Patent Application
20230339856
The present invention lies in the field of the treatment of lysosomal diseases, more specifically the treatment of Pompe disease.
More particularly, the present invention relates to compounds, belonging to the class of iminosugars, capable of selectively interacting with human α-glucosidases and in particular of stabilizing human acid α-glucosidase in its endogenous form (GAA) or of recombinant nature (rhGAA), in a conformation favoring transport thereof to the lysosome, for use thereof as a drug, in particular for treating Pompe disease; and to a pharmaceutical composition containing such compounds. The invention also relates to compounds belonging to the class of iminosugars capable of selectively interacting with human acid α-glucosidase. The invention furthermore relates to a method for preparing such compounds.
Pompe disease, or type 2 glycogenosis, is a rare genetic disease characterized by one or more mutations of the GAA gene encoding the human acid α-glucosidase (GAA) lysosomal enzyme, which causes a deficit in the activity of this enzyme, and consequently the accumulation of glycogen, the hydrolytic biodegradation of which is performed by this enzyme, in the lysosomes, which causes cell malfunctionings in the muscles and heart. The symptoms are diverse, and progression thereof more or less rapid depending on whether the disease manifests at birth or at an adult age. They affect all the muscles, in particular the respiratory muscles and the myocardium. The life expectation of the subjects affected is reduced thereby, especially in babies, who die in the course of their first year of life.
At the present time there is only one treatment for Pompe disease that has received authorization for marketing. This treatment, known as enzyme replacement therapy (ERT), consists in intravenously administering the Myozyme® specialty, the active principle of which is a recombinant human acid α-glucosidase (rhGAA), which makes it possible to stabilize the symptoms of the disease. However, this treatment has numerous drawbacks: it is very expensive, binding for the patient, since it requires perfusion every two weeks, in a hospital environment, and it is of limited efficiency in many cases. It may indeed cause an immune reaction affecting its efficiency and its tolerance by the subject being treated. Furthermore, the recombinant enzyme is relatively unstable in the blood, and the doses that have to be administered to the patients are much greater than those administered for treating other lysosomal diseases.
There thus remains at the present time a need for a satisfactory treatment of Pompe disease.
The present invention aims to propose such a treatment. More particularly, the invention aims to propose compounds affording effective treatment of Pompe disease, this treatment furthermore being easy to administer, and less expensive than the treatment by enzyme replacement therapy proposed by the prior art. An additional objective of the invention is that these compounds, administered at the same time as the enzyme replacement therapy treatment of the prior art, significantly increase the efficiency of the latter.
Seeking to achieve these objectives, the present inventors became interested in a therapeutic approach considered at the present time to be one of the most promising for treating lysosomal diseases: the use of so-called pharmacological chaperone molecules. Chaperone molecules are small molecules promoting the correct folding of mutant enzymes and enabling transport thereof to the lysosomes (Boyd et al., 2013, J. Med. Chem. 56: 2705-2725) rather than degradation at the endoplasmic reticulum. This therapeutic approach was implemented successfully for another lysosomal disease, Fabry disease, against which the migalastat iminosugar (Galafold®) is now prescribed as a pharmacological chaperone of acid α-galactosidase (Markham, 2016, Drugs 76: 1147-1152).
The present inventors have thus sought compounds capable of stabilizing human acid α-glucosidase in its active folded form, as well as increasing the efficacy of the recombinant human acid α-glucosidase used in treatments by enzyme replacement therapy, in order to use them in the treatment of Pompe disease.
Among the existing compounds, deoxynojirimycin (DNJ), an iminosugar of formula:
has been proposed by the prior art as potential chaperone molecule of human acid α-glucosidase (Flanagan et al., 2009, Hum. Mutat. 30: 1683-1692).
A derivative of DNJ, called NB-DNJ, of formula:
has also been proposed by the prior art for the same application (Parenti et al., 2007, Mol. Ther. 15(3): 508-14).
However, these compounds have low selectivity for this enzyme, and they inhibit other human enzymes, both of α- and β-glycosidase type, and glycosyltransferases, which causes many undesirable side effects when they are administered to a subject.
The publication by Boisson et al., Org. Lett., 2015, 17(15), 3662-5 describes particular polyhydroxylated derivatives of indolizidines having good ability to inhibit an α-glucosidase of S. cerevisiae and a rice α-glucosidase.
The publication by Tangara et al., Org. Lett., 2017, 19(18), 4842-5, describes particular aziridinyl-iminosugars.
The publication by Vieira Da Cruz et al., J. Org. Chem., 2017, 82(18), 9866-72 describes particular polyhydroxylated quinolizidines and their ability to inhibit yeast and rice α-glucosidases.
The present inventors have now discovered that specific compounds, belonging to the iminosugars class, complying with a particular structure derived from that of DNJ, bind to human acid α-glucosidase (GAA) and stabilize it, and this in a highly selective manner, i.e. without interacting, at least significantly, with other human glycosidases, in particular with human β-glucocerebrosidases (GBA1 and GBA2), human β-glucosylceramide transferase (GCS) or with the human endoplasmic reticulum α-glucosidase II (GANAB). The effect of stabilizing GAA by these compounds is furthermore particularly great. Thus, administered to patients suffering from Pompe disease, these compounds make it possible to stabilize their endogenous acid α-glucosidase in a correctly folded form to improve transport thereof to the lysosomes and to increase its activity of hydrolysis of glycogen, and therefore to effectively treat this disease, while reducing undesirable side effects during treatment. These compounds also make it possible, associated with enzyme replacement therapy (ERT), to stabilize the recombinant enzyme (rhGAA), thereby increasing the efficacy thereof.
Molecules with a similar, but different, structure, such as DNJ or NB-DNJ, do not make it possible to obtain such a particularly advantageous result on a therapeutic level.
Thus, according to a first aspect, the present invention relates to a compound of general formula (I) below, or one of the pharmaceutically acceptable salts thereof, for use as a drug, in particular as a pharmacological chaperone, in particular for treating Pompe disease, in particular for stabilizing human acid α-glucosidase:
wherein:
and R2 represents a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused, said hydrocarbon radical comprising at least 2 carbon atoms when R1 represents a hydrogen atom,
The term heteroatom designates, in the present description, conventionally per se, any atom belonging to an element other than carbon and hydrogen, such as a nitrogen, oxygen, sulfur, phosphorus, silicon, halogen, etc. atom.
Preferentially, when R1 represents a hydrogen atom, R2 does not represent a methyl radical or a benzyl group.
In the present description, “pharmaceutically acceptable salt”, conventionally per se, means any salt of the compound of general formula (I) comprising, as a counter-ion, a substance that does not produce any adverse, allergic or otherwise undesirable reaction when it is administered to a subject, in particular to a mammal.
Any conventional pharmaceutically acceptable salt of the compound of general formula (I) can be used according to the invention. By way of examples, mention can be made of chlorides, bromides, formiates, acetates, etc.
In the present description, the term “treatment” means the obtaining of a desired pharmacological or physiological effect. The word “treatment”, as used in the present description, comprises the prevention or the partial prevention of one or more of the symptoms of the disease and/or the partial or total curing of the disease and/or the total or partial disappearance of one or more of the symptoms thereof.
Human acid α-glucosidase is a protein of 952 amino acids, of GenBank Accession No. AB153718.1.
The compound used according to the invention, having the general formula (I), is a ligand thereof. It furthermore has particularly strong selectivity with respect to this enzyme. This selectivity is in particular much greater than that of DNJ or NB-DNJ, or other iminosugars interacting with glycosidases.
The characteristics of the compound used according to the invention, relating in particular to the three-dimensional structure thereof, which make it possible to obtain such advantageous properties, will not be prejudged here. It can however be supposed that the following participate therein: the presence of a quaternary center in the a position with respect to the nitrogen of the piperidine nucleus, the D-gluco configuration of the compound, and the particular substituents present on the quaternary carbon in the a position with respect to the nitrogen of the piperidine nucleus and on this nitrogen. Because, at least partly, of these characteristics, the compounds used according to the invention would bind non-covalently with the enzyme and would stabilize the folding thereof, conferring on it a role of chaperone making it possible to restore the transport and the activity of the deficient enzyme in the lysosomes.
The compound used according to the invention thus stabilizes the human acid α-glucosidase at low concentrations, and it has the essential properties of pharmacological chaperones suitable for treating Pompe disease. In particular, at a concentration of 100 μM, it increases, in vitro, the thermal denaturing temperature of the human acid α-glucosidase from 8 to 12° C. at pH 4.0 and the thermal denaturing temperature of the human acid α-glucosidase from 10 to 13° C. at pH 7.4. The chaperone effect of the compound used according to the invention on human acid α-glucosidase is confirmed by experiments on human cells in culture, more precisely fibroblasts of patients suffering from the disease, as well as, in vivo, in mice.
The compound used according to the invention is therefore entirely adapted and advantageous for being used as a pharmacological chaperone for treating Pompe disease, by selective bonding to the human acid α-glucosidase and stabilization thereof, thus restoring the activity of the deficient enzyme of the patient, and/or improving the bioavailability of the rhGAA recombinant enzyme administered during rhGAA enzyme replacement therapy when this therapy is associated with use of the compound used according to the invention.
Thus the compound of general formula (I) can be used for stabilizing human acid α-glucosidase in its active folded form, as well as increasing the efficacy of the recombinant human acid α-glucosidase used in enzyme replacement therapy treatments, the compound of general formula (I) and the recombinant human acid α-glucosidase being co-administered to the patient.
The compound used according to the invention can advantageously be administered to the patient easily, in particular orally, and it is inexpensive to prepare.
It has a low risk of toxicity, and good bioavailability.
Furthermore, in association with enzyme replacement therapy, the compound used according to the invention advantageously improves the bioavailability of the recombinant enzyme injected, which makes it possible to reduce the doses thereof to obtain a same efficacy in treatment, and even superior efficacy.
The compound used according to the invention, of general formula (I), can be administered to any subject in need thereof, i.e. suffering from or likely to contract the disease. This subject may in particular be a mammal, and especially a human.
The compound used according to the invention is preferably administered to the subject in a therapeutically effective amount.
“Therapeutically effective amount” means an amount of the compound which, when it is administered to a subject to treat the disease, is sufficient to ensure such treatment of the disease.
The therapeutically effective amount of the compound used according to the invention depends on several factors, such as the disease and its seriousness, the age, the weight, etc. of the subject to be treated, the particular compound used, the route and form of administration, etc. The therapeutically effective amount of the compound used according to the invention will be determined by the doctor for each individual case.
The compound used according to the invention can be administered to the subject to be treated by any method conventional per se, in particular parenterally, for example subcutaneously, subdurally, intravenously, by intramuscular, intrathecal, intraperitoneal, intracerebral, intra-arterial or intralesion route; intranasally; rectally; by pulmonary route, for example by aerosol or inhalation, or even topically. It is preferentially administered orally. Determining the administration posology of the compound used according to the invention falls within the competence of a doctor. The compound can for example be administered to the subject in need thereof once or twice per day, over a long period, at regular intervals, or in a targeted manner during an associated treatment by enzyme replacement therapy.
As disclosed above, the compound used according to the invention can advantageously be administered to the subject conjointly with an enzyme used for enzyme replacement therapy, in particular conjointly with a recombinant human acid α-glucosidase such as the recombinant enzyme sold under the name Myozyme®. It then advantageously increases the efficacy thereof.
In particular embodiments of the invention, in the general formula (I) R1 and R2 are independent from each other, in the sense that they are not bonded to each other, in particular that they do not form together a ring fused with the piperidine nucleus of the compound.
In particular, in the general formula (I):
and R2 can represent a —CH(R3)—R4 group, wherein R3 and R4, which may be identical or different, each represent a hydrogen atom or a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused, R3 and R4 not simultaneously representing a hydrogen atom when R1 represents a hydrogen atom.
In particular embodiments of the invention, R3 and R4 do not simultaneously represent a hydrogen atom.
In particular embodiments of the invention, R1 and R2 are such that they do not simultaneously represent, respectively, a propyl radical and an ethyl radical. Preferentially, R3 and R4 are such that, when R1 and R3 each represent a hydrogen atom, R4 does not represent a phenyl radical.
In particular embodiments of the invention, R2 represents a —CH(R3)—R4 group, wherein R3 is as defined above and R4 represents a hydrocarbon radical, preferably comprising from 1 to 18, preferentially from 1 to 12 and in particular from 1 to 6, carbon atoms, linear, branched and/or cyclic, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising a single ring or a plurality of rings, optionally fused, and comprising one or more heteroatoms each selected from oxygen, nitrogen, sulfur and silicon and/or one or more groups including at least one heteroatom each selected from the carbonyl, sulf oxide, sulfonyl and silane groups.
Otherwise, R1 representing a hydrogen atom or a hydrocarbon radical, linear, branched and/or cyclic, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused, R2 may represent a —(CHR7)—SO2—Ar1 group wherein Ar1 represents an aryl or heteroaryl radical, optionally substituted, comprising in particular from 5 to 18 atoms, and R7 represents a hydrogen atom or a hydrocarbon radical, preferably comprising from 1 to 18, preferentially from 1 to 12 and in particular from 1 to 6, carbon atoms, linear, branched and/or cyclic, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused. In particular embodiments of the invention, Ar1 represents an aromatic heterocycle, in particular a pyridine ring, optionally substituted, and/or R7 represents a hydrogen atom.
In particular embodiments of the invention, R1 representing a hydrogen atom or a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused, R2 may represent a triazole group, optionally substituted.
In other variants of the invention, R1 representing a hydrogen atom or a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused, R2 represents a —(CH2)2—R8 group, wherein R8 represents:
In particularly preferred embodiments of the invention, R1 represents a hydrogen atom or a C1-C18, preferably C1-C6, for example C1-C3 and more particularly C1-C2, linear, branched and/or cyclic alkyl group, this alkyl group optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom.
The compound used according to the invention can in particular comply with the general formula (I′a):
wherein:
R1 represents a hydrogen atom or C1-C18, preferably C1-C6, linear, branched and/or cyclic alkyl group, optionally interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom, and R8 represents:
R8 not representing a hydrogen atom when R1 represents a propyl radical.
In particular embodiments of the invention, the compound used complies with the general formula (I″a):
wherein:
R1 represents a hydrogen atom or a C1-C18 linear, branched and/or cyclic alkyl group, optionally interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom,
R18 represents a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, comprising 4 to 18 carbon atoms, preferably 4 to 12 carbon atoms, and in particular 5 to 12 carbon atoms, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused.
R18 may in particular represent a butyl, pentyl, hexyl, cyclohexyl, phenyl, benzyl or adamantyl group.
Preferentially, in formula (1″a), R1 represents a hydrogen atom.
Particular compounds that can be used according to the invention comply with the following formulae (IIa), (IIb), (IIb1), (IIb2), (IIb3), (IIb4), (IIb5), (IIb6), (IIb7), (IIc), (IIc1), (IId), (IIe), (IIo), (IIp), (IIp1), (IIq), (IIr), (IIs), (IIt), (IIu), (IIv), (IIw), (IIw1), (IIx), (IIx1), and (IIy):
In alternative embodiments of the invention, the compound used is such that, in the general formula (I), R1 and R2 form together, with the atoms of the piperidine ring to which each is attached, a 6-membered heterocycle fused with the piperidine ring, optionally substituted by one or more radicals, which may be identical or different, each selected from a hydroxyl group, an amino group, a carbonyl group or a carbon radical, preferably comprising from 1 to 18, preferentially from 1 to 12 and in particular from 1 to 6, carbon atoms, linear, branched and/or cyclic, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused.
A particular compound that can be used according to the invention complies with the formula (IIf):
In other embodiments of the invention, the compound used is such that, in the general formula (I), R1 and R2 form together, with the atoms of the piperidine ring to which each is attached, a 5-membered heterocycle fused with the piperidine ring, optionally substituted by one or more radicals, which may be identical or different, each selected from a hydroxyl group, an amino group, a carbonyl group or a carbon radical, preferably comprising from 1 to 18, preferentially from 1 to 12 and in particular from 1 to 6, carbon atoms, linear, branched and/or cyclic, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused.
A particular compound that can be used according to the invention complies with the formula (IIg):
In further alternative embodiments of the invention, the compound used is such that, in the general formula (I), R1 and R2 form together, with the atoms of the piperidine ring to which each is attached, a 4-membered heterocycle fused with the piperidine ring, optionally substituted by one or more radicals, which may be identical or different, each selected from a hydroxyl group, an amino group or a carbon radical, preferably including from 1 to 18, preferentially from 1 to 12 and in particular from 1 to 6, carbon atoms, linear, branched and/or cyclic, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused.
The compound may in particular comply with the general formula (I′b):
wherein
R14 represents a hydrogen atom, a carbonyl radical or a C1-C18, preferably C1-C12 and in particular C1-C16, alkyl, alkenyl, alkynyl, alkylaryl or aryl radical, said radical optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom, and R15 represents a hydrogen atom, a hydroxyl radical, an amino radical, or a C1-C18, preferably C1-C12 and in particular C1-C6, alkyl, alkenyl or aryl radical, said radical optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom. Particular compounds that can be used according to the invention comply in particular with the formulae (IIh), (IIz1), (IIz2), (IIz3), (IIz4), and (IIz5):
In still alternative embodiments of the invention, the compound used is such that, in the general formula (I), R1 and R2 form together, with the atoms of the piperidine ring to which each is attached, a 3-membered heterocycle fused with the piperidine ring, optionally substituted by a —X—R5 group, wherein:
The compound used according to the invention may in particular comply with the general formula (I′c):
wherein R16 represents a hydrogen atom or a C1-C18, preferably C1-C12 and preferentially C1-C6, alkyl radical, said radical optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom.
In particular, R16 may represent a —CH2—OH group, a methyl radical or an ethyl radical.
Particular compounds that can be used according to the invention comply with the formulae (IIj), (IIk), (IIm) and (IIn):
The invention can also be expressed in the terms of a method for the therapeutic treatment of a subject suffering from or likely to contract a disease, in particular Pompe disease, this method comprising a step of administering to said subject in need thereof a therapeutically effective amount of a compound complying with the general formula (I) or one of the pharmaceutically acceptable salts thereof. This method may comply with one or more of the features described above in reference to the therapeutic use of the compound of general formula (I), or one of the pharmaceutically acceptable salts thereof, as a drug.
The invention also relates to the use of a compound of general formula (I), or one of the pharmaceutically acceptable salts thereof, as defined above, for manufacturing a drug, in particular a drug for treating Pompe disease.
According to another aspect, the present invention relates to a pharmaceutical composition containing, as active principle, a compound complying with the general formula (I) or one of the pharmaceutically acceptable salts thereof, in a pharmaceutically acceptable vehicle.
In the present description, “pharmaceutically acceptable vehicle” means any vehicle useful for preparing a pharmaceutical composition and which is generally safe, non-toxic and neither biologically nor otherwise undesirable for the subject to be treated, in particular for mammals and especially humans. The vehicle of the pharmaceutical composition according to the invention may be either solid or semi-solid or liquid. It may be a diluent, an adjuvant or any other vehicle conventional in itself for forming pharmaceutical compositions.
The pharmaceutical composition according to the invention may be in any galenic form, in particular in a form adapted to administration parenterally, intranasally, rectally, or by pulmonary or topical route. Preferentially, it is in a form suitable for oral administration. By way of examples of such galenic forms, non-limitative of the invention, mention can be made of the forms of granules, powder, tablets, capsules, pills, syrup, solution or drinkable suspension, etc. The pharmaceutical composition according to the invention may contain one or more excipients/additives conventional in themselves for forming pharmaceutical compositions, for example selected from preservatives, sweeteners, flavorings, fillers, disintegrators, wetting agents, emulsifiers, surfactants, dispersants, lubricants, stabilizers, buffers, antibacterial agents, antifungal agents, etc., or any one of the mixtures thereof; and/or any compound allowing rapid, prolonged or delayed, and/or targeted, release of the active principle after administration thereof to the subject.
The pharmaceutical composition according to the invention may furthermore contain one or more active principles other than the compound of general formula (I) or one of the pharmaceutically acceptable salts thereof, these active principles being able or not to act synergically with said compound.
The pharmaceutical composition according to the invention is preferably formulated in the form of unit doses.
The invention also relates to the therapeutic use of a pharmaceutical composition according to the invention, as defined above, for treating a disease, in particular Pompe disease. This use may comply with one or more of the features described above in reference to the therapeutic use of the compound of general formula (I) or of one of the pharmaceutically acceptable salts thereof.
According to another aspect, the present invention relates to a compound complying with the following general formula (I′), this general formula (I′) defining a subfamily of the compounds of general formula (I) described above, or one of the pharmaceutically acceptable salts thereof:
wherein:
and R2 represents a —CH(R3)—R4 group, wherein R3 and R4, which may be identical or different, each represent a hydrogen atom or a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused, R3 and R4 being such that, when R1 and R3 each represent a hydrogen atom, R4 does not represent a hydrogen atom or a phenyl radical,
and R1 and R2 being such that they do not simultaneously represent respectively a propyl radical and an ethyl radical,
In particular embodiments of the invention, the compound is such that, in the formula (I′):
R1 represents a hydrogen atom or a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused,
and R2 represents a —CH(R3)—R4 group, wherein R3 is as defined above and R4 represents a hydrocarbon radical, preferably comprising from 1 to 18, preferentially from 1 to 12 and in particular from 1 to 6, carbon atoms, linear, branched and/or cyclic, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising a single ring or a plurality of rings, optionally fused, and including one or more heteroatoms each selected from oxygen, nitrogen, sulfur and silicon and/or one or more groups including at least one heteroatom each selected from the carbonyl, sulfoxide, sulfonyl and silane groups; R4 not representing a phenyl radical when R1 and R3 each represent a hydrogen atom.
In particular, in the formula (I′), when R1 represents a hydrogen atom or a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused, R2 may represent a —(CHR7)—SO2—Ar1 group wherein Ar1 represents an aryl or heteroaryl radical, optionally substituted, preferably comprising from 5 to 18 atoms, and R7 represents a hydrogen atom or a hydrocarbon radical, preferably comprising from 1 to 18, preferentially from 1 to 12 and in particular from 1 to 6, carbon atoms, linear, branched and/or cyclic, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused.
The compound according to the invention may for example comply with the general formula (I′d):
wherein R1, R7 and Ar' are as defined above.
Otherwise, in the formula (I′), when R1 represents a hydrogen atom or a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused, R2 may represent a triazole group, optionally substituted.
The compound according to the invention may in particular comply with the formula (I′e):
wherein R1 is as defined above and R19 represents a hydrogen atom or a C1-C18 alkyl, alkylaryl, trialkylsilyl or aryl radical, said radical optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom.
In variants of the invention, in the general formula (I′), R1 representing a hydrogen atom or a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused, R2 represents a —(CH2)2—R8 group, wherein R8 represents:
optionally, b may be different from 1 when R1 represents a hydrogen atom and Ar2 represents a phenyl radical;
In particularly preferred embodiments of the invention, R1 represents a C1-C18, preferably C1-C6, linear, branched and/or cyclic alkyl group, optionally interrupted and/or substituted by one or more heteroatoms and/or more groups including at least one heteroatom.
The compound according to the invention may for example comply with the general formula (I′a):
wherein:
R1 represents a hydrogen atom or a C1-C18, preferably C1-C12, in particular
C1-C6, for example C1-C3 and more particularly C1-C2, linear, branched and/or cyclic alkyl group, said alkyl group optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom,
R8 represents:
R8 not representing a hydrogen atom when R1 represents a propyl radical. The compound according to the invention may comply with the general formula (I″a):
wherein
R1 represents a hydrogen atom or a C1-C18 linear, branched and/or cyclic alkyl group, optionally interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom,
R18 represents a linear, branched and/or cyclic hydrocarbon radical, saturated or unsaturated, aromatic or not, optionally substituted, comprising from 4 to 18 carbon atoms, preferably from 4 to 12 carbon atoms, and preferentially from 5 to 12 carbon atoms, optionally comprising one or more heteroatoms and/or one or more groups including at least one heteroatom and optionally comprising a single ring or a plurality of rings optionally fused.
R18 may in particular represent a butyl, pentyl, hexyl, cyclohexyl, phenyl, benzyl or adamantyl group.
Particular compounds according to the invention are the compounds of formulae (IIa), (IIb), (IIb1), (IIb2), (IIb3), (IIb4), (IIb5), (IIb6), (IIb7), (IIc), (IIc1), (IId), (IIo), (IIp), (IIp1), (IIq), (IIr), (IIs), (IIt), (IIu), (IIv), (IIw), (IIw1), (IIx), (IIx1), and (IIy) described above.
In alternative embodiments of the invention, the compound complies with the general formula (I′b):
wherein
R14 represents a hydrogen atom, a carbonyl radical or an alkyl, alkenyl, alkynyl, or alkylaryl radical, such as a benzyl or a C1-C18, preferably C1-C12 and in particular C1-C6, aryl radical, said radical optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom; an example of such a radical complies with the formula —CH2-TMS, where TMS represents a trimethylsilyl residue;
and R15 represents a hydrogen atom, a hydroxyl radical, an amino radical or a C1-C18, preferably C1-C12 and in particular C1-C6, alkyl, alkenyl or aryl radical, said radical optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom.
Particular compounds complying with this definition are the compounds of formulae (IIh), (IIz1), (IIz2), (IIz3), (IIz4) and (IIz5) described above.
In other variants of the invention, the compound complies with the general formula (I′c):
wherein R16 represents a hydrogen atom or a C1-C18, preferably C1-C12 and preferentially C1-C6, alkyl radical, said radical optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom.
In particular, R16 may represent a —CH2—OH group, a methyl radical or an ethyl radical.
Particular compounds complying with this definition are the compounds of formulae (IIj) and (IIk) described above.
All the compounds according to the invention, complying with the general formula (I′), can advantageously be used as drugs in accordance with the present invention, in particular for treating Pompe disease.
The compounds of general formula (I) used according to the invention, in particular the compounds complying with the general formula (I′) as defined above, can be synthesized by any method conventional per se for a person skilled in the art. It falls in particular within the competence of a person skilled in the art to determine, for each particular compound, which starting products to use and which synthesis method to apply.
The present invention furthermore relates to novel synthesis methods, which have been developed by the inventors for preparing compounds of general formula (I), and in particular of general formula (I′).
Some of the preparation methods according to the invention make it possible to obtain compounds of general formula (I′) wherein R1 and R2 are independent from one another, in particular complying with the general formula (I′a) or with the general formula (I″a) as above.
One example of such a particular preparation method according to the invention comprises successive steps of:
a/ reacting a compound of general formula (III) or (IV):
wherein Bn represents a benzyl radical,
with a compound of general formula (V):
wherein R8 is as defined above with reference to the compound of general formula (I′) according to the invention, R8 however representing neither a hydrogen atom nor a hydroxyl group —OH, nor an amino group —NH2, in the presence of an organometallic compound,
b/ optionally, reducing the hydroxylamine function into amine,
c/ optionally, alkylation of the nitrogen atom of the piperidine ring,
d/ and hydrogenolysis of the product obtained at the end of step where applicable step b/ or step c/, in particular for achieving the cleavage of the benzyl radicals to form hydroxyl groups, and where applicable the transformation of the hydroxylamine function into amine and the hydrogenation of the triple bond.
The organometallic compound used for the step a/ may be an organozinc, organolithium, organomagnesium, organoalane, organocopper, etc. compound, or any one of the mixtures thereof. Preferentially, in step a/, the reaction is implemented in the presence of dialkylzinc, in particular diethylzinc Et2Zn, or butyllithium.
More generally, the organometallic compound may comply with the general formula (VII):
R17-M (VII)
wherein
R17 represents a C1-C18 alkyl, alkenyl, alkynyl, alkylaryl or aryl radical, said radical optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom, and M represents a metal element such as lithium, zinc, copper, aluminum or magnesium, or M represents Mg—X2, where X2 represents a halogen atom.
The step b/ of reducing the hydroxylamine function into amine and the step c/ of N-alkylation can be implemented according to any method conventional per se for a person skilled in the art.
The step d/ of hydrogenolysis may also be performed by any method conventional per se for a person skilled in the art, in particular by catalytic hydrogenation.
Entirely surprisingly and advantageously, such a method makes it possible to prepare compounds of general formula (I′), in particular of general formula (I′a) or of general formula (I″a), completely stereoselectively.
Examples of such methods can be represented by the synthesis schemes 1 shown on
Compounds of general formula (I′a) or (I″a) may otherwise, for example, be prepared by a method comprising successive steps of:
a/ cycloaddition reaction of a compound of general formula (III) with trimethylsilylacetylene,
b/ reaction of the cycloadduct obtained with a fluoride such as tetrabutylammonium fluoride, for example by a protocol similar to the one described in the publication of Ahn, et al., 1994, J. Org. Chem. 59: 6282-6286,
c/ reduction by a metal hydride such as boron and sodium hydride (NaBH4), lithium and aluminum hydride (LiAlH4) or diisobutylaluminum hydride (DIBAL-H), or by a borane such as a dimethylsulfide-borane complex (BH3Me2S), a borane-tetrahydrofuran complex (BH3THF), a borane-pyridine complex (BH3pyridine), diborane B2H6, lithium and aluminum hydride (LiAIH4) being particularly preferred,
d/ optionally, a step of O-alkylation, O-acylation or O-sulfonylation, which can be implemented according to any method conventional per se for a person skilled in the art,
e/ optionally, a step of N-alkylation, which can be implemented according to any method conventional per se for a person skilled in the art,
f/ and hydrogenolysis of the product obtained at the end of step c/, where applicable step d/ or step e/, in particular for implementing the cleavage of the benzyl radicals to form hydroxyl groups.
This synthesis method is particularly advantageous in that:
Compounds of formula (I′d) above can be prepared by a method comprising the reaction of a compound of formula (III) or (IV) as above with a compound of general formula (VI):
in the presence of a lithiated base such as lithium diisopropylamide (LDA) or lithium bis(trimethylsilyl)amide (LiHMDS), preferably in the presence of LiHMDS at low temperature, preferably −78° C.; then, after optionally a step of reducing the hydroxylamine function into amine, and optionally a step of alkylation of the nitrogen atom of the piperidine ring, hydrogenolysis of the product obtained.
An example of such a method can be represented by the synthesis scheme 3 shown in
Compounds of general formula (I) and in particular of general formula (I′) can otherwise, for example, be prepared, from compounds of formula (III) or (IV), by a method comprising the successive steps of:
R17-M (VII)
wherein
R17 represents a C1-C18 alkyl, alkenyl, alkynyl, alkylaryl or aryl radical, said radical optionally being interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom, and M represents a metal element such as lithium, zinc, copper, aluminum or magnesium, or M represents Mg—X2, where X2 represents a halogen atom.
The synthesis route used for obtaining compounds complying with the general formula (I) falls under the general synthesis scheme 4 shown on
Other preparation methods according to the invention make it possible to obtain compounds of general formula (I′) wherein R1 and R2 form together, with the atoms of the piperidine ring to which each is attached, a 4-membered heterocycle fused with the piperidine ring.
Examples of such methods comprise successive steps of:
or b′/ reducing alkylation of β-lactam carbonyl by reaction with tetramethyldisiloxane in the presence of Vaska's catalyst at ambient temperature, and then addition of a compound of formula (VII) as defined above, at −78° C., according to a protocol similar to the one described in the publication by Xie and Dixon, 2017, Chem. Sci. 8: 7492-7497;
and finally
The step c/ of hydrogenolysis can be implemented according to any method conventional per se for a person skilled in the art, in particular by catalytic hydrogenation.
The steps b/ and b′/ are particularly advantageous, and novel, in that they make it possible to implement the reduction or the reducing alkylation of a β-lactam the nitrogen atom of which is included in a piperidine ring, to form a heterocycle of the conidine type.
Examples of such methods can be represented by the synthesis scheme 5 shown on
Another example of such a method comprises successive steps of:
a/ Kinugasa reaction, as described in the reviews by Stecko et al., 2014, Tetrahedron 70: 7817-7844 or by Khangarot & Kaliappan, 2013, Eur. J. Org. Chem. 7664-7677, of a compound of general formula (III) with a compound of general formula (VIII):
wherein R15 represents a C1-C18 alkyl, alkenyl or aryl radical, optionally interrupted and/or substituted by one or more heteroatoms and/or one or more groups including at least one heteroatom, in the presence of a Cu(I) salt and an amine; then
b/ reducing alkylation of β-lactam carbonyl by reaction with tetramethyldisiloxane in the presence of Vaska's catalyst at ambient temperature, then addition of a compound of formula (VII) as defined above, at −78° C., according to a protocol similar to the one described in the publication by Xie and Dixon, 2017, Chem. Sci. 8: 7492-7497; and finally
c/ hydrogenolysis of the product obtained at the end of the step b/, in particular for implementing the cleavage of the benzyl radicals to form hydroxyl groups. The step c/ of hydrogenolysis can be implemented according to any method conventional per se for a person skilled in the art, in particular by catalytic hydrogenation.
Step b/ is particularly advantageous, and novel, in that it makes it possible to implement the reducing alkylation of a β-lactam the nitrogen atom of which is included in a piperidine ring, to form a heterocycle of the conidine type.
An example of such a method can be represented by the synthesis scheme 6 shown on
Other preparation methods according to the invention make it possible to obtain compounds of general formula (I′) wherein R1 and R2 form together, with the atoms of the piperidine ring to which each is attached, a 3-membered heterocycle fused with the piperidine ring.
An example of such a method comprises successive steps of:
a/ cycloaddition reaction of a compound of general formula (111) or of a compound of general formula (III′):
wherein Ac represents an acetyl radical,
with a compound of general formula (V) as described above, wherein R8 is as defined above with reference to the compound of general formula (I′) according to the invention, R8 however representing neither a hydrogen atom nor a hydroxyl group —OH, nor an amino radical —NH2,
An example of such a method can be represented by the synthesis scheme 7 shown in
An example of a method for preparing compounds of general formula (I′e) as above can be represented by the synthesis scheme 8 shown on
The features and advantages of the invention will emerge more clearly in the light of the following example embodiments, provided by simple way of illustration and in no way !imitative of the invention, with the support of
All the reactions were conducted under inert atmosphere in previously dried glassware, under stirring by a magnetic bar, the assembly being placed above a magnetic stirrer, except in the case of reactions under irradiation by microwave. The latter were implemented in sealed tubes equipped with a magnetic bar placed in a microwave reactor, at a temperature regulated by an internal infrared probe. The toluene, the ether and the dichloromethane used as reaction solvents were previously filtered on an Inert PureSolv® purification system. The acetonitrile and dichloroethane were distilled on CaH2. The THF was distilled on sodium in the presence of benzophenone. The commercial reagents, the methanol and the ethanol were used without purification. The reactions were followed by thin-layer chromatography (TLC) by means of silica on aluminum plates (Merck, Kieselgel 60 F254), with revelation under ultraviolet radiation and then by a 3% solution of potassium permanganate in a 10% solution of potassium hydroxide (w/v). The purifications by column chromatography were implemented by means of Macherey-Nagel® Silica Gel 60 (70-230 mesh). The rotatory powers were measured on a PerkinElmer 341 polarimeter. The infrared spectra were obtained on a Nicolet “Magna 550” spectrometer including an ATR (attenuated total reflexion) module on which the products were deposited pure, in the solid or liquid state. The data are expressed in cm−1. The 1H NMR and 13C NMR {DEPT-Q} were obtained with Avance 500 (1H: 500 MHz, 13C: 125 MHz) or Avance 400 (1H: 400 MHz, 13C: 100 MHz) spectrometers. The chemical shifts for the 1H spectra are expressed with respect to those of the residual solvents contained in CDCl3 (δ7.26 ppm) or CD3OD (δ 3.31 ppm). The chemical shifts for the 13C spectra are expressed with respect to those of the solvents CDCl3 (δ 77.16 ppm) or CD3OD (δ 49.00 ppm). The 1H NMR spectra are reported as follows: chemical shift (ppm), multiplicity (br: broad; s: singulet; d: doublet; dd: doublet of doublets; t: triplet; pst: pseudo triplet; m: multiplet), coupling constants (Hz) and integration. The high-resolution mass spectra (HRMS) were recorded on a Thermo Scientific ESI/LTQ Orbitrap XL® or Waters G2-S Q-TOF mass spectrometer.
The synthesis methods used for obtaining compounds complying with the above general formula (I′a) falls under the general synthesis schemes shown on
A 1M solution of diethyl zinc in hexane (1.5 equiv.) is added dropwise to an alkyne solution of formula (V) (4 equiv.) in anhydrous toluene at 0° C. under argon atmosphere. The resulting mixture is stirred for 30 minutes at 0° C. A solution of ketonitrone of formula (III) (1 equiv.) in anhydrous toluene is then added dropwise at 0° C., and then the reaction mixture is stirred until the reaction is complete (followed by TLC). A saturated aqueous solution of NaHCO3 is added and then the resulting mixture is diluted with diethylether. The organic phase is separated and the aqueous phase is extracted twice with diethylether. The organic phases are washed with brine, dried on MgSO4 and evaporated under reduced pressure. The hydroxylamines of formula A-1 thus formed are purified by silica gel chromatography, before being reduced and debenzylated in accordance with the following general protocol B.
A 1.4 M solution of n-butyllithium in hexane (2.5 equiv.) is added dropwise to a solution of alkyne of formula (V) (2.5 equiv.) in anhydrous THF at −10° C. under argon atmosphere. The resulting solution is stirred for 15 minutes at −10° C. and is then cooled to −78° C. A solution of ketonitrone of formula (III) (1 equiv.) in anhydrous THF is then added dropwise at −78° C., and then the reaction mixture is stirred until the reaction is complete (followed by TLC). A saturated aqueous solution of NH4Cl is added and the mixture is allowed to rise to ambient temperature, and then extracted three times by ethyl acetate. The organic phases are collected and washed with brine, dried on MgSO4 and concentrated under reduced pressure. The hydroxylamines of formula A-1 thus isolated are purified by silica gel chromatography, before being reduced and debenzylated in accordance with the following general protocol B.
1.5 equiv. of hydrochloric acid HCI (2M solution in anhydrous ether) and 0.2 equiv. of Pd/C 10% are added to a solution of O-benzylated iminosugar (1 equiv.) in absolute ethanol. The suspension obtained is stirred at room temperature for 17 hours under pressure of hydrogen (5 bar) and then filtered on Celite®. The Celite® is rinsed with methanol and the filtrate is concentrated at reduced pressure to give an iminosugar hydrochlorate. This salt is purified on Dowex® 50W-X2 ion exchange resin previously activated by HCI, and eluted with aqueous NH4OH, to obtain after evaporation of the solvents the corresponding neutral compound of formula (I′a).
The compound of above formula (IIc) ((2R,3R,4R,5S)-2-(hydroxymethyl)-2-phenethylpiperidine-3,4,5-triol: 36.7 mg; 73% for 2 steps) was prepared in accordance with the general procedures A and B, from nitrone of formula (III) (110.0 mg, 0.205 mmol) and phenylacetylene (Va: R8=Ph, 0.034 mL; 0.307 mmol).
A solid yellow is obtained, with the following characteristics:
[α]20D+4.7 (c 0.98, CH3OH);
IR v 3291, 2918, 2864, 1632, 1494, 1455, 1363, 1270, 1198, 1094, 1076, 1040, 1004 cm−1;
1H NMR (500 MHz, CD3OD) δ 7.31-7.08 (m, 5H), 3.73 (d, J=10.4 Hz, 1H), 3.55 (d, J=10.5 Hz, 1H), 3.55-3.43 (m, 2H), 3.45-3.34 (m, 1H), 3.00-2.90 (m, 1H), 2.68-2.51 (m, 3H), 1.91-1.72 (m, 2H) ppm;
13C NMR (125 MHz, CD3OD) δ 144.4 (ArCq), 129.4 (ArCH), 129.3 (ArCH), 126.7(ArCH), 76.5 (CH), 75.2 (CH), 73.8 (CH), 65.6 (CH2), 60.5 (Cq), 45.9 (CH2), 30.7 (CH2), 29.9 (CH2) ppm;
HRMS (ESI+) Calc. C14H22NO4 [M+H]+: m/z=268.15433; Found m/z=268.15372.
The compound of above formula (IIx1) ((2R,3R,4R,5S)-2-(hydroxymethyl)-2-(2-(trimethylsilyl)ethyl)piperidine-3,4,5-triol: 7.1 mg; 29% for 2 steps) was prepared in accordance with the general procedures A and B, from nitrone of formula (III) (102.1 mg, 0.189 mmol) and trimethylsilylacetylene (Vb: R8=TMS, 0.105 mL; 0.759 mmol).
A white foam is obtained, with the following characteristics:
[hd]20D+4.51 (c 0.71, MeOH);
1H NMR (400 MHz, CD3OD) δ 3.57 (d, J=10.9 Hz, 1H), 3.52 (d, J=9.3 Hz, 1H), 3.44 (t, J=4.0 Hz, 1H), 3.39 (d, J=10.0 Hz, 1H), 3.39-3.33 (m, 1H), 2.85 (dd, J=12.8, 5.5 Hz, 1H), 2.42 (dd, J=12.8, 10.8 Hz, 1H), 1.62-1.51 (m, 2H), 0.56-0.44 (m, 1H), 0.39-0.29 (m, 1H), 0.02 (s, 9H) ppm;
13C NMR (100 MHz, CD3OD) δ 76.6 (CH), 75.5 (CH), 73.8 (CH), 65.6 (CH2), 60.5 (Cq), 45.8 (CH2), 21.0 (CH2), 8.60 (CH2), −1.86 (CH3) ppm;
HRMS (ESI+) Calc. C11H26NO4Si [M+H]+: m/z=264.16256; Found m/z=264.16282.
The compound of above formula (IIr) ((2R,3R,4R,5S)-2-(2-((3R,5R,7R)-adamantan-1-ypethyl)-2-(hydroxymethyl)piperidine-3,4,5-triol: 15.0 mg; 27% for 2 steps) was prepared in accordance with the general procedures A′ and B, from nitrone of formula (III) (100 mg, 0.185 mmol) and adamantylacetylene (Vh: R8=adamantyl, 74.5 mg, 0.465 mmol).
A yellowish lacquer is obtained, with the following characteristics:
[α]20D+7.46 (c 0.63, MeOH);
1H NMR (500 MHz, CD3OD) δ 3.79 (d, J=10.9 Hz, 1H), 3.64-3.48 (m, 4H), 3.11 (br d, J=11.3 Hz, 1H), 2.78-2.66 (m, 1H), 2.00-1.91 (m, 3H), 1.82-1.63 (m, 8H), 1.58-1.49 (m, 6H), 1.19-1.03 (m, 2H) ppm;
13C NMR (100 MHz, CD3OD) δ 74.9 (CH), 72.5 (CH), 70.8 (CH), 63.7 (Cq), 62.5 (CH2), 44.2 (CH2), 43.3 (CH2), 38.2 (CH2), 37.0 (CH2), 30.1 (CH), 20.7 (CH2) ppm;
HRMS (ESI+) Calc. C18H31NO4 [M+H]+: m/z=326.23258; Found m/z=326.23225.
The compound of above formula (IIb7) ((2R,3R,4R,5S)-2-(2-cyclohexylethyl)-2-(hydroxymethyl)piperidine-3,4,5-triol: 57.3 mg; 59% for 2 steps) was prepared in accordance with the general procedures A and B, from nitrone of formula (III) (99.8 mg; 0.185 mmol) and 1-ethynylcyclohexene (Vd: R8=cyclohexene; 87 μL; 0.742 mmol).
A white solid is obtained, with the following characteristics:
[α]20D+3.63 (c 1.43, MeOH);
1H NMR (400 MHz, CD3OD) δ 3.60 (d, J=10.6 Hz, 1H, 7CH2), 3.52-3.43 (m, 2H, 3CH, 4CH), 3.43-3.33 (m, 2H), 2.87 (dd, J=12.8, 5.0 Hz), 2.49 (ps t, J=11.7 Hz, 1H), 1.83-1.62 (m, 5H), 1.60-1.47 (m, 2H), 1.34-1.06 (m, 6H), 1.01-0.86 (m, 2H) ppm;
13C NMR (100 MHz, CD3OD) δ 76.5 (CH), 75.1 (CH), 73.8 (CH), 65.5 (CH2), 60.3 (Cq), 45.8 (CH2), 39.9 (CH), 34.6 (CH2), 34.5 (CH2), 30.6 (CH2), 27.8 (CH2), 27.5 (CH2), 24.8 (CH2) ppm;
HRMS (ESI+) Calc. C14H20NO4 [M+H]+: m/z=274.20128; Found m/z=274.20139.
The compound of above formula (IIb1) ((2R,3R,4R,5S)-2-(hydroxymethyl)-2-pentylpiperidine-3,4,5-triol: 32.8 mg; 67% for 2 steps) was prepared in accordance with the general procedures A and B, from nitrone of formula (III) 96.8 mg; 0.180 mmol) and 1-pentyne (Ve: R8=C3H7; 71 μL; 0.720 mmol).
A yellow solid is obtained, with the following characteristics:
[α]20D−1.29 (c 1.01, MeOH);
1H NMR (400 MHz, CD3OD) δ 3.61 (d, J=10.6 Hz, 1H), 3.52-3.33 (m, 4H), 2.87 (dd, J=12.8, 5.3 Hz, 1H), 2.49 (ps t, J=11.8 Hz, 1H), 1.62-1.43 (m, 2H), 1.43-1.17 (m, 6H), 0.92 (t, J=6.7 Hz, 3H) ppm;
13C NMR (100 MHz, CD3OD) δ 76.5 (CH), 75.2 (CH), 73.9 (CH), 65.7 (CH2), 60.2 (Cq), 45.9 (CH2), 33.9 (CH2), 27.7 (CH2), 23.6 (CH2), 22.9 (CH2), 14.4 (CH3) ppm; HRMS (ESI+) Calc. C11H24NO4 [M+H]+: m/z=234.16998; Found m/z=234.17004.
The compound of above formula (IIb3) ((2R,3R,4R,5S)-2-heptyl-2-(hydroxymethyl)piperidine-3,4,5-triol: 33.2 mg; 69% for 2 steps) was prepared in accordance with the general procedures A and B, from nitrone of formula (III) (100.7 mg; 0.187 mmol) and 1-heptyne (Vf: R8=C5H11; 98 μL; 0.749 mmol).
A yellow solid is obtained, with the following characteristics:
[α]20D+1.04 (c 0.96, MeOH);
1H NMR (400 MHz, CD3OD) δ 3.61 (d, J=10.6 Hz, 1H), 3.51-3.44 (m, 2H), 3.41 (d, J=10.8 Hz), 3.39-3.33 (m, 1H), 2.87 (dd, J=13.0, 5.4 Hz, 1H), 2.49 (ps t, J=11.4 Hz, 1H), 1.58-1.47 (m, 2H), 1.43-1.18 (m, 10H), 0.90 (t, J=6.2 Hz, 3H) ppm;
13C NMR (100 MHz, CD3OD) δ 76.5 (CH), 75.1 (CH), 73.8 (CH), 65.6 (CH2), 60.3 (Cq), 45.9 (CH2), 33.0 (CH2), 31.7 (CH2), 30.4 (CH2), 23.7 (CH2), 23.2 (CH2), 27.8 (CH2), 14.4 (CH3) ppm;
HRMS (ESI+) Calc. C13H28NO4 [M+H]+: m/z=261.20128; Found m/z=261.20131.
The compound of above formula (IIc1) ((2R,3R,4R,5S)-2-(hydroxymethyl)-2-(3-phenylpropyl)piperidine-3,4,5-triol: 20.2 mg; 45% for 2 steps) was prepared according to the general procedures A and B, from nitrone of formula (III) (96.7 mg; 0.179 mmol) and 3-phenyl-1-propyne (Vg: R8=CH2Ph; 89 μL; 0.719 mmol). A yellow lacquer is obtained, with the following characteristics:
[α]20D+1.68 (c 1.37, MeOH);
1H NMR (400 MHz, CD3OD) δ 7.10-7.10 (m, 5H), 3.60 (d, J=10.6 Hz, 1H), 3.49-3.37 (m, 3H), 3.37-3.32 (m, 1H), 2.84 (dd, J=13.1, 5.4 Hz, 1H), 2.69-2.53 (m, 2H), 2.42 (dd, J=13.0, 10.8 Hz, 1 H), 1.75-1.49 (m, 4H) ppm;
13C NMR (100 MHz, CD3OD) δ 143.6 (ArCq), 129.4 (ArCH), 129.3 (ArCH), 126.7 (ArCH), 76.5 (CH), 75.1 (CH), 73.8 (CH), 65.6 (CH2), 60.3 (Cq), 45.8 (CH2), 37.6 (CH2), 27.5 (CH2), 25.3 (CH2) ppm;
HRMS (ESI+) Calc. C15H24NO4 [M+H]+: m/z=282.16998; Found m/z=282.16968.
The synthesis method used for obtaining compounds complying with the general formula (I′a) below falls under the general synthesis scheme 2 shown on
The compound of above formula (IId) (2R,3R,4R,5S)-2-(2-hydroxyethyl)-2-(hydroxymethyl)piperidine-3,4,5-triol is prepared in the following manner.
A mixture of nitrone (III) (566 mg, 1.05 mmol) and of trimethylsilylacetylene alkyne (Vb: R8=SiMe3, 2.2 mL, 15.75 mmol) was stirred at ambient temperature for 21 hours, and then the excess alkyne was evaporated under reduced pressure. The raw isoxazoline B-2 thus obtained (667 mg, 1.05 mmol) was dissolved in anhydrous THF (20 mL) and a solution of TBAF (1M in THF, 1.05 mL, 1.05 mmol) was added at 0° C. The solution was stirred at 0° C. for 45 minutes, and then CH2Cl2 and water were added. The aqueous phase was extracted three times with CH2Cl2. The organic phases were washed with brine, dried on MgSO4, and then the solvents were evaporated under reduced pressure. Purification of the residue obtained by chromatography supplied bicyclic β-lactic C-2 (440.6 mg, 74% in 2 steps). LiAlH4 (9.6 mg, 0.25 mmol) was added at 0° C. to a solution of this β-lactam C-2 (71.3 mg, 0.13 mmol) in ether (1.5 mL). The mixture was stirred at ambient temperature for 2 hours and then diluted with CH2Cl2, aqueous NH4Cl and a few drops of aqueous NaOH up to basic pH. The aqueous phase was extracted (three times) with CH2Cl2. The organic phases were washed with brine, dried on MgSO4, and the solvents were evaporated under reduced pressure to give piperidine D-2 (translucent oil, 61.1 mg, 85%) after purification by chromatography. This compound (26.3 mg, 0.463 mmol) was debenzylated in accordance with the general procedure B to give piperidine (IId) (9.3 mg, 97%). A pale yellow lacquer was obtained, with the following characteristics:
[α]20D+8.2 (c 0.61, CH3OH);
IR v 3287, 2920, 1644, 1431, 1081 cm−1;
1H NMR (500 MHz, CD3OD) δ 1.86 (t, J=6.5 Hz, 2H), 2.65 (dd, J=11.2, 12.6 Hz, 1H), 2.92 (dd, J=5.4, 13.1 Hz, 1H), 3.33-3.43 (m, 2H), 3.48 (d, J=9.1 Hz, 1H), 3.58 (d, J=10.8 Hz, 1H), 3.64-3.76 (m, 3H) ppm;
13C NMR (125 MHz, CD3OD) δ 30.5 (CH2), 45.8 (CH2), 58.8 (CH2), 60.9 (Cq), 65.7 (CH2), 73.5 (CH), 74.6 (CH), 76.3 (CH) ppm;
HRMS (ESI+) Calc. C8H18NO5[M+H]+: m/z=208.1185; Found m/z=208.1184.
The synthesis method used for obtaining compounds complying with the general formula (I′d) below falls under the general synthesis scheme 3 shown on
The compound of above formula (IIw) (2S,3S,4R,5S)-3,4,5-tris(benzyloxy)-2-((benzyloxy)methyl)-2-(pyridin-2-ylsulfonyl)piperidin-1-ol is prepared in the following manner. A solution of nitrone of formula (III) (196 mg; 0.366 mmol) and of 2-pyridyl-methylsulfone (Vla) (74.6 mg; 0.475 mmol) in anhydrous THF (10 mL) under argon atmosphere was cooled to −78° C. 1M LiHMDS in THF (548 μL; 0.548 mmol) was added dropwise and then the reaction mixture was stirred at this same temperature until the reaction was complete. After the addition of water and ethyl acetate, the aqueous phase was extracted (three times) with ethyl acetate. The collected organic phases were washed with brine, dried on MgSO4 and then concentrated at reduced pressure. Purification of the residue obtained by chromatography supplied the compound of formula A-3a (R7=H) 115 mg; 55%). This compound was debenzylated according to a variant of the general procedure B to give piperidine (IIw).
The synthesis methods used for obtaining compounds complying with the general formula (I), in particular compounds complying with the general formula (I′a), below fall under a general synthesis scheme 4 shown on
The compound of above formula (IIa) ((2R,3R,4R,5S)-2-ethyl-2-(hydroxymethyl)piperidine-3,4,5-triol) was prepared in accordance with the general protocol B described for the synthesis method 1, from piperidine D-4b (R17=vinyl, 74.0 mg, 0.137 mmol), obtained as described in the publication by Boisson et al., 2015, Org. Lett. 17: 3662-3665, and was isolated with a yield of 95% (24.9 mg).
A pale yellow lacquer is obtained, with the following characteristics:
[α]20D+1.6 (c 1.22, CH3OH);
IR v 3286, 2939, 1643, 1445, 1096 cm−1;
1H NMR (500 MHz, CD3OD) δ 0.86 (t, J=7.6 Hz, 3H), 1.55-1.68 (m, 2H), 2.51 (dd, J=10.8, 12.8 Hz, 1H), 2.89 (dd, J=5.4, 13.0 Hz, 1H), 3.34-3.40 (m, 1H), 3.42 (d, J=10.7 Hz, 1H), 3.44-3.51 (m, 2H), 3.62 (d, J=10.7 Hz, 1H) ppm; 13C NMR (125 MHz, CD3OD) δ 7.0 (CH3), 20.1 (CH2), 45.7 (CH2), 60.5 (Cq), 65.0 (CH2), 73.6 (CH), 75.1 (CH), 76.4 (CH) ppm;
HRMS (ESI+) Calc. C8H17NO4[M+H]+: m/z=192.1236; Found m/z=192.1237.
The compound of above formula (IIb) ((2R,3R,4R,5S)-2-(hydroxymethyl)-2-propylpiperidine-3,4,5-triol) was prepared in accordance with the general protocol B described for the synthesis method 1, from piperidine D-4a (R17=allyl, 39.7 mg, 0.071 mmol), obtained as described in the publication by Vieira Da Cruz et al., 2017, J. Org. Chem. 82: 9866-9872, and was isolated with a yield of 79% (11.4 mg).
A colorless lacquer is obtained, with the following characteristics:
[α]20D+5.4 (c 0.55, CH3OH);
IR v 3291, 2932, 1625, 1454, 1090 cm−1;
1NMR (500 MHz, CD3OD) δ 0.94 (t, J=7.2 Hz, 3H), 1.22-1.42 (m, 2H), 1.49-1.57 (m, 2H), 2.53 (dd, J=10.8, 13.0 Hz, 1H), 2.89 (dd, J=5.5, 13.0 Hz, 1H), 3.34-3.41 (m, 1H), 3.42 (d, J=10.7 Hz, 1H), 3.45-3.51 (m, 2H), 3.64 (d, J=10.7 Hz, 1H) ppm;
13C NMR (125 MHz, CD3OD) δ 15.3 (CH3), 16.6 (CH2), 30.4 (CH2), 45.7 (CH2), 60.7 (CO, 65.3 (CH2), 73.5 (CH), 74.9 (CH), 76.3 (CH) ppm;
HRMS (ESI+) Calc. C9H20NO4 [M+H]+: m/z=206.1392; Found m/z=206.1396
The compound of above formula (IIe) (2R,3R,4R,5S)-2-ethyl-2-(hydroxymethyl)-1-propylpiperidine-3,4,5-triol is prepared in the following manner.
The piperidine E-4b (R1=allyl, R17=vinyl; 62 mg, 0.105 mmol), obtained as described in the publication by Boisson et al., 2015, Org. Lett. 17: 3662-3665, was dissolved in anhydrous acetonitrile (0.5 mL), treated with 2-nitrobenzenesulfonyl chloride (186 mg, 0.84 mmol) at 0° C., and then with hydrazine monohydrate (0.08 mL, 1.68 mmol), added dropwise at 0° C. This reaction mixture was maintained under stirring at ambient temperature for 28 hours, and then water was added and the aqueous phase was extracted (3 times) with dichloromethane. The collected organic phases were washed with brine, dried over MgSO4 and concentrated under reduced pressure. After purification by chromatography, a mixture of the products of hydrogenation solely of the allyl group and of hydrogenation of the allyl and vinyl groups was isolated (58 mg, 94%, 3:7 mixture). This mixture was dissolved in methanol (1.5 mL), and then treated with Pearlman's reagent (20% Pd(OH)2/C, 16.8 mg, 0.119 mmol) and HCl (2 M solution in ether, 0.2 mL, 0.4 mmol) under hydrogen atmosphere (1 atm) and under vigorous stirring for 40 hours. The mixture was filtered over Celite®, the Celite® was rinsed several times with methanol, then the filtrate was concentrated under reduced pressure. The residue was solubilized in water and purified on cation exchange resin (DOWEX 50W-X8, form H+; elution by an aqueous solution of 1M NH4OH) to provide the product (IIe) (11.9 mg, 49%). A beige solid is obtained, with the following characteristics:
[α]20D−40.7 (c 0.60, CH3OH);
IR v 3363, 2962, 2932, 2874, 2826, 1653, 1464, 1379, 1097, 1048, 1016 cm−1 1H NMR (500 MHz, CD3OD) δ□0.90 (t, J=7.5 Hz, 3H), 0.96 (t, J=7.5 Hz, 3H), 1.39-1.50 (m, 1H), 1.51-1.60 (m, 1H), 1.61-1.75 (m, 2H), 2.26-2.34 (m, 1H), 2.42-2.51 (m, 1H), 2.76-2.85 (m, 1H), 2.93-3.00 (m, 1H), 3.40-3.52 (m, 3H), 3,65 (d, J=11.0 Hz, 1H), 3.79 (d, J=11.0 Hz, 1H) ppm;
13C NMR (125 MHz, CD3OD) δ 9.6 (CH3), 11.9 (CH3), 19.2 (CH2), 23.2 (CH2), 52.0 (CH2), 52.2 (CH2), 62.5 (CH2), 71.5 (CH), 74.9 (CH), 76.4 (CH) ppm;
HRMS (ESI+) Calc. C11H24NO4 [M+H]+: m/z=234.1700; Found m/z=234.1696.
The compound of formula (IIf) as above (1R,2R,3S,9aR)-9a-(hydroxymethyl)octahydro-1H-quinolizine-1,2,3-triol is prepared in the following manner.
Piperidine E-4a (R1=R17=allyl; 35 mg, 0.06 mmol), obtained as described in the publication by Vieira Da Cruz et al., 2017, J. Org. Chem. 82: 9866-9872, was dissolved in anhydrous CH2Cl2 (3 mL). This solution was carefully degassed, and then the Grubbs II catalyst (1.3 mg, 2.5 mol %) was added. The mixture was maintained under stirring at ambient temperature for 20 hours, and then filtered over silica. The silica was rinsed with ethyl acetate and methanol, and the solvents were evaporated under reduced pressure. After purification of the residue thus obtained by chromatography, the product of cyclization by metathesis of F-4a olefins (32 mg, 95%) was isolated. The latter (34 mg, 0.06 mmol) was dissolved in methanol (1 mL), and then treated with Pearlman's reagent (20% Pd(OH)2/C, 14 mg, 0.10 mmol) and HCl (2M solution in ether, 0.045 mL, 0.09 mmol) under hydrogen atmosphere (5 bar) and under vigorous stirring for 17 hours. The mixture was filtered over Celite®, the Celite®was rinsed several times with methanol, and then the filtrate was concentrated under reduced pressure. The residue was dissolved in water and purified over cation exchange resin (DOWEX® 50W-X8, form H+; elution by an aqueous solution of 1M NH4OH) to provide the product (IIf) (9 mg, 73%).
A colorless oil is obtained, with the following characteristics:
[α]20D+16.6 (c 0,41, CH3OH);
IR v 3336, 2942, 2869, 1053 cm−1;
1H NMR (500 MHz, CD3OD) δ 1.28 (br d, J=11.6 Hz, 1H), 1.37-1.44 (m, 1H), 1.62-1.76 (m, 3H), 1.81-1.94 (m, 1H), 2.66 (dd, J=3.0, 14.0 Hz, 1H), 2.72 (dd, J=4.9, 11.3 Hz, 1H), 3.12 (t, J=11.0 Hz, 1H), 3.20 (pstd, J=3.0, 14.0 Hz, 1H), 3.38 (pst, J=9.3 Hz, 1H), 3.45-3.52 (m, 2H), 3.68 (d, J=11.1 Hz, 1H), 3.95 (d, J=11.1 Hz, 1H) ppm;
13C NMR (125 MHz, CD3OD) δ 18.7 (CH2), 19.2 (CH2), 20.7 (CH2), 48.5 (CH2), 53.3 (CH2), 60.7 (CH2), 62.0 (Cq), 71.4 (CH), 74.1 (CH), 75.9 (CH) ppm; HRMS (ESI+) Calc. C10H20NO4 [M+H]+: m/z=218.1392; Found m/z=218.1396.
The compound of formula (IIg) as above (6S, 7R, 8R, 8aR)-8a-(hydroxymethyl)-octahydroindolizine-6,7,8-triol is prepared in the following manner.
Piperidine E-4b (R1=allyl, R17=vinyl; 74 mg, 0.125 mmol), obtained as described in the publication by Boisson et al., 2015, Org. Lett. 17: 3662-3665, was dissolved in anhydrous CH2Cl2 (6.3 mL). This solution was carefully degassed, and then the Grubbs II catalyst (5.3 mg, 0.006 mmol) was added and the mixture was heated at 40° C. for 3 hours, under stirring. After cooling to ambient temperature, the reaction mixture was filtered over silica, the silica was rinsed with ether, and the solvents were evaporated under reduced pressure. After purification of the residue thus obtained by chromatography, the product of cyclization by metathesis of olefins F-4b (65 mg, 93%) was isolated. The latter (50 mg, 0.089 mmol) was dissolved in methanol (1.4 mL), then treated with Pearlman's reagent (20% Pd(OH)2/C, 17.9 mg, 0.025 mmol) and HCl (2M solution in ether, 0.066 mL, 0.133 mmol) under hydrogen atmosphere (5 bar) and under vigorous stirring for 17 h. The mixture was filtered over Celite®, the Celite® was rinsed several times with methanol, and then the filtrate was concentrated under reduced pressure. The residue was dissolved in water and purified over cation exchange resin (DOWEX 50W-X8, form H+; elution by an aqueous solution of 1M NH4OH) to provide the product (IIg) (16 mg, 89%).
A beige solid is obtained, with the following characteristics:
[α]20D+27.9 (c 0.41, CH3OH);
IR v 3330, 2924, 1652, 1031 cm−1;
1H NMR (500 MHz, CD3OD) δ 1.46-1.53 (m, 1H), 1.67-1.76 (m, 1H), 1.88-1.96 (m, 1H), 1.98-2.08 (m, 1H), 2.46 (pst, J=11.0 Hz, 1H), 2.80-2.89 (m, 2H), 3.09-3.18 (m, 1H), 3.33-3.41 (m, 2H), 3.43-3.49 (m, 1H), 3.53 (d, J=11.5 Hz, 1H), 3.76 (d, J=9.5 Hz, 1H) ppm;
13C NMR (125 MHz, CD3OD) δ 21.0 (CH2), 26.1 (CH2), 53.8 (CH2), 55.9 (CH2), 63.6 (CH2), 71.0 (CH), 73.0 (CH), 73.2 (Cq), 76.5 (CH) ppm;
HRMS (ESI+) Calc. C9H18NO4 [M+H]+: m/z=204.1236; Found m/z=204.1234.
The synthesis method used for obtaining the compound complying with the general formula (I′b) as above falls under a general synthesis scheme 5 shown on
The compound of above formula (IIh) ((3S,4R,5R,6R)-6-(hydroxymethyl)-1-azabicyclo[4.2.0]octane-3,4,5-triol) is prepared in the following manner.
[Rh(COD)2]BF4 (2.5 mg, 0.006 mmol), 1,3-bis(diphenylphosphino)propane (2.7 mg, 0.006 mmol) and PhSiH3 (25 μL, 0.20 mmol) were added to a solution of β-lactam C-5 (corresponding to the compound C-2 obtained as intermediate in synthesis method 2, as described in the protocol for preparing the compound (IId) above) (57.1 mg, 0.10 mmol) in distilled THF. The solution was stirred at 50° C. for 4 hours. After cooling, the reaction mixture was diluted with EtOAc and aqueous NaOH (1 M). The aqueous phase was extracted (three times) with EtOAc. The organic phases were washed with brine, dried over MgSO4, and the solvents were evaporated under reduced pressure to give conidine G-5 (33.2 mg, 60%) after purification by chromatography. This compound (20.0 mg, 0.04 mmol) was debenzylated in accordance with the general procedure B to give the compound (IIh) (6.3 mg, 93%).
A pale yellow oil is obtained, with the following characteristics:
[α]20D−5.17 (c 0.58, CH3OH);
IR v 3234, 2917, 1429, 1030 cm−1;
1H NMR (500 MHz, CD3OD) δ 2.18-2.26 (m, 1H), 2.55-2.63 (m, 1H), 2.89 (dd, J=5.9, 13.3 Hz, 1H), 3.21 (dd, J=5.1, 13.3 Hz, 1H), 3.62 (d, J=11.7 Hz, 1H), 3.69-3.80 (m, 5H), 3.99 (pst, J=6.5 Hz, 1H) ppm;
13C NMR (125 MHz, CD3OD) δ 21.3 (CH2), 51.4 (CH2), 52.9 (CH2), 65.5 (CH2), 71.2 (CH), 72.7 (CH), 73.2 (Cq), 76.8 (CH) ppm;
HRMS (ESI+) Calc. C8H16NO4 [M+H]+m/z=190.1074; Found m/z=190.1074.
General Protocol C—reducing alkylation of β-lactam C-5 leading to conidines of general formula (I′b)
A solution of β-lactam C-5 (1 equiv.) and of Vaska's catalyst (4% mol) in anhydrous dichloromethane under argon atmosphere is added to tetramethyldisiloxane (TMDS, 2 equiv.) at ambient temperature. The mixture is stirred for 45 minutes and then cooled to −78° C. Grignard reagent (2 equiv.) is added dropwise and then the mixture is stirred for 7-10 minutes at this same temperature before increasing to ambient temperature. It is next stirred for 21 hours. A saturated aqueous solution of NH4Cl is added and then the resulting mixture is diluted with CH2Cl2. The organic phase is separated and the aqueous phase is extracted twice with CH2Cl2. The organic phases are washed with brine, dried over MgSO4 and evaporated under reduced pressure. The conidines of formula (I′b) thus formed are purified by chromatography over silica gel before being reduced and debenzylated in accordance with the general protocol B above.
The compound of above formula (1Iz2) ((3S,4R,5R,6R,8R)-8-benzyl-6-(hydroxymethyl)-1-azabicyclo[4.2.0]octane-3,4,5-triol: 20.3 mg; 65% for 2 steps) was prepared in accordance with the general procedures C and B, from β-lactam C-5 (70.1 mg; 0.124 mmol) and 2M benzylmagnesium chloride in THF (R14=Bn; 36.3 μL; 0.248 mmol).
A translucent lacquer is obtained, with the following characteristics:
1H NMR (500 MHz, CD3OD) δ 7.39-7.20 (m, 5H), 4.45-4.34 (m, 1H), 3.95 (t, J=5.7 Hz, 1H), 3.80-3.74 (m, 2H), 3.70 (d, J=5.6 Hz, 1H), 3.56 (d, J=12.1 Hz, 1H), 3.21-3.12 (m, 2H), 3.00 (dd, J=13.6, 8.4 Hz, 1H), 2.94 (dd, J=13.1, 6.5 Hz, 1H), 2.60 (dd, J=11.9, 9.0 Hz, 1H), 2.32-2.20 (m, 1H) ppm;
13C NMR (125 MHz, CD3OD) δ 137.1 (ArCq), 130.1 (ArCH), 129.7 (ArCH), 128.0 (ArCH), 76.5 (CH), 71.5 (CH), 71.1 (CH), 65.7 (CH), 64.1 (CH2), 51.4 (CH2), 41.0 (CH2), 27.3 (CH2) ppm;
HRMS (ESI+) Calc. C15H22NO4 [M+H]+: m/z=280.15433; Found m/z=280.15437.
The compound of above formula (IIz3) ((3S,4R,5R,6R,8R)-6-(hydroxymethyl)-8-methyl-1-azabicyclo[4.2.0]octane-3,4,5-triol: 8.5 mg; 46% for 2 steps) was prepared in accordance with the general procedures C and B, from β-lactam C-5 (192.6 mg; 0.342 mmol) and 3M methylmagnesium chloride in THF (R14=Me; 228 μL; 0.683 mmol).
A yellowish solid is obtained, with the following characteristics:
[60 ]20D−22.9 (c 1.07, MeOH);
1H NMR (500 MHz, CD3OD) δ 3.99 (t, J=6.7 Hz, 1H), 3.85-3.75 (m, 1H), 3.75-3.69 (m, 1H), 3.67 (d, J=7.6 Hz, 1H), 3.54 (d, J=11.2 Hz, 1H), 3.47 (d, J=11.2 Hz, 1H), 3.10 (dd, J=13.9, 5.5 Hz, 1H), 2.71 (dd, J=13.8, 4.0 Hz, 1H), 2.58 (dd, J=11.5, 8.9 Hz, 1H), 1.64 (dd, J=10.9, 8.0 Hz, 1H), 1.21 (d, J=6.1 Hz, 3H) ppm;
13C NMR (100 MHz, CD3OD) δ 77.2 (CH), 74.7 (CH), 71.7 (CH), 68.3 (Cq), 67.4 (CH2), 58.4 (CH), 52.4 (CH2), 29.1 (CH2), 22.0 (CH3) ppm; HRMS (ESI+) Calc. C9H18NO4 [M+H]+: m/z=204.12303; Found m/z=204.12232.
The compound of above formula (IIz4) ((3S,4R,5R,6R,8S)-8-cyclopentyl-6-(hydroxymethyl)-1-azabicyclo[4.2.0]octane-3,4,5-triol: 14.6 mg; 62% for 2 steps) was prepared in accordance with the general procedures C and B, from β-lactam C-5 (70.0 mg; 0.124 mmol) and 2M cyclopentylmagnesium chloride in ether (R14=cPent; 36.4 μL; 0.248 mmol).
A yellowish solid is obtained, with the following characteristics:
1H NMR (500 MHz, CD3OD) δ 3.98 (t, J=7.0 Hz, 1H), 3.76-3.63 (m, 2H), 3.58-3.36 (m, 3H), 3.10 (dd, J=14.5, 5.0 Hz, 1H), 2.76 (dd, J=14.5, 4.0 Hz, 1H), 2.50 (t, J=11.8 Hz, 1H), 2.06-1.95 (m, 1H), 1.89-1.76 (m, 1H), 1.74-1.47 (m, 6H), 1.36-1.22 (m, 1H), 1.15-1.00 (m, 1H) ppm;
13C NMR (125 MHz, CD3OD) δ 77.7 (CH), 75.0 (CH), 71.9 (CH), 67.5 (CH2), 67.2 (CH), 66.3 (Cq), 54.2 (CH2), 48.9 (CH), 31.0 (CH2), 29.1 (CH2), 27.0 (CH2), 26.4 (CH2), 25.9 (CH2) ppm;
HRMS (ESI+) Calc. C13H24NO4 [M+H]+: m/z=258.16998; Found m/z=258.16987.
The compound of above formula (IIz5) ((3S,4R,5R,6R,8R)-6-(hydroxymethyl)-8-pentyl-1-azabicyclo[4.2.0]octane-3,4,5-triol: 19.1 mg; 45% for 2 steps) was prepared in accordance with the general procedures C and B, from β-lactam C-5 (70.0 mg; 0.124 mmol) and 2M pentylmagnesium chloride in THF (R14=n-Pent; 33.6 μL; 0.248 mmol).
A yellow solid is obtained, with the following characteristics:
[α]20D−31.1 (c 0.46, MeOH);
1H NMR (500 MHz, CD3OD) δ 3.99 (t, J=7.0 Hz, 1H), 3.74-3.64 (m, 2H), 3.64-3.55 (m, 1H), 3.51 (d, J=11.1 Hz, 1H), 3.46 (d, J=11.1 Hz, 1H), 3.09 (dd, J=13.7, 5.4 Hz, 1H), 2.70 (dd, J=13.7, 4.0 Hz, 1H), 2.52 (dd, J=11.4, 8.9 Hz, 1H), 1.68-1.53 (m, 2H), 1.51-1.39 (m, 1H), 1.39-1.21 (m, 6H), 0.90 (t, J=6.7 Hz, 3H) ppm;
13C NMR (100 MHz, CD3OD) δ 77.5 (CH), 75.2 (CH), 71.9 (CH), 67.8 (CH2), 66.4 (Cq), 62.4 (CH), 53.8 (CH2), 38.8 (CH2), 33.0 (CH2), 27.9 (CH2), 26.5 (CH2), 23.7 (CH2), 14.3 (CH3) ppm;
HRMS (ESI+) Calc. C13H26NO4 [M+H]+: m/z=260.18563; Found m/z=260.18561.
The synthesis method used for obtaining the compounds complying with the general formula (I′c) below falls under the general synthesis scheme 7 shown on
The compound of formula (IIk) as above ((3S,4R,5R,6R,7S)-6,7-bis(hydroxymethyl)-1-azabicyclo[4.1.0]heptane-3,4,5-triol) is obtained in the following manner.
A mixture of nitrone (III) (P=Bn; 193 mg, 0.360 mmol) and of alkyne Vc (R8=NBnTs; 308 mg, 1.079 mmol) in solution in CH2Cl2 (0.4 mL) was stirred at ambient temperature for 3 days, and then the reaction mixture was concentrated under reduced pressure. The residue (raw cycloadduct B-7c) was dissolved in ethanol (0.1M), transferred into a sealed tube, and heated at 110° C. (IR probe) under microwave irradiation for 15 minutes. After concentration under reduced pressure, the residue was purified by chromatography to give acylaziridine H-7c (P=Bn, R8=NBnTs; 238 mg, 80% on 2 steps). A fraction of this compound (58 mg, 0.070 mmol) was dissolved in THF (1 mL), and then LiAlH4 (6 mg, 0.157 mmol) was added at 0° C. The mixture was stirred for 1.5 hours at ambient temperature. After adding water (0.1 mL) and a 10% aqueous solution of NaOH (0.1 mL), the reaction mixture was stirred at ambient temperature for 2 hours and then filtered over Celite®. The filtrate, concentrated under reduced pressure, was purified by chromatography to isolate the aziridine-alcohol J-7c (P=Bn, R16=H; 34.2 mg, 86%). A solution of this aziridine-alcohol J-7c (28 mg, 0.067 mmol) in THF (2 mL) was added to a solution of lithium (54 mg, 7.78 mmol) in liquid ammonia (10 mL) at −78° C. The mixture was stirred for 45 minutes at -78° C., and then Milli-Q water (0.5 mL) and MeOH (2 mL) were added. After returning the solution to ambient temperature, the solvents were evaporated under reduced pressure. The residue was dissolved in Milli-Q water and neutralized with Amberlite® IR-120 (22.8 g) previously treated with 1M HCl (15 mL). The resin was introduced into a column, washed with water, and then the compound (IIk) was eluted with a 1M solution of aqueous NH4OH. The solution obtained after evaporation of the solvents under reduced pressure was purified by chromatography to give the pure iminosugar (IIk) (R16=H; 7 mg, 60%).
A white solid is obtained, with the following characteristics:
[α]20D=+2.90 (c 0.34, CH3OH);
IR v 3409, 3010, 2926, 1084, 1032 cm−1;
1H NMR (500 MHz, CD3OD) δ 4.16 (d, J=7.9 Hz, 1H), 3.79 (d, J=11.6 Hz, 1H), 3.60-3.50 (m, 3H), 3.47-3.40 (m, 1H), 3.33-3.25 (m, 2H), 2.55 (dd, J=10.6, 12.4 Hz, 1H), 2.19 (dd, J=6.8, 5.3 Hz, 1H) ppm;
13C NMR (125 MHz, CD3OD) 576.0 (CH), 73.2 (CH), 70.6 (CH), 63.2 (CH2), 62.0 (CH2), 57.0 (CH2), 51.9 (Cq), 51.6 (CH) ppm;
HRMS (ESI+) Calc. C8H16NO5 [M+H]+ m/z=206.1028; Found m/z=206.1028.
The compound of above formula (IIj) ((3S,4R,5R,6R,7S)-7-((S)-1,2-dihydroxyethyl)-6-(hydroxymethyl)-1-azabicyclo[4.1.0]heptane-3,4,5-triol) is obtained as follows.
Acylaziridine H-7d (P=Bn, R8=CH2OAc; 33 mg, 0.052 mmol), obtained as described in the publication by Tangara et al., 2017, Org. Lett. 19: 4842-4845, was dissolved in THF (1 mL), and then LiAlH4 (38 mg, 0.165 mmol) was added at 0° C. and the mixture was stirred for 1 hour at ambient temperature. After adding water (0.1 mL) and a 10% aqueous solution of NaOH (0.1 mL), the reaction mixture was stirred at ambient temperature for 2 hours and then filtered over Celite®. The filtrate, concentrated under reduced pressure, was purified by chromatography to isolate a translucent oil J-7d (R16=CH2OH; 26 mg, 83%). A solution of this aziridine-alcohol J-7d (40 mg, 0.067 mmol) in THF (2 mL) was added to a solution of lithium (25 mg, 3.571 mmol) in liquid ammonia (10 mL) at −78° C. The mixture was stirred for 45 minutes at −78° C. and then Milli-Q water (0.5 mL) and MeOH (2 mL) were added. After returning the solution to ambient temperature, these solvents were evaporated under reduced pressure. The residue was dissolved in Milli-Q water and neutralized with Amberlite® IR-120 (8.5 g) previously treated with 1M HCl (5 mL). The resin was introduced into a column, washed with water, and then the compound (IIj) was eluted with a 1M solution of aqueous NH4OH. The solid obtained after evaporation of the solvents under reduced pressure was purified by chromatography to give the pure iminosugar (IIj) (R16=CH2OH; 10.1 mg, 64%).
A white solid is obtained, with the following characteristics:
[α]20D=+5.90 (c 0.34, CH3OH);
IR: v 3249, 2939, 2885, 1748, 1656, 1404, 1031, 995 cm−1;
1H NMR (500 MHz, CD3OD) δ 4.16 (d, J=8.0 Hz, 1H), 3.91 (d, J=11.6 Hz, 1H), 3.71 (dd, J=3.5, 11.4 Hz, 1H), 3.61 (dd, J=5.8, 11.4 Hz, 1H), 3.54-3.48 (m, 1H), 3.48-3.41 (m, 1H), 3.39 (d, J=11.6 Hz, 1H), 3.30-3.25 (m, 2H), 2.52 (dd, J=10.2, 12.2 Hz, 1H), 2.10 (d, J=8.7 Hz, 1H) ppm;
13C NMR (125 MHz, CD3OD) δ 75.9 (CH), 73.1 (CH), 72.4 (CH), 70.7 (CH), 66.1 (CH2), 63.8 (CH2), 56.5 (CH2), 52.0 (Cq), 51.2 (CH) ppm;
HRMS (ESI+) Calc. C9H18NO6 [M+H]+m/z=236.1129; Found m/z=236.1124.
The compound of above formula (IIm) (3S,4R,5R,6R,7S)-7-((S)-hydroxy(phenyl)methyl)-6-(hydroxymethyl)-1-azabicyclo[4.1.0]heptane-3,4,5-triol is obtained as follows.
The cycloadducts B-7e (P=Ac, R8=Ph; 15.2 mg, 0.030 mmol, 4:1 mixture of diastereoisomers), obtained as described in the publication by Tangara et al., 2018, New J. Chem. 42: 16735-16743, were dissolved in dichloroethane (0.1M solution) in a sealed tube. The solution was heated at 110° C. (IR probe) under irradiation by microwave for 45 minutes (30+15). After evaporation of the solvent under reduced pressure, purification of the residue by chromatography made it possible to isolate the acylaziridines H-7e (P=Ac, R8=Ph; 12 mg, 79%) in the form of a 9:1 mixture of two diastereoisomers. These acylaziridines (30 mg, 0.067 mmol) were dissolved in THF (1.2 mL), the solution was cooled to 0° C. and then LiAlH4 (5.6 mg, 0.147 mmol) was added. The reaction mixture was stirred at ambient temperature for 1.5 hours, and then water (0.1 mL) and a 10% aqueous solution of NaOH (0.1 mL) were added thereto. After stirring at ambient temperature for 1.5 hours, this mixture was filtered over Celite®, the filtrate being concentrated under reduced pressure, and the residue was purified by chromatography to provide the aziridinyl iminosugar (IIm) (R16=Ph; 15 mg, 82%) in the form of a single diastereoisomer.
A colorless oil is obtained, with the following characteristics:
[α]20D−4.1 (c 1.58, CH3OH);
IR v 3296, 2931, 1557, 1409, 1053, 697 cm−1;
1H NMR (500 MHz, CD3OD) δ 7.49-7.44 (m, 2H), 7.40-7.35 (m, 2H), 7.31-7.25 (m, 1H), 4.29 (d, J=8.7 Hz, 1H), 4.18 (d, J=7.9 Hz, 1H), 4.01 (d, J=11.6 Hz, 1H), 3.56 (d, J=11.6 Hz, 1H), 3.48-3.37 (m, 2H), 3.26 (dd, J=9.1, 8.0 Hz, 1H), 2.26-2.18 (m, 2H) ppm;
13C NMR (125 MHz, CD3OD) δ 144.6 (ArCq), 129.5 (ArCH), 128.6 (ArCH), 127.1 (ArCH), 75.8 (CH), 73.8 (CH), 73.1 (CH), 70.7 (CH), 63.6 (CH2), 56.4 (CH2), 56.0 (CH), 52.8 (Cq)
ppm;
HRMS (ESI+) Calc. C14H20NO5 [M+H]+: m/z=282.1341; Found m/z=282.1346.
The compound of above formula (IIn) (3S,4R,5R,6R,7S)-7-((R)-cyclohexyl(hydroxy)methyl)-6-(hydroxymethyl)-1-azabicyclo[4.1.0]heptane-3,4,5-triol is obtained as follows.
Acylaziridine H-7f (P=Bn, R8=c-Hex; 77 mg, 0.119 mmol), obtained as described in the publication by Tangara et al., 2017, Org. Lett. 19: 4842-4845, was dissolved in ethanol (1.4 mL), cooled to 0° C., and then NaBH4 (13 mg, 0.342 mmol) was added. The mixture was stirred at the same temperature for 3.3 hours, and then water, a 10% aqueous solution of NaOH and CH2Cl2 were added. The aqueous phase was extracted (3 times) by CH2Cl2, the organic phases were collected, dried on MgSO4 and concentrated under reduced pressure. The residue thus obtained was filtered on silica to isolate the aziridine-alcohol J-7f (R16=c-Hex; 72 mg, 94%) in the form of an oil. A solution of this aziridine-alcohol J-7f (42 mg, 0.064 mmol) in THF (4 mL) was added to a solution of lithium (12 mg, 2.0 mmol) in liquid ammonia (10 mL) at −78° C. The reaction mixture was stirred for 45 minutes at −78° C., and then Milli-Q water (0.5 mL) and MeOH (2 mL) were added. After returning the solution to ambient temperature, the solvents were evaporated under reduced pressure. The residue was dissolved in Milli-Q water and neutralized with Amberlite® IR-120 (3.5 g) previously treated with 1M HCl (2 mL). The resin was introduced into a column, washed with water, and then the compound (IIn) was eluted with a 1M solution of aqueous NH4OH. The solid obtained after evaporation of the solvents under reduced pressure was purified by chromatography to give the pure iminosugar (IIn) (R16=c-Hex; 13 mg, 72%).
A white solid is obtained, with the following characteristics:
[α]20D+2.8 (c 0.80, CH3OH);
IR v 3335, 2916, 2846, 1451, 1087, 1054, 1008, 660 cm−1;
1H NMR (500 MHz, CD3OD) δ 4.17 (d, J=7.8 Hz, 1H), 3.88 (d, J=11.7 Hz, 1H), 3.50-3.44 (m, 2H), 3.42 (d, J=11.7 Hz, 1H), 3.33-3.29 (m, 1H), 2.95 (dd, J=8.6, 6.6 Hz, 1H), 2.51 (dd, J=11.8, 9.0 Hz, 1H), 2.08 (d, J=8.7 Hz, 1H), 1.99-1.66 (m, 5H), 1.53-1.45 (m, 1H), 1.36-1.03 (m, 5H) ppm;
13C NMR(125 MHz, CD3OD) δ 76.3 (CH), 76.0 (CH), 73.3 (CH), 70.8 (CH), 64.1 (CH2), 56.0 (CH2), 52.9 (CH), 51.1 (Cq), 45.3 (CH), 30.1 (CH2), 29.7 (CH2), 27.7 (CH2), 27.4 (CH2), 27.2 (CH2) ppm;
HRMS (ESI+) Calc. C14H26NO5 [M+H]+: m/z=288.1811; Found m/z=288.1810.
A synthesis method used for obtaining a compound complying with the general formula (I′e) below falls under the general synthesis scheme 8 shown on
The compound of above formula (IIw1) ((2R,3R,4R,5S)-2-(hydroxymethyl)-2-(1H-1,2,3-triazol-4yl)piperidine-3,4,5-triol is prepared in the following manner. A solution of hydroxylamine of formula A-1a (240.0 mg; 0.377 mmol) and of zinc powder (247.0 mg; 3.77 mmol) in a 4:1 mixture EtOH/AcOH (5 mL) was stirred at 65° C. under ultrasound until the reaction was complete. The reaction mixture is filtered over Celite and then evaporated under reduced pressure. The raw product is redissolved in CH2Cl2 and then treated with aqueous 1M NaOH. The aqueous phase was extracted (three times) with CH2Cl2. The organic phases were washed with brine, dried over MgSO4 and then evaporated under reduced pressure. Purification of the residue obtained by chromatography provided the corresponding piperidine D-8 (178.0 mg; 76%). A mixture of this piperidine D-8 (176.0 mg; 0.284 mmol), of benzyl chloroformiate (121 μL; 0.852 mmol) and of K2CO3 (157.0 mg; 1.14 mmol) in anhydrous THF (1.7 mL) was stirred at ambient temperature until the reaction was complete. Methanol (2 mL) was added and then the reaction mixture was stirred at ambient temperature until the reaction was complete. Purification of the residue obtained by chromatography provided the compound of formula K-8 (164.0 mg; 78% over 2 steps). Copper iodide (3.5 mg; 0.018 mmol) and DIPEA (108 μL; 0.618 mmol) were added to a solution of this same compound K-8 (46.8 mg; 0.062 mmol) and of benzyl azide (247 μL; 0.124 mmol) in DMF (1.5 mL) under inert atmosphere. The reaction mixture was stirred at ambient temperature until the reaction was complete. The raw product was diluted in ethyl acetate and washed several times with brine. The organic phase was dried over MgSO4 and evaporated at reduced pressure. Purification of the residue obtained by chromatography provided the compound of formula L-8 (38.2 mg; 76%). This compound (32.1 mg; 0.039 mmol) was debenzylated in accordance with the general procedure B to give piperidine (IIw1) (11.0 mg; 100%).
A colorless lacquer is obtained, with the following characteristics:
[α]20D−17.2 (c 1.09, CH3OH);
1H NMR (500 MHz, CD3OD) δ 7.90 (s, 1H), 3.80 (d, J=10.9 Hz, 1H), 3.69 (d, J=9.7 Hz, 1H), 3.59 (d, J=11.1 Hz, 1H), 3.52-3.45 (m, 1H), 3.27 (t, J=9.3 Hz, 1H), 2.99 (dd, J=12.6, 5.5 Hz, 1H), 2.54 (dd, J=12.3, 11.2 Hz, 1H) ppm;
13C NMR (125 MHz, CD3OD) δ 144.5 (Cq), 131.0 (CH), 76.9 (CH), 74.5 (CH), 73.0 (CH), 68.1 (CH2), 62.5 (Cq), 47.2 (CH2) ppm;
HRMS (ESI+) Calc. C8H15N4O4 [M+H]+: m/z=231.10878; Found m/z=231.10861.
The following various biological activity tests are implemented on the compounds (IIa) to (IIn) described above.
The inhibiting activity on recombinant human acid α-glucosidase (rhGAA) of the compounds is implemented using the Fluopol-ABPP method (Fluorescence Polarization Activity Based Protein Profiling) described in the publication of Lahav et al., 2017, J. Am. Chem. Soc. 139: 14192-14197. This technique, based on the competition between an inhibitor and a fluorescent probe capable of binding covalently to the active site of an enzyme, makes it possible to measure the affinity of this inhibitor for the active site of the rhGAA enzyme used for these experiments by the laboratory of Professors Herman S. Overkleeft and Johanes M. F. G. Aerts, Leiden Institute of Chemistry, Leiden University (NL), is the enzyme marketed under the name Myozyme®. The median inhibiting concentrations (IC50) are determined in the Mcllvaine buffer (citrate-phosphate) 150 mM at pH 5.0, in the presence of 0.1% bovine gamma-globulin (p/v) and 0.5 mg/mL of Chaps detergent (Sigma) in 96-well plates (Griener). The rhGAA enzyme (10 μg/mL) is pre-incubated with solutions of inhibitor (containing 2.5% DMSO that was used to prepare the mother solutions of the compounds) at various concentrations [I] in the buffer, for 45 minutes at 37° C. The tetraaminomethylrhodamine (TAMRA) fluorescent probe in solution (25 nM) in the buffer is next added to the mixture. After 4 hours, the samples are irradiated with a polarized light (λ=530 nm) and the fluorescence emitted (λ=580 nm) is measured by means of an Infinite® M1000Pro (Tecan) spectrofluorimeter. For each concentration of inhibitor, the percentage of inhibition of the enzyme is determined by the formula:
%Inhibition=[Fmeasured−Fcontrol1)/Fcontro2)×100
where Fmeasured corresponds to the fluorescence measured in the presence of the iminosugars; Fcontrol1 corresponds to the fluorescence measured in the presence of a powerful inhibitor of human acid α-glucosidase acid serving as a positive control (100% inhibition), CF 022 ((1S,2R,3S,4R,5R,6R)-2,3,4-trihydroxy-5-(hydroxymethyl)-7-(8-azidooctyl)-7-aza-bicyclo[4.1.0]heptane), and Fcontrol2 represents the fluorescence of the probe measured in the absence of the inhibitor (0% inhibition).
The IC50 values are calculated by a non-linear regression of the % inhibition as a function of the concentration [I] by means of the GraphPad Prism 6.0 software. The results are a mean of three identical experiments (triplicates).
In a complementary way, the activity of inhibition of recombinant human acid α-glucosidase (rhGAA) of the compounds was evaluated by the team of Professor Marco Moracci, Department of Biology, University of Naples Federico II (IT), using the method described in the publication of Porto et al., 2012, Molecular Therapy 20: 2201-2211. The rhGAA enzyme sold under the name Myozyme® used comes from residues of perfusions of the recombinant enzyme used for treating, by enzyme therapy, the patients with Pompe disease in the Department of Translational Medical Sciences, University of Naples Federico II (IT). The compounds are solubilized at various concentrations in a 100 mM sodium acetate buffer, pH 4.0, as well as the 4-nitrophenyl-α-
The stabilization of rhGAA in the presence of the compounds is determined in accordance with the method described in the publication of Niesen et al., 2007, Nat. Protoc. 2: 2212-2221. The rhGAA enzyme (2.5 μg, 0.1 mg/mL) is incubated in the absence and in the presence of each compound (at a concentration [I]=100 μM), of SYPRO® orange dye (Life Technologies), and of sodium phosphate buffer (25 mM) and of NaCI (150 mM), pH 7.4 or of sodium acetate buffer (25 mmol) and of NaCl (150 mM), pH 4.0. The thermal stability of the enzyme in these various conditions is evaluated by DSF (differential scanning fluorimetry) by varying the temperature by 1° C./min over an interval of 25 to 95° C., and measuring the fluorescence of the SYPRO® orange dye every minute by means of a Real-Time Cycler spectrofluorimeter (Biorad). The relative fluorescence is determined by comparing each fluorescence value measured with that of the maximum fluorescence value of the SYPRO® orange dye for each scan. The results are a mean of three identical experiments (triplicates).
The selectivity of the compounds was evaluated in the laboratory of Professors Herman S. Overkleeft and Johanes M. F. G. Aerts, Leiden Institute of Chemistry, Leiden University (NL). The enzymes used for determining the selectivity of inhibition of the compounds with respect to various human enzymes are the α-glucosidase II of the endoplasmic reticulum (GANAB), recombinant human lysosomal β-glucocerebrosidase (GBA1), human non-lysosomal β-glucosylceramidase (GBA2) and β-glucosylceramide synthase (GCS). The inhibiting activity of the compounds on these various enzymes is determined as described in the publication by Artola et al., 2017, ACS Cent. Sci. 3: 784-793. The enzyme GBA1 sold under the name Cerezyme® and the enzyme rhGAA sold under the name Myozyme® are used. The human GANAB enzyme used for this study is that of fibroblasts of patients suffering from Pompe disease diagnosed as lacking active GAA and voluntary donors. These fibroblasts were cultivated on an HAMF12-DMEM medium (Sigma) supplemented by 10% (v/v) FCS (fetal calf serum). The GBA2 enzyme was over-expressed in HEK298T cells cultivated on a DMEM medium enriched with glucose (Gibco) supplemented by 10% NBS (newborn bovine serum) and 100 units/mL of penicillin/streptomycin (Gibco) and 5% CO2 at 37° C. The activity of the compounds on human β-glucosylceramide synthase (GCS) is evaluated in situ on cells of the RAW 264.7 type cultivated on an RPMI medium (Gibco) supplemented by 10% FCS, 1 mM of glutamax, 100 units/mL of penicillin/streptomycin (Gibco) and 5% CO2 at 37° C.
The IC50 values for the GANAB, GBA2 and GCS enzymes are determined from cell lysates prepared in a buffer (20 mM hepes, 2 mM DTT, 0.25 M sucrose, 1 mM MgCl2, 2.5 U/mL benzonase) at pH 7.0, and placed on ice for 30 minutes. These cell lysates are homogenized using a SilentCrusher grinder (Heidolph®), and then subjected to ultracentrifugation at 32,000 rpm for 30 minutes at 4° C. The total protein concentration is determined in accordance with the Bradford method (Bradford, 1976, Anal. Biochem. 72: 248-254), using a Bradford BioRad Quick Start® kit (Pierce) and BSA (Sigma). The lysates are next aliquoted and stored at −80° C. before use.
The IC50 values for the GANAB enzyme are determined on cell lysates of fibroblasts of patients suffering from Pompe disease, using as a buffer the Mcllvaine 150 mM, pH 7.0, 0.1% bovine serum albumin (BSA) (p/v), a substrate (4-methyl-umbelliferone-α-
The IC50 values for the GBA1 enzyme are determined using as buffer the Mcllvaine 150 mM, pH 5.2, 0.2% taurocholate (p/v), 0.1% Triton X-100 (v/v), 0.1% bovine serum albumin (BSA) (p/v), an enzyme concentration of 0.7 nM, a substrate (4-methyl-umbelliferone-β-
The residual activity of GBA2 in the presence of the compounds is determined after pre-incubation for 30 minutes of the homogenates of HEK298T cells over-expressing GBA2 with an inhibitor of GBA1, conduritol β-epoxide (Sigma), at a concentration of 1 mM. The IC50 values are determined on cell lysates, using as a buffer the Mcllvaine 150 mM, pH 5.8, 0.1% bovine serum albumin (BSA) (p/v), a substrate (4-methyl-umbelliferone-β-
The residual activity of GCS in the presence of the compounds is determined after pre-incubation for 1 hour of the homogenates of RAW 264.7 cells with an inhibitor of GBA1, conduritol β-epoxide (Sigma), at a concentration of 300 μM.
The I50 values for the GCS enzyme are determined in situ in the cell culture medium at pH 7.0 using 1 μM of NBD-ceramide (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-D-erythro-sphingosine) as substrate, as described in the aforementioned publication of Lahav et al.
The results obtained for all these tests are set out in tables 1 and 2 below. In these tables, by way of comparison, the values obtained for the compounds of the prior art DNJ and NB-DNJ, as described in the literature, are also indicated. In these tables: a indicates Porto et al., 2012, Mol. Ther. 20: 2201-2211; b indicates Bruckmann et al., 2012, ChemMedChem 7: 1943-1953; c indicates Flanagan et al., 2009, Human Mut. 30: 1683-1692; d indicates D'Alonzo et al., 2017, J. Med. Chem. 60: 9462-9469; * indicates ΔTm measured at [compound]=10 times the value of Ki; e indicates Asano et al., 1995, J. Med. Chem. 38: 2349-56; f indicates Wennekes et al., 2010, J. Med. Chem. 53: 689-698.
All these results demonstrate a selectivity of the compounds used in accordance with the invention for human acid α-glucosidase, compared with the other human enzymes tested. This selectivity is much greater than that of the molecules proposed by the prior art DNJ and NB-DNJ. The compounds used in accordance with the invention furthermore have an important effect of stabilization of rhGAA, comparable to that of DNJ and NB-DNJ.
Furthermore, the compounds (IIb7), (IIb3), (IIc1), (IIr) and (Ic), complying with the general formula (I″a), as well as the compound (IIx1), have performances superior to those of the other compounds according to the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR2004065 | Apr 2020 | FR | national |
This application is the U.S. national phase of International Application No. PCT/EP2021/060572 filed Apr. 22, 2021, which designated the U.S. and claims priority to FR Patent Application No. 2004065 filed Apr. 23, 2020, the entire contents of each of which are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/060572 | 4/22/2021 | WO |
