WO2017137895A1 - Nouvelles pyrrolidines dendrimères, leur synthèse et utilisation médicale - Google Patents

Nouvelles pyrrolidines dendrimères, leur synthèse et utilisation médicale Download PDF

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WO2017137895A1
WO2017137895A1 PCT/IB2017/050670 IB2017050670W WO2017137895A1 WO 2017137895 A1 WO2017137895 A1 WO 2017137895A1 IB 2017050670 W IB2017050670 W IB 2017050670W WO 2017137895 A1 WO2017137895 A1 WO 2017137895A1
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mmol
valency
triazole
formula
scaffold
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PCT/IB2017/050670
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English (en)
Inventor
Francesca CARDONA
Camilla PARMEGGIANI
Camilla MATASSINI
Andrea GOTI
Amelia MORRONE
Serena CATARZI
Giampiero D'ADAMIO
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Universita' Degli Studi Di Firenze
Azienda Ospedaliero Universitaria Meyer
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Publication of WO2017137895A1 publication Critical patent/WO2017137895A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to the field of organic compounds having pharmacological activity, in particular it relates to dendrimeric pyrrolidines, their synthesis (and related synthetic intermediates) and their medical application as inhibitors/chaperones of lysosomal enzymes responsible for lysosomal storage diseases (LSDs).
  • LSDs lysosomal storage diseases
  • Lysosomal storage diseases are a group of about 50 rare metabolic diseases caused by complete or partial deficiency of lysosomal enzymes, resulting in accumulation of natural substrates within the lysosomes and in the onset of clinical and heterogeneous multisystemic manifestations, including neurological symptoms and scarce patients' life expectation.
  • the Enzyme Replacement Therapy (ERT) only available for 7 LSDs, requires "wild-type” recombinant enzyme periodic infusions but, besides being extremely expensive, is not resolutive especially for those LSDs with involvement of the central nervous system (CNS), because the recombinant enzyme does not cross the blood-brain barrier, and, moreover, patients may develop an antibody response to the enzyme with serious health risks, and discontinuation of the treatment.
  • the bone marrow transplantation which is effective only if made within the first 6 months of life of patients, is often not applicable because many of these diseases manifest their effects later in life.
  • the substrate reduction therapy (SRT), which is available for Gaucher disease and Niemann-Pick disease type C, exploits the administration of an inhibitor of the synthesis of the substrate which tends to accumulate, thus leading to only slowing the disease course.
  • PCs pharmacological chaperones
  • ASSCs active-site specific chaperones
  • the PCs-ASSCs are molecules acting as competitive inhibitors of the deficient enzyme, but if used in sub-inhibitory amounts (with minimization of side effects) can correct the folding and/or stabilize the catalytic activity and half-life of the enzymes compromised by conformational changes.
  • the therapy based on PCs has considerable advantages compared to the above mentioned ones, such as oral administration, restoration of the endogenous enzyme activity and low production costs.
  • PCs can also be used in a combined ERT/PC therapy, thus stabilizing the exogenous enzyme administered with the ERT and increasing its half-life.
  • the synergy between ERT and PC today represents a new therapeutic frontier, aimed to improve the pharmacological properties and exogenous enzyme stability by reducing the number of infusions with significant improvement in patients' life quality.
  • dendrimers bearing 3 or 4 deoxynojirimycin (DNJ) units have shown chaperonic properties towards the ⁇ -glucosidase enzyme deficient in Gaucher disease.
  • DNJ deoxynojirimycin
  • the present invention solves the above mentioned problems by means of a dendrimeric iminosugar of formula (I)
  • p is an integer number comprised between 2 and 12;
  • havin valency a 9 and where Y is selected from the group consisting of
  • the scaffold Q is a second-generation scaffold derived from any of the previous first generation scaffolds by elongation of each value (a') with a valency multiplier, having valency k, chosen between
  • the molecules object of the present invention inhibit the enzymes N- acetylgalactosamine-6-sulfatase (GALNS), iduronate-2-sulfatase (IDS), a- mannosidase and ⁇ -glucosidase all deficient in lysosomal storage diseases, more precisely, in the deficit syndrome Morquio A, in the Hunter syndrome, the a- mannosidosis and in Gaucher disease, respectively.
  • GALNS N- acetylgalactosamine-6-sulfatase
  • IDS iduronate-2-sulfatase
  • a- mannosidase and ⁇ -glucosidase all deficient in lysosomal storage diseases, more precisely, in the deficit syndrome Morquio A, in the Hunter syndrome, the a- mannosidosis and in Gaucher disease, respectively.
  • the presentation of multivalent iminosugars on one dendrimeric scaffold is a prerequisite for the inhibitory activity
  • the inhibitory activity reported is at the basis of the use of the molecules of the invention also as active site-specific pharmacological chaperones (ASSPCs) in the treatment of the above mentioned pathologies.
  • ASSPCs active site-specific pharmacological chaperones
  • the inhibitory activity is reversible; by lowering the dose to sub-inhibitory amounts, it is potentially possible that the molecules of the invention will work as ASSPCs or as stabilizers of recombinant enzymes.
  • the subject-matter of the present invention is thus also a dendrimeric iminosugar of formula (I) as described above for use as a medicament, in particular as pharmacological active-site specific chaperone (ASSC) or as recombinant enzymes stabilizer.
  • ASSC pharmacological active-site specific chaperone
  • the activity as an inhibitor of lysosomal enzymes makes the molecules of the invention useful in mechanicistic studies of LSDs.
  • the present invention relates to a dendrimeric iminosugar of formula (I) as described above for use in the treatment of lysosomal storage diseases, in particular for the treatment of Morquio A syndrome, Hunter syndrome, the - mannosidosis and Gaucher disease.
  • a further subject-matter of the present invention is also a pharmaceutical composition comprising a compound as described above and at least another pharmaceutically acceptable ingredient.
  • the present invention also relates to a synthetic strategy for the preparation of a compound according to the invention, based on the click chemistry reaction between an iminosugar of formula (II)
  • the present invention also relates to new alkynes of formula (III) in which Q and a are selected from the roup consisting of
  • the molecules of the invention have the pyrrolidine portion of the compounds having the following stereochemical configuration: arabinose configuration or ribose configuration.
  • All multivalent molecules can be obtained through a synthetic strategy based on the Cu-catalyzed click-chemistry cycloaddition reaction of an alkyne (III) with an azide (II).
  • the biomolecules in use are iminosugars with different configurations and are properly modified to allow scaffold functionalization.
  • the scaffolds are molecules that allow the covalently insertion of bioactive compounds in different number. Examples of alkynes scaffolds of the first generation of formula (III) are hereinafter reported indicating for each of them the CAS Number if commercially available, the reference (if already known), or by suggesting the respective synthetic preparation (for new ones):
  • the aforementioned new hexavalent scaffold can be prepared according to the following proposed summary:
  • alkyne-scaffolds defined as first generation scaffolds
  • second generation alkyne-scaffolds as shown in the diagram below thus obtaining alkyne-scaffolds of higher valency:
  • FIG. 1 - a Reversibility test of compound 114 towards GALNS and corresponding IC50, in which the inhibitor concentration corresponding to subsequent dilutions were reported
  • b Reversibility test of compound 115 towards GALNS and corresponding IC50, in which the inhibitor concentrations corresponding to subsequent dilutions were reported.
  • FIG: 2 Increased activity of Vimizim®,once coincubated with 114 (10 nM) and of Elaprase®, once coincubated with 115 (10 nM).
  • FIG. 3 Evaluation of the thermal denaturation of Vimizim®, coincubated without (C) or with different concentrations of the compound 114.
  • Example 3 Azido derivative of pyrrolidine with D-ribose configuration, synthesis and characterization (106, (2/?,3/?,4S)-1-(6-azidohexyl)-2- (hydroxymethyl) pyrrolidine-3,4-diol)
  • GALNS N-acetyl galactosamine 6-sulfatase
  • IDS iduronate 2-sulfate sulfatase
  • ⁇ -glucosidase enzymes in leukocytes homogenates isolated from healthy blood donors.
  • the leukocyte pellet isolated from whole blood of healthy donors, was lysed by sonication in water, and the protein content was quantified with the BCA method. Enzyme inhibition test on IDS
  • the enzymatic activity was evaluated by setting up the test in triplicate in 0.2 ml tubes as follows:
  • the eppendorf tubes are moved into ice and the content of each tube is transferred in a well of a flat-bottomed 96 well plate at 0 °C and the reaction is stopped by the addition of 200 ⁇ _ of NaHCO3-Na2CO3buffer (0.5 M, 0.0025% triton X100, pH 10.7).
  • the fluorescence is measured in SpectraMax M2 microplate reader (Molecular-Devices) using an excitation wavelength of 365 nm and an emission wavelength of 435 nm.
  • inhibitor solution 3 ⁇ of inhibitor solution, 7 ⁇ of leukocytes homogenate (0,5 mg/ml) and 20 ⁇ of substrate solution (methylumbelliferyl-p-d-galactoside-6-sulphate Et3N) for 17 h at 37 °C.
  • the eppendorf tubes were placed at 0 °C and the reaction is stopped by addition of 5 ⁇ of stop buffer (Na- phosphate 0.9M, pH 4.3) stirring with vortex.
  • stop buffer Na- phosphate 0.9M, pH 4.3
  • the Eppendorf tubes are placed in ice and the content of each tube is transferred in a well of a flat-bottomed 96 well plate at 0 °C.
  • the fluorescence is measured in SpectraMax M2 microplate reader (Molecular-Devices) using an excitation wavelength of 365 nm and an emission wavelength of 435 nm.
  • the enzymatic activity was evaluated by setting up the test in triplicate in a flat— bottomed 96 well plate.
  • SpectraMax M2 microplate fluorescence is measured in SpectraMax M2 microplate reader (Molecular-Devices) using an excitation wavelength of 365 nm and an emission wavelength of 435 nm. The percentage of a-mannosidase inhibition is given with respect to the control without inhibitors.
  • the enzymatic activity was evaluated by setting up the test in triplicate in a flat— bottomed 96 well plate,
  • Table 1 Inhibition values of compounds towards human enzymes of interest.
  • An alternative application for the compounds of the invention is their use as stabilizers for the recombinant enzymes currently employed in the ERT, respectively Vimizim ® for Morquio A syndrome and I'Elaprase ® for 'Hunter disease.
  • compounds 114 and 115 were tested as inhibitors of both Vimizim ® and Elaprase ® enzymes.
  • Vimizim ® remaining in injection vials after administration to patients were recovered, pooled, and used without purification.
  • the same protocol was applied also for Elaprase ® .
  • the inhibitory activity was measured following the protocol described above for the inhibition of GALNS and IDS, simply replacing the 7 ⁇ of leukocytes homogenate with 7 ⁇ of the recombinant enzyme at the proper concentration. The percentage of inhibition and IC50 values are reported in Table 2.
  • Vimizim ® was incubated with different concentrations of compounds 114 and 115 and we found that compound 114 is able to enhance the Vimizim ® activity of 1 .15 fold (14%) at 10nM.
  • the same experiment carried out with Elaprase ® showed that compound 115 is able to enhance the Elaprase ® activity of 1 .16 fold (1 1 %) at l OnM ( Figure 2).
  • Vimizim ® aliquots were incubated with different concentrations of compound 114 (ranging from 10 ⁇ to 500 ⁇ ) at 48 °C and then the substrate (methylumbelliferyl-p-D-galactoside-6-solfato Et3N) was added after 20, 40 and 60 min.
  • Vimizim ® The activity of the Vimizim ® was then determined using the fluorimetric assay above described. The enzymatic activity, indicated relatively to the non-heated enzyme (37 °C), showed that Vimizim ® incubated for 40 min (grey bars) and for 60 min (light grey bars) at 48 °C with compound 114 (500 ⁇ ) is doubly active than alone (Figure 3).

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

L'objet de la présente invention concerne la synthèse de nouvelles molécules dendrimères à base de pyrrolidines polyhydroxylées obtenues au moyen de réactions de chimie click. Les molécules proposées inhibent les enzymes N-acétylgalactosamine-6-sulfatase (GALNS), iduronate-2-sulfatase (IDS), a-mannosidase et β-glucosidase, déficiente dans des maladies du stockage lysosomal. La présentation d'iminosucres multivalents sur un échafaudage est une condition préalable pour l'activité inhibitrice étant donné que les monomères correspondants ne sont pas actifs. L'activité inhibitrice rapportée est la base du développement des premiers chaperons pharmacologiques ASSC proposés pour le traitement des pathologies mentionnées ci-dessus.
PCT/IB2017/050670 2016-02-09 2017-02-08 Nouvelles pyrrolidines dendrimères, leur synthèse et utilisation médicale WO2017137895A1 (fr)

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ITUB2016A000605A ITUB20160605A1 (it) 2016-02-09 2016-02-09 Nuove pirrolidine dendrimeriche, loro sintesi e uso medico
IT102016000013155 2016-02-09

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020021032A1 (fr) * 2018-07-26 2020-01-30 Universite De Reims Champagne Ardenne Composes de type dendrimere, leurs procedes de preparation et leurs utilisations

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BRISSONNET ET AL.: "Correction to "Topological effects and binding modes operating with multivalent iminosugar-based glycoclusters and mannosidases"", J. AM. CHEM. SOC., vol. 136, 29 April 2014 (2014-04-29), pages 6773 - 6773 *
BRISSONNET ET AL.: "Topological Effects and binding modes operating with multivalent iminosugar-based glycoclusters and mannsidases", J. AM. CHEM. SOC., vol. 135, 13 November 2013 (2013-11-13), pages 18427 - 18435, XP002759786 *
COMPAIN ET AL.: "The multivalent effect in glycosidase inhibition: a new, rapidly emerging topic in glycoscience", CHEMBIOCHEM, vol. 15, 2014, pages 1239 - 1251, XP002759789 *
JOOSTEN ET AL.: "A systematic investigation of iminosugar Click Clusters as pharmacological chaperones for the treatment of Gaucher disease", CHEMBIOCHEM, vol. 15, 2014, pages 309 - 319, XP002759787 *
ZELLI ET AL.: "Synthesis and biological properties of multivalent iminosugars", NEW J. CHEM., vol. 39, 2015, pages 5050 - 5074, XP002759788 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020021032A1 (fr) * 2018-07-26 2020-01-30 Universite De Reims Champagne Ardenne Composes de type dendrimere, leurs procedes de preparation et leurs utilisations
FR3084364A1 (fr) * 2018-07-26 2020-01-31 Universite De Reims Champagne Ardenne Composes de type dendrimere, leurs procedes de preparation et leurs utilisations

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