 LD50 (Lemley, P. V.; Amanatides, P.; Wright, D. C. Hybridoma 1994, 13, 417. Guo, J. W.; Shen, B. F.; Feng, J. N.; Sun, Y. X.; Yu, M.; Hu, M. R. Hybridoma 2005, 24, 263. Furukawa-Stoffer, T. L.; Mah, D. C.; Cherwonogrodzky, J. W.; Weselake, R. J. Hybridoma 1999, 18, 505).
Several studies are in the process of being carried out in order to develop neutralizing “humanized” antibodies that can be used in the event of ricin poisoning. However, immunotherapy has several disadvantages: (i) its efficacy is linked to it being rapidly administered since the antibodies cannot rescue the poisoned cells, they act only on extracellular ricin, and (ii) it appears to be relatively ineffective in the event of aerial or digestive poisoning, since the antibodies are unable to reach the affected pulmonary or digestive epithelium.
Studies based on the use of soluble receptor mimics for trapping ricin have also been carried out. This involves sugar derivatives (including dendrimers) that do not have an inhibitory effect greater than that of lactose alone (Rivera-Sagredo, A.; Solis, D.; Diaz-Mauriro, T.; Jimenez-Barbero, J.; Martin-Lomes, M. Eur. J. Biochem. 1991, 197, 217. and Dawson, R. M.; Alderton, M. R.; Wells, D.; Hartley, P. G. J. Appl. Toxicol. 2006, 26, 247).
Lactose
Galactose
Galactose with anomeric lipid chain
Other sugars carrying a water-insoluble lipid chain in the anomeric position have also been synthesized. They form a self-assembled lyotropic gel which is capable of sequestering ricin by means of the galactose-based surfactants.
A certain number of vaccines have been described in particular in patents by the US ARMY. They claim protection with respect to ricin via the administration of an immunogenic amount of RTA or RTB derivatives (U.S. Pat. No. 6,869,787).
However, a vaccine approach for countering the effects of ricin does not appear, at the current time, to be an effective response against this threat.
This is because only a few categories of individuals, identified as potentially exposed to ricin, could be preventively vaccinated. As things stand, it is not envisioned to vaccinate the population against this bioagent.
More recently, Haslam et al. (Saenz, J. B.; Doggett, T. A.; Haslam, D, B. Identification and Characterization of Small Molecules That Inhibit Intracellular Toxin Transport”, Infect. Immun. 2007, 75, 4552-4561) have described a high-throughput screening on a cell assay (Vero monkey kidney cells) for searching for ricin inhibitors, and have presented the following compounds:
The mode of action of these compounds is not specified, but these molecules appear to block the transport of the toxin inside the cells (endosomes and Golgi apparatus). However, these compounds, in particular compound A, which is a benzodiazepine derivative, have been tested and it has been noted that they do not protect A549 human pulmonary epithelial cells, unlike the compounds which are subjects of the present invention.
Thus, to date, these strategies have not made it possible to identify compounds capable of effectively protecting cells or animals exposed to ricin. The antibodies which are effective in the case of ricin injection are not affected in the case of inhalation or ingestion.
In conclusion, no specific treatment is yet available in humans for combating ricin poisoning.
In this perspective, the inventors have identified, by high-throughput screening, molecules capable of protecting cells in culture brought into contact with ricin. The mode of action of these compounds remains unknown even though they appear to act at the cellular level—and not directly on the toxin—probably by modifying one or more steps of the intracellular trafficking of ricin. Chemical optimization of these compounds has made it possible to progress to compounds having a greater cell protection capacity.
It should be emphasized that these compounds are the first inhibitors active on human pulmonary and digestive epithelial cells with respect to the toxic activity of ricin that have been identified to date.
Given that the compounds identified act at the cellular level by modifying the intracellular trafficking of ricin, these compounds may also be capable of blocking the internalization of other AB toxins and of viruses. This is because AB toxins and viruses (Sieczkarski, S. B., Whittaker, G. R. Dissecting virus entry via endocytosis, J. Gen. Virol. 2002, 83, 1535) exploit cellular trafficking pathways which are partly in common with those used by ricin. Thus, cell protection has been obtained with respect to diphtheria toxin and verotoxin-2 (Shiga-like toxin) in the presence of some of the compounds identified by screening.
The subject of the present invention is thus compounds having the property of protecting eukaryotic cells against the effects of toxins with intracellular activity, such as ricin, botulinum toxins, diphtheria toxins, anthrax toxins, cholera toxin, pertussis toxin, Shiga toxin (verotoxin-2) Escherichia coli thermolabile toxins, the major clostridial toxins, dermonecrotic factors and viruses which use the internalization pathway for infecting cells, for example RNA viruses, Flaviviridae (such as dengue or yellow fever), Orthomyxoviridae (such as the flu) or else Rhabdoviridae (such as rabies). This property is particularly advantageous since, during ricin poisoning via the respiratory route (inhalation) or by absorption (ingestion), these cells are the first that come into contact with the toxin.
Firstly, the invention relates to the use of compounds of general formula (I)
in which:
Cy represents a group chosen from:
W is chosen from a hydrogen atom or a halogen atom, Y is chosen from a hydrogen atom or a hydroxyl function and Z is a carbon atom or a bond (Cy is then a noradamantyl nucleus),
it being understood that, when Cy is an adamantyl nucleus, the chain
is attached thereto in position 1 or 2,
p represents 0 or 1;
X represents either:
R1 represents a radical containing 1 to 21 carbon atoms, which is optionally branched and/or cyclic, and saturated or unsaturated, in which one or more of the carbon atoms can be replaced with a nitrogen, oxygen and/or sulfur atom; said radical being optionally monosubstituted or disubstituted with a halogen atom, a —COOH, —OH or —NO2 function, a C1-C3 alkyl radical, a C1-C3 alkoxy radical or a C1-C3 acyloxy radical;
R2 either represents a bond or is chosen from a hydrogen atom, an optionally unsaturated, optionally branched, C1-C3 alkyl radical, a C2-C4 acyl radical or the radical
it being understood that, when R2 is a bond, then the nitrogen atom bearing R2 and X or the adjacent carbon atom (when p=1) are linked by a double bond,
it being understood that X, R1 and R2 can form, with the adjacent nitrogen atom, an imidazole, oxazole, triazole or benzimidazole ring, which is optionally partially saturated, such as in particular dihydroimidazole, optionally substituted with a phenyl or pyridine radical;
and the pharmaceutically acceptable salts thereof,
for the preparation of a pharmaceutical composition intended for the prevention and/or treatment of poisonings with at least one toxin with an intracellular mode of action or with at least one virus that uses the internalization pathway for infecting mammalian eukaryotic cells.
The invention also relates to the pharmaceutically acceptable salts of these compounds, such as hydrochlorides, hydrobromides, sulfurtes or bisulfurtes, phosphates or hydrogen phosphates, acetates, oxalates, benzoates, succinates, fumarates, maleates, lactates, citrates, tartrates, gluconates, methanesulfonates, benzenesulfonates and para-toluenesulfonates.
The term “halogen atom” is intended to mean the chemical elements of group VII of the Periodic Table of Elements, in particular fluorine, chlorine, bromine and iodine. The halogen atoms that are preferred for implementing the present invention are bromine (Br) and fluorine (F).
The term “C1-C3 alkyl chain or radical” denotes, respectively, a linear or branched hydrocarbon-based chain or radical; mention may be made, for example, of methyl, ethyl, propyl or isopropyl.
The term “C1-C3 alkoxy radical” is intended to mean an —OCnH2n+1 radical, n being an integer between 1 and 3; mention may be made, for example, of the methoxy, ethoxy, propyloxy or isopropyloxy radical. Preferably, n is 1.
The term “C1-C3 acyloxy radical” is intended to mean an —O(CO)CnH2n+1 or —(CO)OCnH2n+1 radical, n being an integer between 1 and 3; mention may be made, for example, of the acetyl radical. Preferably, n is 1.
The term “C1-C3 acyl radical” is intended to mean a —(CO)CnH2n+1 radical, n being an integer between 1 and 3.
According to one particular embodiment of the invention, the R1 radical containing 1 to 21 carbon atoms, which is optionally branched and/or cyclic, and saturated or unsaturated, in which one or more of the carbon atoms can be replaced with a nitrogen, oxygen and/or sulfur atom, is chosen from: a linear or branched, saturated or unsaturated alkyl radical containing from 1 to 8 carbon atoms, a saturated or unsaturated cyclic radical containing 5 or 6 carbon atoms, a saturated or unsaturated bicyclic radical containing 9 or 10 carbon atoms, a saturated or unsaturated tricyclic radical containing 14 carbon atoms, a saturated or unsaturated heterocyclic radical containing 5 atoms, a saturated or unsaturated heterocycle containing 6 atoms, and a saturated or unsaturated biheterocyclic radical containing 9 or 10 atoms.
More specifically, the linear or branched, saturated or unsaturated alkyl radical containing from 1 to 8 carbon atoms is chosen from: a tert-butyl, 2,4,4-trimethylpentanyl, 3-hydroxy-2-methylpropanoate and hydroxymethylpropane-1,3-diol radical.
The cyclic radicals listed above are preferably chosen from the radicals: cyclopentylmethanol, cyclomethanoate, phenyl, cyclohexyl, pyridine, furan, thiophene, imidazole, quinoline, indole, benzofuran, adamantyl, naphthalene, anthracene, and the following cyclic radicals:
with W′ being —H or —COOCnH2n+1, with n being between 1 and 3,
Preferably, the compounds of general formula (I) are such that R1 is an optionally substituted phenyl radical and/or X is —CH2— and/or R2 is a hydrogen atom and/or W represents a Br atom.
According to one particular variant of the invention, the compounds of general formula (I) are such that R1 is the radical:
in which:
According to another variant of the invention, the compounds of general formula (I) are defined by general formula (I′):
where W, p, R3 and R4 are as defined above and X is either a bond or —CO—.
More particularly, the compounds of general formula (I) are chosen from:
The present invention also relates to the compounds of general formula (I) as defined above and the pharmaceutically acceptable salts thereof as such, except for compounds 1, 6, 8, 11, 18, 29, 30, 34, 74, 78, 98, 100, 113, 114, 116, 121, 122, 124, 128, 135, 145 and 161.
The compounds which are aminoadamantane derivatives were prepared by reductive amination according to the reaction which follows:
Specifically, the invention relates to the process for preparing the compounds of general formula (Ia):
in which:
In a more detailed manner, the compounds of general formula (Ia) which appear in tables A1 and A2 according to example 1 are prepared in methanol by treatment of 1-adamantylamine (for the compounds of table A1) or of 2-adamantylamine (for the compounds of table A2) in the presence of an aromatic aldehyde (1 equiv.) according to the compound to be prepared. Supported cyanoborohydride is used (BH3CN on resin, 1.5 equiv.) as reducing agent in the presence of acetic acid (3 equiv.). The whole is stirred for 2 days at ambient temperature, filtered, washed with methanol, evaporated, and then purified according to methods known to those skilled in the art.
This same process makes it possible to prepare the derivatives of general formula (Ib):
in which:
it being understood that, when R2 is nothing, then the nitrogen atom and X are linked by a double bond.
The invention therefore also relates to a process for preparing the compounds of general formula (Ib), characterized in that it comprises the following steps:
In a more detailed manner, the compounds (Ib) that appear in table A3 and in table A4 are prepared by adding, with stirring, a suspension of an amine (1 equiv.) chosen according to the compound to be prepared (see tables A3 and A4 according to example 1) in methanol to an aldehyde (1 equiv.) for the compounds of table A3 or of 3-bromobenzylamine (1 equiv.) and an aldehyde in order to obtain the compounds of table A4, in the presence of 1.5 equiv. of BH3CN on resin and of acetic acid (3 equiv.). The whole is stirred for 2 days at ambient temperature, filtered, washed with methanol, evaporated, and then purified according to methods known to those skilled in the art.
The formation of the salts is carried out by treatment of the amine compound (corresponding to the desired salt) in CH2Cl2 with a solution of the corresponding acid in a solvent (for example, HCl in ether).
The precipitate is filtered off and dried under vacuum so as to give the salt of the acid (for example, hydrochloride in the case of HCl). They appear in table B of example 1.
The 1-aminoadamantane, 2-aminoadamantane or noradamantylamine derivatives of general formula (Ic)
in which:
with Z being a carbon atom or nothing (noradamantyl nucleus), with the nitrogen atom in position 1 or 2 when Cy is the adamantyl nucleus;
Thus, the invention relates to the process for preparing the 1-aminoadamantane, 2-aminoadamantane or noradamantylamine derivatives of general formula (Ic), characterized in that it comprises the following steps:
The preparation of the imines is carried out by adding a solution of the amine in MeOH to the aldehyde, the amine and the aldehyde being chosen according to the imine to be prepared, and stirring the mixture for 2 days. After evaporation and purification, the imines are obtained.
The imines are reduced as follows: BH3CN on resin (3 equiv.) and AcOH are added to a solution of the imine in MeOH. After 3 days at ambient temperature, the mixture is filtered, washed with methanol, and then concentrated under vacuum. The resulting crude compound is purified according to conventional methods.
The invention also relates to a process for preparing the imines of general formula (I), characterized in that it comprises the following steps:
The invention also relates to a process for preparing a reduced imine of general formula (I), characterized in that it comprises the following steps:
The N-1-adamantylbenzamide and N-2-adamantylbenzamide compounds are prepared from the corresponding amines (1- or 2-adamantylamine (1 equiv.)) via treatment with a base such as NaH (1.1 equiv.) in DMF then addition of benzoyl chloride (1.2 equiv.) at 0° C. The mixture is stirred for 24 hours at ambient temperature, evaporated, and then washed with cyclohexane.
Finally, the invention relates to a process for preparing the N-1-adamantylbenzamide and N-2-adamantylbenzamide compounds comprising the following steps:
The invention also relates to a process for preparing a urea derivative of general formula (I), characterized in that it comprises the following steps:
The alcohols are firstly treated with iodine (2.5 equiv.) and a base such as potassium carbonate in tert-butanol and are heated for 24 h at 70° C. The nucleophile (amine, alcohol) is then added (1.5 equiv.). After conventional treatment and purification, the alkylated compounds are obtained.
The invention also relates to a process for preparing triazole derivatives of general formula (I), characterized in that it comprises the following steps:
The invention also relates to 2-amino-N-phenylbenzamide as synthesis intermediate for the amines of general formula (I).
The present invention also relates to the use of compounds which are benzodiazepine derivatives of general formula (II):
where
A and B represent a carbon atom or a nitrogen atom with the proviso that, if A=N then B=C, and if A=C then B=N;
R3 is chosen from a hydrogen or halogen atom; a C1-C6 alkyl radical, a C1-C6 alkoxy radical or a C1-C6 acyloxy radical, these radicals being optionally substituted with a C1-C6 alkoxy radical; an aryloxy radical or a heteroaryloxy radical;
R4 either represents a bond or is chosen from a hydrogen atom, a C1-C3 acyloxy radical, a C1-C3 alkoxy radical or a phenyl;
R5 either represents a bond or is chosen from a hydrogen atom; a C1-C3 alkyl radical; a C1-C3 alkoxy radical; a C1-C3 acyloxy radical or a phenyl radical which is optionally substituted with an —OH function and/or a halogen atom, a C1-C3 alkyl radical, a C1-C3 alkoxy radical, a C1-C3 acyloxy radical, an —NO2 or —CF3 function, or a radical
it being understood that R4 and R5 cannot simultaneously represent a bond and that, when one of the two is a bond, then A and B are linked by a double bond; and that, when B is a carbon atom, R5 can also form, with the hydrogen atom borne by the carbon adjacent to B, a ring of 5 or 6 atoms, optionally substituted with a phenyl radical, optionally interrupted with a nitrogen, sulfur or oxygen atom; preferably, it is a ring containing 5 atoms, interrupted with an oxygen atom; for the preparation of a pharmaceutical composition intended for the prevention and/or treatment of poisonings with at least one toxin with an intracellular mode of action or with at least one virus that uses the internalization pathway for infecting mammalian eukaryotic cells.
The invention also relates to the pharmaceutically acceptable salts of these compounds.
The term “C1-C6 alkyl radical” denotes a linear or branched hydrocarbon-based radical containing from 1 to 6 carbon atoms; mention may be made, for example, of methyl, ethyl, propyl, or isopropyl.
The term “C1-C6 alkoxy radical” is intended to mean an —OCmH2m+1 radical, m being an integer between 1 and 6.
The term “C1-C6 acyloxy radical” is intended to mean an —O(CO)CmH2m+1 or —(CO)OCmH2m+1 radical, m being an integer between 1 and 6.
The term “aryloxy radical” is intended to mean an aryl group linked to the rest of the compound by an oxygen atom.
The term “heteroaryloxy radical” is intended to mean a heteroaryl group linked to the rest of the compound by an oxygen atom.
Preferably, the compounds of general formula (II) are such that R3 is a bond and/or R4 represents a hydrogen atom and/or R5 represents a phenyl radical and/or, when A is a carbon atom, then R4 is a phenyl radical, and/or, when B is a carbon atom, then R5 is a phenyl radical.
More particularly, the compounds of general formula (II) are chosen from:
The present invention also relates to the compounds of general formula (II) and the pharmaceutically acceptable salts thereof as such, except for compounds 162, 163, 164, 165, 166, 167, 169, 170, 171 and 193.
The synthesis of the compounds of general formula (II) according to the invention is described in example 1.
More particularly, the benzo[e][1,4]diazepine derivatives and the benzo[b][1,4]diazepine derivatives of general formula (II) are prepared as follows:
The invention also relates to the compounds which are of use as synthesis intermediates for the compounds of general formula (II), chosen from: tert-butyl N-[(phenylcarbamoyl)methyl]carbamate, tert-butyl(4-methoxyphenylcarbamoyl)methylcarbamate, 2-amino-N-phenylacetamide, 2-amino-N-(4-methoxyphenyl)acetamide and 2-benzoyl-4-bromoaniline.
The compounds according to the invention are pharmacologically active substances and are of value by virtue of their inhibitory effect on toxins with an intracellular mode of action, in particular on ricin.
According to another of its subjects, the invention relates to the compounds of general formulae (I) and (II) and the pharmaceutically acceptable salts thereof except for compounds 1, 6, 8, 11, 18, 29, 30, 34, 74, 78, 98, 100, 113, 114, 116, 121, 122, 124, 128, 135, 145, 161, 162, 163, 164, 165, 166, 167, 169, 170, 171 and 193, for use as a medicament, in particular as an active ingredient.
The invention also relates to pharmaceutical compositions or medicaments comprising one or more compounds of general formula (I) or (II) and the pharmaceutically acceptable salts thereof except for compounds 1, 6, 8, 11, 18, 29, 30, 34, 74, 78, 98, 100, 113, 114, 116, 121, 122, 124, 128, 135, 145, 161, 162, 163, 164, 165, 166, 167, 169, 170, 171 and 193, in a pharmaceutically acceptable vehicle.
The term “pharmaceutically acceptable” is intended to mean compatible with administration to an individual, preferably a mammal, by any route of administration.
Those skilled in the art will be capable of adapting the formulation of the compounds of general formulae (I) and (II) according to their physicochemical properties and their route of administration.
The medicament may be administered by the oral, parenteral, pulmonary, ocular, nasal, etc., route. The modes of administration of the compounds (I) and (II) that are preferred are those which use the aerial (inhalation), oral (ingestion), parenteral or local (topical) routes.
The amount of compound of formula (I) or (II) to be administered to the mammal depends on the actual activity of this compound, it being possible for said activity to be measured by means which are disclosed in the examples. This amounts also depends on the seriousness of the pathological condition to be treated, in particular on the amount of ricin absorbed and on the route via which it was absorbed; finally, it depends on the age and the weight of the individual to be treated.
The use of the compounds of general formula (I) or (II) is particularly advantageous for preventing and/or treating disorders caused by AB toxins with an intracellular mode of action and viruses that use the internalization pathway for infecting cells.
More specifically, the AB toxins or toxins with an intracellular mode of action are in particular: ricin, botulinum toxins, diphtheria toxins, anthrax toxins, cholera toxin, pertussis toxin, Shiga toxin (verotoxin-2), Escherichia coli thermolabile toxins, the major clostridial toxins, and dermonecrotic factors; examples of these toxins are listed in the table below:
C. diphtheriae (+)
P. aeruginosa (−)
C. Botulinum (+)
C. tetani (+)
C. sordellii (+)
C. difficile (+)
C. novyi (+)
B. pertussis (−)
E. coli (−)
V. cholerae (−)
E. coli (−)
B. pertussis (−)
S. dysenteriae (−)
E. coli (−)
B. anthracis (+)
C. botulinum (+)
C. perfringens (+)
C. spiroforme (+)
C. difficile (+)
B. cereus (−)
Various Activities, Molecular Targets and Structures of the Main Bacterial Toxins with an Intracellular Action
The viruses that use the internalization pathway for infecting cells, hereinafter also denoted viruses, are, for example, RNA viruses, Flaviviridae (such as dengue or yellow fever), Orthomyxoviridae (such as the flu) or else Rhabdoviridae (such as rabies).
This use proves to be effective whether the individual touches, ingests or inhales the toxin or the virus, or else whether the toxin or the virus is injected into said individual.
Thus, these compounds may be used for the preparation of a pharmaceutical composition intended for treating the effects of AB toxins or toxins with an intracellular mode of action and of viruses that use the internalization pathway for infecting cells.
Concretely, a toxin such as ricin, when it is inhaled, produces signs of ocular irritation (burning sensation, watering of the eyes, more or less severe conjunctivitis) and pharyngeal irritation and also a more or less marked respiratory irritation: cough, dyspnea, pulmonary edema which can result in acute respiratory distress syndrome (ARDS). It should be noted that there is a risk of anaphylactic reaction. The lethal dose is 1 mg/kg (Ministry of Health, France).
Thus, the invention relates to the use of a compound of general formula (I) or (II), for the preparation of a pharmaceutical composition intended for protection against the effects of ricin, of other AB toxins and of viruses that use the internationalization pathway for infecting eukaryotic cells, especially epithelial, ocular, pharyngeal, tracheal, bronchial, skin or muscle cells, in particular pulmonary and digestive, preferably intestinal, epithelial cells, of mammals, preferably of humans.
The invention relates more specifically to the use of the compounds of general formulae (I) and (II) and the pharmaceutically acceptable salts thereof, for the preparation of a pharmaceutical composition for preventing and/or treating poisoning with ricin toxin; preferably, the compounds of general formulae (I) and (II) and the pharmaceutically acceptable salts thereof are chosen from compounds 1, 3, 5, 9, 15, 19, 24, 28, 34, 36, 39, 59, 65, 67, 68, 69, 75, 86, 89, 105, 106, 109, 110, 111, 112, 117, 118, 122, 128, 131, 138, 139, 140, 142, 143, 148, 154, 161 and 193.
According to one preferred variant, the invention relates to the use of compounds of general formula (I) which are defined by general formula (I.1):
in which:
Cy represents a group chosen from:
Z is a carbon atom or a bond (Cy is then a noradamantyl nucleus),
it being understood that, when Cy is an adamantyl nucleus, the nitrogen atom is attached thereto in position 1 or 2,
R1 represents:
Preferably, the compounds of general formula (I.1) are chosen from compounds 1, 3, 5, 9, 15, 19, 24, 28, 34, 36, 39, 59, 65, 86, 109, 110, 111, 112, 138, 139, 140, 142, 143 and 148.
According to another preferred variant, the invention relates to the use of compounds of general formula (I) which are defined by general formula (I.2):
in which X represents —(CH2)2—O—CH2—, —(CH2)3—, —CO— or —SO2—,
and the pharmaceutically acceptable salts thereof,
for the preparation of a pharmaceutical composition for preventing and/or treating poisoning with ricin toxin.
Preferably, the compounds of general formula (I.2) are chosen from compounds 68, 69, 122 and 128.
According to yet another preferred variant, the invention relates to the use of compounds of general formula (I) which are defined by general formula (I.3):
in which W represents a halogen atom,
and the pharmaceutically acceptable salts thereof,
for the preparation of a pharmaceutical composition for preventing and/or treating poisoning with ricin toxin.
Preferably, the compounds of general formula (I.3) are chosen from compounds 117 and 118.
Another preferred variant of the invention relates to the use of the compounds of general formula (I.4):
in which Y is O or S,
and the pharmaceutically acceptable salts thereof,
for the preparation of a pharmaceutical composition for preventing and/or treating poisoning with ricin toxin.
Preferably, the compounds of general formula (I.4) are chosen from compounds 154 and 161.
The present invention also relates to the use of the compounds of general formulae (I) and (II) and the pharmaceutically acceptable salts thereof, for the preparation of a pharmaceutical composition for preventing and/or treating poisoning with diphtheria toxin.
The compounds suitable for the prevention and/or treatment of poisoning with diphtheria toxin are in particular those of general formula (I.5):
in which:
Cy represents a group:
to which the nitrogen atom is attached in position 1 or 2,
W and W′ are, independently of one another, chosen from a hydrogen atom, a halogen atom and a C1-C3 alkoxy radical,
and the pharmaceutically acceptable salts thereof.
Preferably, the compounds of general formula (I.5) are chosen from compounds 5, 9, 39, 110, 111, 112, 139 and 140.
In addition to the above arrangements, the invention also comprises other arrangements which will emerge from the description that follows, which refer to examples of implementation of the present invention, and also to the appended figures in which:
The commercial reactants were purchased from Sigma-Aldrich and were used without prior purification. All the reactions were carried out under nitrogen with freshly distilled dry solvents and oven-dried glassware.
The purification methods used for preparing the compounds are specified in the “Purification method” column of the tables and coded as follows:
2: Short pad (small column with silica already packaged)
3: Silica gel chromatography column
6: Aqueous treatment then separation.
The 1H NMR was carried out with a Brucker Advance 400 MHz instrument with a BBO probe. The solvents are specified for each experiment. The chemical shifts are given in parts per million (ppm), relative to the internal reference (TMS). The data are listed in the following order: δ, chemical shift; multiplicity (with s singlet, d doublet, t triplet, q quadruplet, m, multiplet), integration, coupling constants (J in Hertz, Hz).
The LC/MS analyses were carried out by HPLC (High Pressure Liquid Chromatography) coupled with a Waters® Autopurif mass spectrometer.
The ionization is obtained either by electron impact or by electrochemical ionization.
The data are obtained in m/z form.
Column: Xbridge C18 3-5 μM, 4.6 mm×100 mm
Flow rate: 1 ml/min
Detectors:
Injection volume: 1 μl with the Waters 2767 autosampler.
Method: 95% solution A (99.99% water, 0.01% formic acid), 5% B (100% acetonitrile) to 0% A, 100% B on a gradient of 8 minutes, then 5-minute stage.
The column chromatographies were carried out with a
Merck silica gel (particle size: 230-400 mesh). All the reactions were monitored by thin-layer chromatography with plates precoated with 0.2 mm-thick silica gel 60G-264 (Merck). Developing was carried out with a UV lamp or with diode.
The 1-aminoadamantane and 2-aminoadamantane derivatives of general formula (Ia) as described above and which appear, respectively, in tables A1 and A2 are prepared as follows: a suspension, in methanol, of 1-adamantylamine or 2-adamantylamine (0.5 mmol; 75 mg; 1 equiv.) is added, with stirring, to an aromatic aldehyde (1 equiv.) according to the compound to be prepared, in the presence of BH3CN on resin (0.75 mmol; 1.5 equiv.) and of acetic acid (1.5 mmol; 84 μl; 3 equiv.). The whole is stirred for 2 days at ambient temperature, filtered, washed with methanol, evaporated and then purified.
Tables A1 and A2 give the number of the compound, the aldehyde used, the name of the compound obtained, the characteristics of the compound and also the code of the purification method used, the appearance of the compound obtained and its yield.
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (DMSO-d6,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3.
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3, 400 MHz): δ
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3, 400 MHz): δ
1H NMR (CDCl3, 400 MHz): δ
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3, 400 MHz): δ
13C NMR (CDCl3, 100 MHz):
1H NMR (CDCl3, 400 MHz): δ
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3, 400 MHz): δ
13C NMR (CDCl3, 100 MHz):
1H NMR (CDCl3,
Similarly, the derivatives and the alkylamines of general formula (Ib) as described above and which appear in table A3 and in table A4 are prepared by adding, with stirring, a suspension of an amine (1 equiv.) chosen according to the compound to be prepared (see tables A3 and A4) in methanol to 3-bromobenzaldehyde or 3-fluorobenzaldehyde (1 equiv.) for the compounds of table A3, or 3-bromobenzylamine or 3-fluorobenzylamine (1 equiv.) for obtaining the compounds of table A4, in the presence of BH3CN on resin (0.75 mmol; 1.5 equiv.) and of acetic acid (1.5 mmol; 84 μl; 3 equiv.). The whole is stirred for 2 days at ambient temperature, filtered, washed with methanol, evaporated, and then purified according to methods known to those skilled in the art.
Tables A3 and A4 give the number of the compound, the reactants used, the name of the compound obtained, the characteristics of the compound and also the code of the purification method used, the appearance of the compound obtained and its yield.
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
1H NMR (CDCl3, 400 MHz):
1H NMR (CDCl3, 400 MHz):
1H NMR (CDCl3, 400 MHz):
1H NMR (CDCl3, 400 MHz):
1H NMR (CDCl3, 400 MHz):
13C NMR (CDCl3,
1H NMR (CDCl3,
13C NMR (CDCl3,
13C NMR (CDCl3,
13C NMR (CDCl3,
The compounds of table A5 are prepared according to process A, adding a microwave heating step.
1H NMR (CDCl3, 400 MHz): δ 7.38 (s, 1H),
13C NMR (CDCl3, 100 MHz): δ 139.2,
1H NMR (CDCl3, 400 MHz): δ 7.47 (s, 1H),
13C NMR (CDCl3, 100 MHz): δ 135.3,
1H NMR (CDCl3, 400 MHz): δ 7.80 (s, 1H),
13C NMR (CDCl3, 100 MHz): δ 138.6,
1H NMR (CDCl3, 400 MHz): δ 7.73 (bs,
13C NMR (CDCl3, 100 MHz): δ 140.7,
1H NMR (CDCl3, 400 MHz): δ 8.14 (d, 1H,
13C NMR (CDCl3, 100 MHz): δ 133.7,
1H NMR (CDCl3, 400 MHz): δ 8.74 (m, 1H),
13C NMR (CDCl3, 100 MHz): δ 131.0,
1H NMR (CDCl3, 400 MHz): δ 7.83 (d, 2H,
13C NMR (CDCl3, 100 MHz): δ 170.4,
A solution of HCl in Et2O (2N) is added to a solution of the amine (corresponding to the desired hydrochloride salt) in CH2Cl2. The salt is obtained after filtration and drying of the precipitate under vacuum.
1H NMR (CDCl3, 400 MHz): δ 9.55 (bs, 1H),
1H NMR (CDCl3, 400 MHz): δ 8.94 (bs, 1H),
13C NMR (DMSO-d6, 100 MHz): δ 155.7, 133.2,
1H NMR (CDCl3, 400 MHz): δ 9.84 (bs, 1H),
13C NMR (CDCl3, 100 MHz): δ 1133.3, 132.5,
The 1-aminoadamantane, 2-aminoadamantane or noradamantylamine derivatives of general formula (Ic) as described above and which appear, respectively, in tables C1, C2 and C3 are prepared as follows: a stirred suspension of 1-aminoadamantane, 2-aminoadamantane or noradamantylamine (1 equiv.) in methanol is added to an aldehyde (1 equiv.) chosen according to the compound to be prepared (see tables C1, C2 and C3). The whole is mixed for two days at ambient temperature and then evaporated.
1H NMR (DMSO,
1H NMR (DMSO,
1H NMR (DMSO,
13C NMR (CDCl3,
1H NMR (DMSO,
1H NMR (CDCl3,
1H NMR (CDCl3,
The N-1-adamantylbenzamide and N-2-adamantylbenzamide compounds are prepared as follows: 1-adamantylamine or 2-adamantylamine (1 equiv.) and benzoyl chloride (1.2 equiv.) are added, at 0° C., to a suspension of NaH (1.1 equiv.) in DMF. The mixture is stirred for 24 hours at ambient temperature, evaporated, and then washed with cyclohexane.
1H NMR (CDCl3, 400 MHz): δ
13C NMR (CDCl3, 100 MHz):
1H NMR (CDCl3, 400 MHz): δ
The sulfonylation is carried out according to the following process:
Et3N (138 μl; 1 mmol; 5 equiv.) and benzenesulfonyl chloride (76 al; 0.6 mmol; 3 equiv.) are added to a solution of 1-adamantylamine hydrochloride or 2-adamantylamine hydrochloride (0.2 mmol; 1 equiv.) in CH2Cl2 (1 ml). The mixture is stirred at ambient temperature for 24 h, and hydrolyzed with NH4Cl. The organic phase is washed three times with water, dried over MgSO4, filtered and evaporated to give the expected product.
Starting from 1-adamantylamine, the product obtained is a brown solid (86%).
1H NMR (CDCl3, 400 MHz): δ 7.91 (dd, 2H, J=1.6 and 7.2 Hz), 7.53 (m, 3H), 2.01 (s, 3H), 1.79 (m, 6H), 1.58 (m, 6H).
13C NMR (CDCl3, 100 MHz): δ 143.9, 132.0, 128.8, 126.8, 55.2, 43.0, 35.0, 29.4.
Starting from 2-adamantylamine, the product obtained is a yellow solid (86%).
1H NMR (CDCl3, 400 MHz): δ 7.89 (dd, 2H, J=1.9 and 7.2 Hz), 7.55 (m, 3H), 3.43 (s, 1H), 1.79-1.53 (m, 14H).
13C NMR (CDCl3, 100 MHz): δ 141.2, 132.3, 129.0, 126.8, 57.9, 37.3, 37.1, 32.7, 31.1, 26.8, 26.7.
Phenylisocyanate or phenylthioisocyanate (0.75 mmol; 1.5 equiv.) is added, at 0° C., to a stirred suspension of 1-adamantylamine (0.5 mmol; 1 equiv.) in THF (3 ml). The mixture is stirred for 24 hours at ambient temperature and then evaporated;
or
phenylisocyanate or phenylthioisocyanate (0.75 mmol; 1.5 equiv.) and triethylamine (1.05 equiv.) are added, at 0° C., to a stirred suspension of 2-adamantylamine hydrochloride (0.5 mmol; 1 equiv.) in DMF (3 ml). The mixture is stirred for 24 hours at ambient temperature, then evaporated, placed in solution in Et2O, and filtered, and then the filtrate is evaporated.
This process makes it possible to prepare the following compounds:
Starting from 1-adamantylamine and phenylisocyanate, a white solid is obtained (95%).
1H NMR (CDCl3, 400 MHz): δ 7.35 (m, 3H), 7.11 (m, 2H), 2.18 (s, 3H), 2.00 (s, 6H), 1.68 (s, 6H).
13C NMR (CDCl3, 100 MHz): δ 160.5, 1387, 129.2, 123.7, 121.0, 51.5, 42.2, 36.3, 29.5.
ESI+MS: calcd for C17H22N2O: 270.17; found: 271.1 (MH+).
Phenylisocyanate or phenylthioisocyanate (0.75 mmol; 1.5 equiv.) and triethylamine (1.05 equiv.) are added, at 0° C., to a stirred suspension of 2-adamantylamine hydrochloride (0.5 mmol; 1 equiv.) in DMF (3 ml). The mixture is stirred for 24 h at ambient temperature, evaporated, placed in solution in Et2O and filtered, and then the filtrate is evaporated.
A white solid is obtained (69%).
1H NMR (CDCl3, 400 MHz): δ 7.34 (m, 4H), 7.11 (t, 1H, J=6.8 Hz), 3.96 (s, 1H), 1.96 (s, 2H), 1.84-1.61 (m, 12H).
ESI+MS: calcd for C17H22N2O: 270.17; found: 271.1 (MH+).
The process which follows makes it possible to prepare the compounds below: phenyl chloroformate (0.6 mmol; 1.2 equiv.) and triethylamine (2.5 mmol; 5 equiv.) are added, at ambient temperature, to a stirred suspension of 1-adamantylamine hydrochloride or 2-adamantylamine hydrochloride (0.5 mmol; 1 equiv.) in CH2Cl2 (4 ml). The mixture is stirred for 24 h at ambient temperature, washed with water, placed in a saturated solution of NH4Cl, dried over Na2SO4, filtered, evaporated and short-pad-purified.
Starting from 1-adamantylamine and phenyl chloroformate, a white solid is obtained (13%).
1H NMR (CDCl3, 400 MHz): δ 7.35 (t, 2H, J=8 Hz), 7.18 (t, 1H, J=7.2 Hz), 7.12 (d, 2H, J=7.6 Hz), 4.88 (bs, 1H), 2.11 (s, 3H), 2.01 (s, 6H), 1.69 (s, 6H).
13C NMR (CDCl3, 100 MHz): δ 150.9, 129.1, 125.0, 121.7, 51.2, 41.7, 36.3, 29.4.
ESI+MS: calcd for C17H21NO2: 271.16; found: 272.1 (MH+).
Starting from 2-adamantylamine hydrochloride and phenyl chloroformate, a white solid is obtained (17%).
1H NMR (CDCl3, 400 MHz): δ 7.36 (t, 2H, J=7.6 Hz), 7.20 (t, 1H, J=7.6 Hz), 7.14 (d, J=8 Hz), 5.36 (bs, 1H), 3.88 (s, 1H), 2.03 (s, 2H), 1.87-1.67 (m, 12H).
13C NMR (CDCl3, 100 MHz): δ 129.2, 125.1, 121.5, 114.9, 55.1, 37.4, 37.0, 32.0, 31.7, 27.1, 27.0.
ESI+MS: calcd for C17H21NO2: 271.16; found: 272.1 (MH+).
Benzoyl chloride (0.47 mmol; 55 μl; 3 equiv.) and triethylamine (109 μl; 5 equiv.) are added, at ambient temperature, to a stirred suspension of 1-adamantylamine (0.156 mmol; 50 mg; 1 equiv.) in CH2Cl2 (3 ml). The mixture is stirred for 24 h at ambient temperature, washed with water, placed in a saturated solution of NH4Cl, dried over Na2SO4, filtered, evaporated and short-pad-purified.
Starting from acetyl chloride, a colorless oil is obtained (26%).
1H NMR (CDCl3, 400 MHz): δ 7.39 (dd, 1H, J=1.2 and 10.8 Hz), 7.25 (t, 1H, J=7.6 Hz), 7.17 (d, 1H, J=7.6 Hz), 4.58 (s, 2H), 2.20 (s, 6H), 2.07 (s, 6H), 1.63 (m, 6H).
13C NMR (CDCl3, 100 MHz): δ 172.2, 142.2, 130.3, 130.1, 128.7, 124.2, 123.0, 59.3, 48.0, 39.9, 36.3, 30.1, 25.7.
ESI+MS: calcd for C19H24BrNO: 361.10; found: 362.0 (MH+).
Phenylisocyanate (19 μl; 1.1 equiv.) is added, at 0° C., to a stirred suspension of 1-adamantylamine (0.156 mmol; 50 mg; 1 equiv.) in THF (3 ml). The mixture is stirred for 24 h at ambient temperature, evaporated and short-pad-purified.
Starting from phenylisocyanate, a colorless oil is obtained (51%).
1H NMR (CDCl3, 400 MHz): δ 7.52-6.98 (m, 9H), 6.13 (bs, 1H), 4.64 (s, 2H), 2.25 (s, 6H), 2.11 (s, 3H), 1.68 (m, 6H).
13C NMR (CDCl3, 100 MHz): δ 155.5, 141.1, 138.9, 130.8, 130.6, 128.9, 128.7 124.2, 123.4, 122.9, 120.0, 58.2, 47.2, 40.4, 36.3, 30.1.
ESI+MS: calcd for C24H27 BrN2O: 438.13; found: 439.0 (MH+).
Benzoyl chloride (0.47 mmol; 55 μl; 3 equiv.) and triethylamine (109 μl; 5 equiv.) are added, at ambient temperature, to a stirred suspension of 2-adamantylamine (0.156 mmol; 50 mg; 1 equiv.) in CH2Cl2 (2 ml). The mixture is stirred for 24 h at ambient temperature, washed with water, placed in a saturated solution of NH4Cl, dried over Na2SO4, filtered, evaporated and short-pad-purified.
Starting from benzoyl chloride, a white oil is obtained (17%).
1H NMR (CDCl3, 400 MHz): δ 7.43 (m, 2H), 7.35 (m, 4H), 7.19 (s, 1H), 7.16 (t, 1H, J=8 Hz), 7.05 (d, 1H, J=7.6 Hz), 4.78 (s, 2H), 4.17 (s, 1H), 2.32 (s, 2H), 2.02-1.66 (m, 12H).
13C NMR (CDCl3, 100 MHz): δ 175.4, 142.1, 137.8, 130.2, 130.0, 129.7, 128.8, 128.4, 127.1, 124.2, 122.8, 60.2, 49.5, 38.0, 37.4, 32.8, 30.0, 27.3, 26.9.
ESI+MS: calcd for C24H26BrNO: 423.12; found: 424.8 (MH+).
Et3N (0.83 mmol: 1.1 equiv.), phenylacetylene (0.83 mmol; 1.1 equiv.) or ethynylpyridine (0.83 mmol; 1.1 equiv.), and the 1-adamantylamine-derived azide (0.75 mmol; 1 equiv.) are added to a suspension of Cu/C (50 mg) in 1,4-dioxane (1.5 ml). The mixture is stirred at 60° C. for 2 days, filtered through a short pad, washed with EtOAc and then evaporated. This process makes it possible to prepare the following compounds:
Starting from 1-azidoadamantane and phenylacetylene, a brown oil is obtained (96%).
1H NMR (CDCl3, 400 MHz): δ 7.84 (dd, 3H, J=1.2 and 8.4 Hz), 7.42 (t, 2H, J=8 Hz), 7.32 (t, 1H, J=7.2 Hz), 2.30 (s, 9H), 1.82 (s, 6H), 1.43 (m, 4H).
13C NMR (CDCl3, 100 MHz): δ 146.5, 140.0, 128.6, 127.6, 125.4, 116.0, 59.4, 42.8, 35.7, 29.3.
ESI+MS: calcd for C18H21N3: 279.17; found: 280.2 (MH+).
Starting from 1-azidomethyladamantane and phenylacetylene, a brown oil is obtained (68%).
1H NMR (CDCl3, 400 MHz): δ 7.86 (dd, 2H, J=1.2 and 8.4 Hz), 7.68 (s, 1H), 7.44 (t, 2H, J=7.2 Hz), 7.34 (t, 1H, J=7.6 Hz), 4.08 (s, 2H), 2.02 (s, 3H), 1.58 (m, 12H).
13C NMR (CDCl3, 100 MHz): δ 147.0, 130.7, 128.7, 127.9, 125.6, 120.9, 62.2, 40.2, 36.4, 34.1, 28.0.
ESI+MS: calcd for C19H23N3: 293.19; found: 294.2 (MH+).
Starting from 1-azidoadamantane and 2-ethynylpyridine, a brown oil is obtained (39%).
1H NMR (CDCl3, 400 MHz): δ 8.58 (d, 1H, J=4.4 Hz), 8.22 (m, 2H), 7.79 (t, 1H, J=7.6 Hz), 7.23 (t, 1H, J=6 Hz), 2.30 (s, 8H), 1.81 (m, 7H).
13C NMR (CDCl3, 100 MHz): δ 150.7, 149.2, 147.3, 136.9, 122.6, 120.1, 118.6, 59.8, 42.9, 35.9, 29.4.
ESI+MS: calcd for C17H20N4: 280.17; found: 281.2 (MH+).
Potassium carbonate (415 mg; 3 mmol; 3 equiv.) and iodine (635 mg; 2.5 mmol; 2.5 equiv.) are added to a solution of 1-adamantanemethanol (166 mg; 1 mmol) in tert-butanol (8 ml). The mixture is stirred at 70° C. for 16 h and the amine (1.5 equiv.) diluted in tert-butanol is added. The mixture is refluxed at 70° C. for 2 h, diluted with a saturated solution of Na2SO3 (5 ml), and extracted with chloroform (20 ml). The organic phases are washed with 2N NaOH (20 ml) and brine (20 ml), dried over Na2SO4, filtered and evaporated.
Starting from 1-adamantanemethanol and ethylenediamine, a colorless oil is obtained (66%).
1H NMR (CDCl3, 400 MHz): δ 3.59 (s, 4H), 2.04 (s, 3H), 1.89 (s, 6H), 1.73 (m, 6H).
Starting from 1-adamantanemethanol and 2-aminoethanol, a colorless oil is obtained (2.4%).
1H NMR (CDCl3, 400 MHz): δ 4.23 (t, 2H, J=9.6 Hz), 3.83 (t, 2H, J=9.6 Hz), 2.02 (s, 3H), 1.91 (s, 6H), 1.73 (m, 6H).
Starting from 1-adamantanemethanol and N-phenylethylenediamine, a yellow oil is obtained (15%).
1H NMR (CDCl3, 400 MHz): δ 7.40-7.18 (m, 5H), 3.82 (t, 2H, J=9.1 Hz), 3.68 (t, 2H, J=9.6 Hz), 1.85 (s, 3H), 1.81 (s, 6H), 1.54 (m, 6H).
13C NMR (CDCl3, 7100 MHz): δ 172.0, 145.0, 129.2, 127.1, 58.2, 52.1, 40.5, 37.5, 36.4, 28.2.
ESI+MS: calcd for C19H24N2: 280.19; found: 281.2 (MH+).
Starting from 1-adamantanemethanol and (1R,2R)-1,2-cyclohexanediamine, a yellow oil is obtained (10%).
1H NMR (CDCl3, 400 MHz): δ 4.67 (bs, 1H), 2.84 (bs, 2H), 2.03 (s, 3H), 1.87 (s, 6H), 1.72 (m, 6H).
ESI+MS: calcd for C17H26N2: 258.21; found: 259.2 (MH+).
The synthesis of the amine compounds requires the prior preparation of the following synthesis intermediate:
2-nitro-N-phenylbenzamide (1 eq., 5 mmol, expected m=1.06 g) is dissolved in 12 ml of methanol. A few drops of acetic acid are added to the reaction medium. Decaborane (0.3 eq., 1.5 mmol, 183 mg) is then added, with palladium on carbon (100 mg, 10% of the mass of the 2-nitro-N-phenylbenzamide). After 3 hours at reflux, the mixture is filtered and then purified by silica chromatography (cyclohexane/ethyl acetate in 70:30 proportions), which gives the compound 2-amino-N-phenylbenzamide with a yield of 88% (933 mg).
1H NMR (400 MHz, DMSO d6)
δ: 9.95 (s, 1H, NH), 7.70-7.68 (d, J=7.6 Hz, 2H, HAr2′), 7.62-7.59 (d, J=8 Hz, 1H, HAr 3), 7.33-7.29 (t, J=7.6 Hz, 2H, HAr 3′), 7.20-7.16 (t, J=8.4 Hz, 1H, HAr 4), 7.08-7.04 (t, J=7.2 Hz, 1H, HAr 4′), 6.75-6.73 (d, J=8.4 Hz, 1H, HAr 6), 6.60-6.56 (t, J=8 Hz, 1H, HAr 5), 6.29 (2H, NH2).
MS (ES) m/z 213 (M+H+), 120.
The 2-amino-N-phenylbenzamide (1 eq., 42 mg, 0.2 mmol) is dissolved in 2 ml of methanol with 3-bromobenzaldehyde (1 eq., 23 μl, 0.2 mmol). After an overnight period, the reaction mixture is evaporated (disappearance of the 2-amino-N-phenylbenzamide is observed by NMR) and the imine 149 is formed quantitatively.
1H NMR (400 MHz, DMSO d6) δ : 8.40 (s, 1H, CH), 7.70-6.60 (m, 13H, HAr).
The 2-amino-N-phenylbenzamide (1 eq., 21 mg, 0.1 mmol) is dissolved in 1 ml of methanol with 3-fluorobenzaldehyde (1 eq., 10 al, 0.1 mmol). After an overnight period, the reaction mixture is evaporated (disappearance of the 2-amino-N-phenylbenzamide is observed by NMR) and the imine 151 is formed quantitatively.
1H NMR (400 MHz, DMSO d6) δ : 9.43 (s, 1H, CH), 7.62-6.72 (m, 13H, HAr).
DCC (10.9 g; 52.7 mmol; 1.1 equiv.), DMAP (1.17 g, 9.6 mmol, 0.2 equiv.) and aniline (6.1 ml; 67.1 mmol; 1.4 equiv.) are added, at ambient temperature, to a solution of nitro acid (8 g; 47.9 mmol; 1 equiv.) in CH2Cl2 (100 ml). The mixture is stirred for 4 days and evaporated. The solid is then solubilized in acetone and then filtered.
The brown filtrate is evaporated, to give a brown solid which is washed with Et2O to give 2-nitro-N-phenylbenzamide in the form of an orange solid (11.6 g; 71%).
Furthermore, palladium on carbon (550 mg) and decaborane (757 mg, 6.2 mmol; 0.3 equiv.) are added, at ambient temperature, to a solution of 2-nitro-N-phenylbenzamide (5 g; 20.7 mmol; 1 equiv.) in MeOH (65 ml). The mixture is heated at 60° C. for 6 h, filtered through celite, washed with EtOAc and evaporated to give 2-amino-N-phenylbenzamide (brown solid, 4.2 g; 96%), which is used without purification.
An aldehyde (0.2 mmol; 1 equiv.) is added to a solution of 2-amino-N-phenylbenzamide (42 mg; 0.2 mmol; 1 equiv.) in MeOH (2 ml), and the mixture is stirred for 2 days at ambient temperature, evaporated, and purified by a suitable method if necessary.
This process makes it possible to prepare the following compounds:
Starting from furan-2-carbaldehyde, a yellow solid is obtained (99%).
1H NMR (CDCl3, 400 MHz): δ 8.02 (dd, 1H, J=1.2 and 8 Hz), 7.40-7.24 (m, 7H), 6.91 (t, 1H, J=8 Hz), 6.70 (d, 1H, J=8 Hz), 6.33 (d, 1H, J=3.2 Hz), 6.25 (dd, 1H, J=2 and 3.2 Hz), 6.06 (d, 1H, J=2 Hz), 4.94 (bs, 1H).
13C NMR (CDCl3, 100 MHz): δ 162.6, 152.2, 145.1, 142.6, 140.6, 133.7, 128.9, 128.8, 126.7, 126.1, 119.7, 117.1, 115.1, 110.3, 109.0, 68.4.
ESI+MS: calcd for C18H14N2O2: 290.11; found: 291.1 (MH+).
Starting from 5-methylfuran-2-carbaldehyde, a yellow solid is obtained (100%).
ESI+MS: calcd for C19H16N2O2: 304.12; found: 305.0 (MH+).
Starting from furan-3-carbaldehyde, a yellow solid is obtained (97%).
1H NMR (CDCl3, 400 MHz): δ 8.02 (d, 1H, J=7.6 Hz), 7.39-7.23 (m, 7H), 6.93 (t, 1H, J=7.6 Hz), 6.70 (d, 1H, J=8 Hz), 6.28 (s, 1H), 6.04 (s, 1H), 4.70 (bs, 1H).
13C NMR (CDCl3, 100 MHz): δ 162.7, 145.5, 143.6, 140.8, 140.4, 133.8, 129.0, 128.9, 126.9, 126.7, 125.5, 119.7, 117.1, 115.2, 108.7, 67.7.
ESI+MS: calcd for C18H14N2O2: 290.11; found: 291.1 (MH+).
Starting from 2-fluorobenzaldehyde, a yellow solid is obtained (53%).
ESI+MS: calcd for C20H15FN2O: 318.12; found: 319.1 (MH+).
Starting from 3-fluorobenzaldehyde, a yield of 100% is obtained.
ESI+MS: calcd for C20H15FN2O: 318.12; found: 319.1 (MH+).
Starting from 3-bromobenzaldehyde, a yield of 100% is obtained.
1H NMR (DMSO-d6, 400 MHz): δ 8.40 (s, 1H), 7.70-6.60 (m, 13H, H).
Starting from 4-fluorobenzaldehyde, a yellow oil is obtained (8%).
Purification mode: Short pad
1H NMR (DMSO d6, 400 MHz): δ 9.43 (s, 1H), 7.62-6.72 (m, 13H)
ESI+MS: calcd for C20H15BrN2O: 318.12; found: 319.2 (MH+).
Starting from 5-fluoro-2-nitrobenzaldehyde, a yellow oil is obtained (2.9%).
Purification mode: Short pad
ESI+MS: calcd for C20H14FN3O3: 363.34; found: 364.1 (MH+).
Starting from 5-bromo-2-hydroxybenzaldehyde, a yellow oil is obtained (92%).
ESI+MS: calcd for C20H15BrN2O2: 394.03; found: 395.0 (MH+).
Starting from 1-methyl-1H-indole-2-carbaldehyde, a yellow oil is obtained (12%).
Purification mode: Short pad
ESI+MS: calcd for C23H19N3O: 353.15; found: 354.2 (MH+).
Starting from (E)-2-(3-fluorobenzylideneamino)-N-phenylbenzamide.
Yield=100%.
Starting from (E)-2-(2-fluorobenzylideneamino)-N-phenylbenzamide, a colorless oil is obtained (1%).
Purification mode: Short pad
ESI+MS: calcd for C20H17FN2O: 320.13; found: 321.0 (MH+).
The synthesis of the compounds of general formula (II) requires the prior preparation of the following synthesis intermediates:
N-boc-glycine (1 eq., 5.71 mmol, 1.0 g) is dissolved in 10 ml of dichloromethane at 0° C. DCC (1.1 eq., 6.28 mmol, 1.3 g) is then added in small portions. Finally, aniline (1.4 eq., 8.0 mmol, 0.73 ml) is added at ambient temperature. After two days at ambient temperature, the content of the round-bottom flask is filtered and then evaporated. Silica gel column purification (mixture of ethyl acetate/N-hexane in a gradient of 50:50 to 80:20) makes it possible to obtain 740 mg of purified tert-butyl N-[(phenylcarbamoyl)-methyl]carbamate (51%).
1H NMR (400 MHz, CDCl3)
δ: 9.89 (s, 1H, NH), 7.57-7.55 (d, J=7.6 Hz, 2H, HAr), 7.30-7.26 (1, J=7.6 Hz, 2H, HAr), 7.02-7.00 (d, J=7.2 Hz, 1H, HAr), 5.19 (s, 1H, NH), 3.70-3.69 (d, J=6 Hz, 2H, CH2), 1.38 (s, 9H, (CH3)3).
MS (ES) m/z 251 (M+H+), 195, 151, 94.
N-boc-glycine (1 eq., 7.2 mmol, 1.26 g) is dissolved in 10 ml of dichloromethane at 0° C. DCC (1.1 eq., 8.0 mmol, 1.65 g) is then added in small portions. Finally, para-anisidine (1.4 eq., 10 mmol, 1.23 g) is added at ambient temperature. After two days at ambient temperature, the content of the round-bottom flask is filtered and then evaporated. Silica gel column purification (98:2 mixture of acetone/dichloromethane) makes it possible to obtain 1.154 g of purified tert-butyl (4-methoxyphenylcarbamoyl)methylcarbamate compound (57%).
1H NMR (400 MHz, CDCl3)
δ: 9.74 (s, 1H, NH), 7.48-7.45 (d, J=9.2 Hz, 2H, HAr), 7.00 (s, 1H, NH), 6.87-6.84 (d, J=8.8 Hz, 2H, HAr), 3.70 (s, 3H, OCH3), 3.67-3.66 (d, J=6 Hz, 2H, CH2), 1.38 (s, 9H, (CH3)3).
MS (ES) m/z 281 (M+H+), 225, 181, 124.
The tert-butyl N-[(phenylcarbamoyl)methyl]carbamate compound (1 eq., 3 mmol, 450 mg) is dissolved in a 2:1 mixture of dichloromethane/trifluoroacetic acid. After one hour, the 2-amino-N-phenylacetamide product is quantitatively obtained after evaporation and used directly in Pictet-Spengler reaction tests.
MS (ES) m/z 151 (M+H+), 94.
The tert-butyl(4-methoxyphenylcarbamoyl)methylcarbamate compound (1 eq., 3 mmol, 540 mg) is dissolved in a 2:1 mixture of dichloromethane/trifluoroacetic acid. After one hour, 2-amino-N-(4-methoxyphenyl)acetamide is quantitatively obtained after evaporation and used directly in Pictet-Spengler reaction tests.
MS (ES) m/z 181 (M+H+), 124.
Pathway 1: 4-bromoaniline (1 eq., 0.05 mol, 8.6 g) is dissolved in benzoyl chloride (2.7 eq., 0.135 mol, 15.7 ml). The reaction mixture is heated to 180° C. and then zinc chloride (1.25 eq., 0.063 mol, 8.5 g) is added. After heating for two hours at 205° C., the mixture is cooled to 120° C. and then 60 ml of 3N hydrochloric acid are added. After refluxing and separation of the hot acid layer by settling out, the water-insoluble residue is dissolved in 80 ml of 70% sulfuric acid, brought to reflux for 8 hours, and then poured into a large amount of ice-cold water. After the addition of ethyl acetate (3×50 ml), the organic phases are combined and then evaporated. After purification by HPLC, the yield is less than 1% (the mass obtained was approximately 200 mg).
Pathway 2: 2-aminobenzophenone (1 eq., 5 mmol, 0.986 g) is dissolved in dichloromethane at 0° C. N-bromosuccinimide (1 eq., 5 mmol, 0.890 g) is then added in small portions. The temperature of the reaction mixture is allowed to return to ambient temperature over approximately two hours. The reaction mixture is then evaporated and 2-benzoyl-4-bromoaniline is quantitatively obtained.
1H NMR (400 MHz, CDCl3)
δ: 7.64-7.63 (d, J=7.2 Hz, 2H, 2′), 7.56 (s, 1H, 6), 7.55-7.54 (d, J=2.4 Hz, 1H, 4′), 7.50-7.47 (t, J=7.6 Hz, 2H, 3′), 7.38-7.35 (q, J=8.8-2.4 Hz, 1H, 4), 6.66-6.64 (d, J=8.4 Hz, 1H, 3), 6.09 (s, 2H, NH2).
MS (ES) m/z 276 (M+H+), 198, 105.
2-amino-N-(4-methoxyphenyl)acetamide (1 eq., 0.5 mmol, 140 mg) is dissolved in 2 ml of acetonitrile. Benzaldehyde (1.5 eq., 0.75 mmol, 76 μl) is added to the reaction medium, followed by 1 ml of trifluoroacetic acid. After refluxing for 3 hours, the mixture is evaporated and then purified by silica gel chromatography (mixture of cyclohexane/ethyl acetate in a gradient of 80:20 to 50:50), which makes it possible to obtain 16 mg of compound 167, i.e. a yield of 12%.
1H NMR (400 MHz, CDCl3)
δ: 7.50-6.60 (m, 8H, HAr), 6.44 (s, 1H, NH), 5.30 (s, 1H, CH), 3.82-3.72 (dd, J=10 et 15.6 Hz, 2H, CH2), 3.73 (s, 3H, OCH3), 3.49 (s, 1H, NH).
MS (ES) m/z 269 (M+H+), 222, 204, 145, 106.
In a round-bottomed flask fitted with a Dean-Stark apparatus and a condenser, aminobenzophenone (2.85 eq., 5 mmol, 1.0 g) is dissolved in 15 ml of pyridine containing 4 Å molecular sieve. Ethyl glycinate hydrochloride (1 eq., 1.75 mmol, 244 mg) is then added, and the mixture is then brought to reflux for 3 hours. Approximately 5 ml of pyridine are withdrawn from the Dean-Stark apparatus and replaced with 5 ml of fresh pyridine. After an overnight period at 110° C., the pyridine is evaporated off, while adding 10 ml of toluene, and the residue is then dissolved in 30 ml of dichloromethane and 30 ml of 2.5% Na2CO3. The combined organic phases are washed with a saturated solution of sodium chloride and then dried with anhydrous Na2SO4 and, finally, evaporated to give the crude product. Purification on a silica gel column (mixture of dichloromethane/acetone in a concentration gradient of from 95:5 to 80:20) makes it possible to obtain 217 mg of purified product 161, i.e. a yield of 52.5%.
1H NMR (400 MHz, CDCl3)
δ: 8.45 (s, 1H, NH), 7.64-7.26 (m, 9H, HAr), 4.34 (s, 2H, CH2).
MS (ES) m/z 237 (M+H4).
In a round-bottomed flask fitted with a Dean-Stark apparatus and a condenser, 4-chloro-2-aminobenzophenone (2.85 eq., 5 mmol, 1.16 g) is dissolved in 15 ml of pyridine containing 4 Å molecular sieve. Methyl glycinate hydrochloride (1 eq., 1.75 mmol, 220 mg) is then added, and the mixture is then brought to reflux for 3 hours. After an overnight period at 110° C., the pyridine is evaporated off, while adding 10 ml of toluene, and the residue is then dissolved in 30 ml of dichloromethane and 30 ml of 2.5% Na2CO3. The combined organic phases are washed with a saturated solution of sodium chloride, then dried with anhydrous Na2SO4 and, finally, evaporated to give the crude product. Purification on a silica gel column (mixture of cyclohexane/ethyl acetate in a gradient of from 90:10 to 50:50) makes it possible to obtain 80 mg of product 170 (17%).
1H NMR (400 MHz, CDCl3)
δ: 9.42 (s, 1H, NH), 7.78-7.16 (m, 8H, HAr), 4.33 (s, 2H, CH2).
MS (ES) m/z 271 (M+H+).
In a round-bottomed flask fitted with a Dean-Stark apparatus and a condenser, 2-benzoyl-4-bromoaniline (2 eq., 0.47 mmol, 130 mg) is dissolved in 5 ml of pyridine. Methyl glycinate hydrochloride (1 eq., 1.24 mmol, 30 mg) is then added and the mixture is then brought to reflux for 3 hours. Approximately 2 ml of pyridine are withdrawn from the Dean-Stark apparatus and replaced with 2 ml of fresh pyridine. After an overnight period at 110° C., the pyridine is evaporated off, while adding 5 ml of toluene, and the residue is then dissolved in 20 ml of dichloromethane and 20 ml of 2.5% Na2CO2. The combined organic phases are washed with a saturated solution of sodium chloride, then dried over anhydrous Na2SO4 and, finally, evaporated to give the crude product. Purification on a silica gel column (mixture of cyclohexane/ethyl acetate in a concentration gradient of from 90:10 to 50:50) makes it possible to obtain 100 mg of product 163, i.e. 26%.
1H NMR (400 MHz, CDCl3)
δ: 9.26 (s, 1H, NH), 7.90-7.32 (m, 8H, HAr), 4.37 (s, 2H, CH2).
MS (ES) m/z 315 (MH+).
Compound 162 (1 eq., 0.42 mmol, 100 mg) is dissolved in 2 ml of methanol. The reducing agent on polymer beads (3 eq., 1.27 mmol, loading: 4.4 mmol/g, 290 mg) is added, as are a few drops of acetic acid (50 μl). The reaction takes place at ambient temperature for 3 days. After filtration and then evaporation, the product obtained is purified by silica gel chromatography (cyclohexane/ethyl acetate in a gradient of from 80:20 to 1:2), and the desired product 169 is obtained with a yield of 60%.
1H NMR (400 MHz, CDCl3)
δ: 9.12 (s, 1H, NH), 7.35-6.67 (m, 9H, HAr), 5.21 (s, 1H, CH), 3.35-3.25 (q, J=10-14.8 Hz, 2H, CH2), 3.21 (s, 1H, NH).
MS (ES) m/z 239 (M+H+), 182, 132, 91.
Compound 170 (1 eq., 0.22 mmol, 60 mg) is dissolved in 2 ml of methanol. The reducing agent on polymer beads (3 eq., 0.66 mmol, loading: 4.4 mmol/g, 150 mg) is added, as are a few drops of acetic acid (50 μl). The reaction takes place at ambient temperature for 5 days. After filtration and then evaporation, the product obtained is purified by silica gel chromatography (cyclohexane/ethyl acetate in a gradient of from 80:20 to 1:2), and the desired product 171 is obtained with a yield of 55%.
1H NMR (400 MHz, CDCl3)
δ: 9.15 (s, 1H, NH), 7.40-6.86 (m, 8H, HAr), 5.20 (s, 1H, CH), 3.45-3.36 (q, J=10-13.6 Hz, 2H, CH2), 3.10 (s, 1H, NH).
MS (ES) m/z 273 (M+H+), 221, 91.
Compound 163 (1 eq., 0.25 mmol, 80 mg) is dissolved in 2 ml of methanol. The reducing agent on polymer beads (3 eq., 0.75 mmol, loading: 4.4 mmol/g, 170 mg) is added, as are a few drops of acetic acid (50 μl). The reaction takes place at ambient temperature for 3 days. After filtration and then evaporation, the product obtained is purified by silica gel chromatography (cyclohexane/ethyl acetate in a gradient of from 80:20 to 1:2), and the desired product 164 is obtained with a yield of 52%.
1H NMR (400 MHz, CDCl3)
δ: 8.48 (s, 1H, NH), 7.52-6.86 (m, 8H, HAr), 5.18 (s, 1H, CH), 3.45-3.35 (q, J=10-14.8 Hz, 2H, CH2), 2.86 (s, 1H, NH).
MS (ES) m/z 317 (M+H+), 91.
1,2-Diaminobenzene (1 eq., 2 mmol, 216 mg) and ethyl 3-oxo-3-phenylpropanoate (1 eq., 2 mmol, 384 mg) are mixed together in a pill bottle flask. The reaction mixture is then heated at 150° C. for 2 hours. The residue is then diluted in ethyl acetate and hydrochloric acid (pH˜5), and then the organic phase is washed with water and then with a saturated solution of sodium chloride. Finally, the solution is dried with Na2SO4 and then evaporated. Purification on a silica gel column (70:30 cyclohexane/ethyl acetate) makes it possible to obtain 325 mg of 165, i.e. a yield of 69%.
1H NMR (400 MHz, CDCl3)
δ: 12.88 (s, 1H, NH), 8.17-6.64 (m, 9H, HAr), 5.50 (s, 2H, CH2).
MS (ES) m/z 237 (M+H+), 195.
Compound 165 (1 eq., 0.42 mmol, 100 mg) is dissolved in 2 ml of methanol. The reducing agent on polymer beads (3 eq., 1.27 mmol, loading: 4.4 mmol/g, 290 mg) is added, as are a few drops of acetic acid (50 μl). The reaction takes place at ambient temperature for 3 days. After filtration then evaporation, the product obtained is purified by silica gel chromatography (cyclohexane/ethyl acetate in a gradient of from 80:20 to 1:2), and the desired product 166 is obtained with a yield of 77%.
The second process that follows also allows the synthesis of compound 166: 1,2-diaminobenzene (1 eq., 10 mmol, 1.08 g) is mixed with cinnamic acid (1 eq., 10 mmol, 1.48 g) in a pill bottle flask. The reaction mixture is then heated at 150° C. without solvent for two hours. The residue is diluted in dichloromethane and a 5% Na2CO3 solution. The organic phase is extracted several times with the Na2CO3 solution, and then with a saturated solution of sodium chloride. Finally, the solution is dried with Na2SO4 and then evaporated. Purification on a silica gel column (cyclohexane/ethyl acetate in a gradient of from 80:20 to 1:1) makes it possible to isolate 166 with a yield of 12% (286 mg).
1H NMR (400 MHz, CDCl3)
δ: 9.15 (s, 1H, NH), 7.40-6.72 (m, 9H, HAr), 5.76 (s, 1H, NH), 4.91-4.89 (m, 1H, CH), 2.54-2.52 (d, J=6 Hz, 2H, CH2).
MS (ES) m/z 239 (M+H+), 197, 131.
Compound 166 (1 eq., 0.218 mmol, 52 mg) is dissolved with ethyl chloroformate (1.2 eq., 0.262 mmol, 25 μl) in a solution composed of dichloromethane and triethylamine in 10:1 proportions. After two hours at ambient temperature, the content of the pill bottle flask is evaporated. Purification on a silica gel column (cyclohexane/ethyl acetate in a gradient of from 90:10 to 1:1) makes it possible to isolate 15 mg of 168, i.e. a yield of 21%.
1H NMR (400 MHz, CDCl3)
δ: 9.10 (s, 1H, NH), 7.62-6.72 (m, 9H, HAr), 5.10 (s, 1H, CH), 4.24 (m, 2H, CH2), 2.62-2.61 (d, J=6 Hz, 2H, CH2), 1.46 (t, J=8 Hz, 3H, CH3).
MS (ES) m/z 311 (M+H+), 269, 207, 135.
A β-keto ester (0.5 mmol; 1 equiv.) is added to a stirred suspension of diamine (0.5 mmol; 54 mg; 1 equiv.) in toluene (2 ml). The mixture is stirred at reflux (120° C.) for 3 h. The mixture is diluted in EtOAc, acidified (ph 5), extracted with EtOAc, filtered, evaporated and washed with Et2O to give the desired compound.
Starting from ethyl 3-oxo-3-m-tolylpropanoate, a brown solid is obtained (31%).
1H NMR (CDCl3, 400 MHz): δ 7.94 (s, 1H), 7.91 (d, 1H, J=8 Hz), 7.69 (bs, 1H), 7.54 (d, 1H, J=8.8 Hz), 7.38 (t, 1H, J=7.6 Hz), 7.31 (m, 2H), 7.05 (dd, 1H, J=1.6 and 7.6 Hz), 3.59 (s, 2H), 2.45 (s, 3H).
ESI+MS: calcd for C16H14N2O: 250.11; found: 250.1 (MH+).
Starting from ethyl 3-(3-methoxyphenyl)-3-oxopropanoate, a brown solid is obtained (20%).
1H NMR (CDCl3, 400 MHz): δ 7.68 (m, 3H), 7.55 (dd, 1H, J=2 and 8 Hz), 7.40 (t, 1H, J=8.4 Hz), 7.28 (m, 1H), 7.06 (dt, 2H, J=2.8 and 8 Hz), 3.91 (s, 3H), 3.58 (s, 2H).
13C NMR (CDCl3, 100 MHz): δ 167.3, 159.9, 158.8, 139.8, 138.9, 129.7, 128.9, 128.3, 126.5, 125.2, 121.6, 120.4, 117.7, 112.2, 55.5, 39.9.
ESI+MS: calcd for C16H14N2O2: 266.11; found: 267.0 (MH+).
Starting from ethyl 3-(3-nitrophenyl)-3-oxopropanoate, a brown solid is obtained (23%).
1H NMR (CDCl3, 400 MHz): δ 9.00 (t, 1H, J=2 Hz), 8.40 (d, 1H, J=8 Hz), 8.35 (dd, 1H, J=1.6 and 8 Hz), 7.73 (bs, 1H), 7.68 (t, 1H, J=8.4 Hz), 7.55 (m, 1H), 7.32 (m, 2H), 7.09 (m, 1H), 3.62 (s, 2H).
ESI+MS: calcd for C15H11N3O3: 281.08; found: 282.0 (MH+).
Starting from ethyl 3-(3-chlorophenyl)-3-oxopropanoate, a brown solid is obtained (15%).
1H NMR (CDCl3, 400 MHz): δ 8.14 (t, 1H, J=1.6 Hz), 7.96 (d, 1H, J=7.6 Hz), 7.76 (bs, 1H), 7.53-7.41 (m, 3H), 7.29 (m, 2H), 7.07 (dd, 1H, J=1.2 and 6.8 Hz), 3.56 (s, 2H).
13C NMR (CDCl3, 100 MHz): δ 167.1, 157.3, 139.7, 139.3, 135.0, 131.0, 129.9, 128.9, 128.4, 127.8, 126.9, 125.8, 125.3, 121.7, 39.7.
Starting from ethyl 3-(3-bromophenyl)-3-oxopropanoate, a brown solid is obtained (7%).
1H NMR (CDCl3, 400 MHz): δ 8.30 (t, 1H, J=1.6 Hz), 8.00 (d, 1H, J=8 Hz), 7.77 (bs, 1H), 7.63 (dd, 1H, J=0.8 and 8 Hz), 7.52 (dd, 1H, J=2 and 7.6 Hz), 7.36 (t, 1H, J=8 Hz), 7.27 (m, 1H), 7.07 (dd, 1H, J=1.6 and 7.2 Hz), 3.55 (s, 2H).
13C NMR (CDCl3, 100 MHz): δ 166.9, 157.3, 139.7, 139.3, 133.9, 130.7, 130.2, 128.8, 128.4, 126.9, 126.3, 125.3, 123.1, 121.7, 39.7.
Starting from ethyl 3-(3-(trifluoromethyl)phenyl)-3-oxopropanoate, a brown solid is obtained (34%).
1H NMR (CDCl3, 400 MHz): δ 8.42 (s, 1H), 8.26 (d, 1H, J=7.6 Hz), 7.78 (bs, 1H), 7.76 (d, 1H, J=8.4 Hz), 7.63 (t, 1H, J=7.6 Hz), 7.54 (dd, 1H, J=2.4 and 8 Hz), 7.33-7.29 (m, 2H), 7.08 (dd, 1H, J=2.4 and 7.2 Hz), 3.60 (s, 2H).
A β-keto ester (0.5 mmol; 1 equiv.) is added to a stirred suspension of diamine (0.5 mmol; 94 mg; 1 equiv.) in toluene (2 ml).
The mixture is stirred at reflux (120° C.) for 3 h. The mixture is diluted with EtOAc, acidified (pH 5), extracted with EtOAc, filtered, evaporated and washed with Et2O.
Starting from ethyl 3-oxo-3-m-tolylpropanoate, a brown solid is obtained (28%).
1H NMR (CDCl3, 400 MHz): δ 7.89 (m, 3H), 7.40-7.32 (m, 4H), 3.58 (s, 2H), 2.44 (s, 3H).
ESI+MS: calcd for C16H13BrN2O: 328.02; found: 328.9 (MH+).
Starting from ethyl 3-(3-methoxyphenyl)-3-oxopropanoate, a brown solid is obtained (10%). ESI+MS: calcd for C16H13BrN2O2: 344.02; found: 344.9 (MH+).
Starting from 3-(3-nitrophenyl)-3-oxopropanoate, a brown solid is obtained (6.5%).
ESI+MS: calcd for C15H10BrN3O3: 358.99; found: 359.8 (MH+).
Starting from ethyl 3-(3-chlorophenyl)-3-oxopropanoate, a brown solid is obtained (23%).
ESI+MS: calcd for C15H10BrClN2O: 347.97; found: 348.8 (MH+).
Starting from ethyl 3-(3-bromophenyl)-3-oxopropanoate, a brown solid is obtained (10%).
ESI+MS: calcd for C15H10Br2N2O: 391.92; found: 392.8 (MH+).
Starting from ethyl 3-(3-(trifluoromethyl)phenyl)-3-oxopropanoate, a brown solid is obtained (29%). ESI+MS: calcd for C16H10BrF3N2O: 381.99; found: 382.8 (MH+).
A cell assay was developed in order to demonstrate the cytotoxic activity of ricin while at the same time being suitable for the constraints of high-throughput screening (see
(i) selection of molecules which can act on the various stages of cell poisoning (receptor binding, internalization, intracellular trafficking, enzymatic activity, etc.);
(ii) selection of molecules which penetrate the cell;
(iii) elimination of cytotoxic compounds.
In addition, the assay used directly measures the ability of the cells to synthesize proteins, which makes it an excellent screening assay since this biosynthetic pathway is stopped by ricin.
The principle of the cell assay is the following: the compounds of combinatorial libraries are preincubated with ricin and the whole is added to the culture medium of human lung epithelial cells (A549 cells, 60 000 cells/well, [ricin]=10−10M) cultured on plates having a solid scintillant bottom (Scintiplates, GE). After incubation, the medium is removed and replaced with a leucine-free culture medium containing the radioactive tracer, which is [14C]-leucine (0.05 μCi/well). The incorporation of [14C]-leucine is then measured after a second incubation. The presence of the tracer in the cells reflects the presence of an inhibitor (C=50 μM) in the well (
The yellow-colored wells are positive controls (A549 treated with ricin (10−10M) in the presence of 20 mM of lactose, which is an inhibitor of ricin binding to cells), whereas the green wells are negative controls (cells treated with ricin alone). The red wells are cells in contact with the compounds of the libraries in the presence of ricin (80 different compounds per plate, 50 μM final concentration).
The results of the screening carried out are represented in the graph of
The compounds were tested on A549 cells at the concentration of 30 μM by incubation with various ricin concentrations (10−9 to 10−12M), with the protocol described previously for the high-throughput screening. The radioactivity measured is then proportional to the cell survival rate. Analysis of the data by nonlinear regression makes it possible to estimate the EC50, i.e. the effective concentration for which 50% radioactive leucine assimilation is observed, which corresponds to 50% of viable cells. The higher the EC50 value, the greater the cell protection, since a higher concentration of ricin is then necessary in order to generate the same cytotoxicity.
The results, presented in the table below, are indicated in the form of a protective index (PI=EC50 compound/EC50 ricin ratio): the higher it is, the greater the protection of the cells against the action of ricin (the effect is protective if the ratio is greater than 1).
It is clearly observed that these compounds inhibit the cytotoxic action of ricin. Furthermore, they are the only inhibitors known at this time to protect A549 cells (human pulmonary epithelium) against ricin.
These compounds are the first inhibitors that are active on human pulmonary and digestive epithelial cells with respect to the toxic activity of ricin.
The compounds were also tested on Vero cells (ATCC No. CCL-81) at the concentration of 30 μM by incubation with various concentrations of diphtheria toxin (10−9 to 10−12M) (Sigma), with the protocol described in point 2.1.
The results, presented in the table below, also represent a protective index (PI=EC50 compound/EC50 ricin ratio): the higher it is, the greater the protection of the cells against the action of diphtheria toxin.
| Number | Date | Country | Kind |
|---|---|---|---|
| 08290570.4 | Jun 2008 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12999576 | Mar 2011 | US |
| Child | 14810342 | US |
