New VASHs inhibitors, conjugates thereof and their uses as drugs or as research tools
20240391887 ยท 2024-11-28
Assignee
- Centre National de Recherche Scientifique (CNRS) (Paris, FR)
- Universite De Montpellier (Montpellier, FR)
- Ecole Nationale Superieure De Chimie De Montpellier (Montpellier, FR)
- MT ACT (Montpellier Cedex 02, FR)
Inventors
- Krzysztof ROGOWSKI (MONTPELLIER, FR)
- Muriel Amblard-Caussil (Saint Vincent de Barbeyrargues, FR)
- Siem VAN DER LAAN (CAZILHAC, FR)
- Lubomir VEZENKOV (Montpellier, FR)
- Guillaume MARCELLIN (Grabels, FR)
- Yoann LANNAY (Mas De Londres, FR)
- Khaled HACHED (Montpellier, FR)
Cpc classification
C07D303/48
CHEMISTRY; METALLURGY
International classification
C07D303/48
CHEMISTRY; METALLURGY
Abstract
The present invention concerns a compound of formula (I) or a pharmaceutically acceptable salt and/or solvate thereof, wherein X, R1, R2, R3 are defined, a conjugate thereof and their uses as drug or research tools.
##STR00001##
Claims
1. A compound of formula (I): ##STR00119## or a pharmaceutically acceptable salt and/or solvate thereof, in which X is ##STR00120## R.sup.1 is OC.sub.1-C.sub.6 alkyl, OC.sub.2-C.sub.6 alkenyl, NR.sup.1aR.sup.1b or ##STR00121## R.sup.1a is H or a C.sub.1-C.sub.6 alkyl, R.sup.1b is OH or C.sub.1-C.sub.6 alkyl, said alkyl being optionally substituted with C(O)OH, C(O)OC.sub.1-C.sub.6 alkyl or an aryl, R is OR.sup.2 or NHS(O).sub.2R.sup.9, R.sup.2 is H, a C.sub.1-C.sub.6 aliphatic chain, aryl, heteroaryl or C.sub.1-C.sub.6 alkyl-aryl, wherein up to 4 methylene units of said aliphatic chain are optionally replaced by O, C(O), NH or NC.sub.1-C.sub.6alkyl, said aliphatic chain, aryl, heteroaryl or alkyl-aryl being optionally substituted, R.sup.3 is OH, OC.sub.1-C.sub.6 aliphatic chain, O-aryl, OC.sub.1-C.sub.6 alkyl-aryl, O-heteroaryl, OC.sub.1-C.sub.6 alkyl-heteroaryl or NHOH, wherein up to 4 methylene units of said aliphatic chain are optionally replaced by O, C(O), NH or NC.sub.1-C.sub.6alkyl, said aliphatic chain, aryl, heteroaryl, alkyl-heteroaryl or alkyl-aryl being optionally substituted, R.sup.4 is H or a C.sub.1-C.sub.12 aliphatic chain wherein up to 4 methylene units are optionally replaced by O, C(O), NH or NC.sub.1-C.sub.6alkyl, said C.sub.1-C.sub.12 aliphatic chain being optionally substituted, ##STR00122## Y is (CH.sub.2).sub.m or R.sup.5 is OH, OC.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, heteroaryl, OC.sub.1-C.sub.6 alkyl-aryl, OC.sub.1-C.sub.6 alkyl-heteroaryl, C(O)OH, C(O)OC.sub.1-C.sub.6 alkyl, C(O)NHOH, C(O)NH.sub.2, C(O)NHC.sub.1-C.sub.6 alkyl, C(O)NHOC.sub.1-C.sub.6 alkyl, NHC.sub.1-C.sub.6 alkyl, N(C.sub.1-C.sub.6 alkyl).sub.2, NHC(O)C.sub.1-C.sub.6 alkyl or NHS(O).sub.2R.sup.9, said alkyl, alkenyl, alkynyl, aryl, heteroaryl, alkyl-aryl or alkyl-heteroaryl being optionally substituted, R.sup.6 is OH, OC.sub.1-C.sub.6 aliphatic chain, NHOH or NHCH(R.sup.7)(CH.sub.2).sub.nR.sup.8, said aliphatic chain being optionally substituted, R.sup.7 is H, C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, aryl, heteroaryl, C.sub.1-C.sub.6 alkyl-aryl or C.sub.1-C.sub.6 alkyl-heteroaryl, R.sup.8 is C(O)NH.sub.2, C(O)NHC.sub.1-C.sub.6 alkyl, aryl, heteroaryl, SH, NH.sub.2 or SC.sub.1-C.sub.6alkyl, and m and n are each independently an integer ranging from 0 to 6, R.sup.9 is a C.sub.1-C.sub.6 aliphatic chain or an aryl, said aliphatic chain or aryl being optionally substituted, provided that when X is ##STR00123## and R.sup.3 is OH, R.sup.1 is not OC.sub.1-C.sub.6 alkyl, and compound of formula (I) is not ##STR00124##
2. The compound according to claim 1, wherein R is OR.sup.2.
3. The compound according to claim 2, wherein R.sup.2 is H, an optionally substituted C.sub.1-C.sub.6 alkyl or an optionally substituted C.sub.1-C.sub.6 alkyl-aryl.
4. The compound according to claim 1, wherein R.sup.3 is OH, OC.sub.1-C.sub.6 aliphatic chain, OC.sub.1-C.sub.6 alkyl-aryl, or NHOH, said aliphatic chain being optionally substituted.
5. The compound according to claim 1, wherein R.sup.1 is OC.sub.1-C.sub.6 alkyl, OC.sub.2-C.sub.6 alkenyl or NR.sup.1aR.sup.1b, R.sup.1a and R.sup.1b being as defined in claim 1.
6. The compound according to claim 1, wherein R.sup.1 is ##STR00125## with Y, R.sup.5, and R.sup.6 being as defined in claim 1.
7. The compound according to claim 6, wherein Y is ##STR00126## R.sup.5 is OH, OC.sub.1-C.sub.6 alkyl, OC.sub.1-C.sub.6 alkyl-aryl, C(O)NHOC.sub.1-C.sub.6 alkyl, or NH(S(O).sub.2R.sup.9, and R.sup.6 is OH or OC.sub.1-C.sub.6 alkyl.
8. The compound according to claim 6, wherein Y is (CH.sub.2).sub.m, R.sup.5 is C(O)OH, C(O)OEt or C(O)NHOH, R.sup.6 is NHCH(R.sup.7)(CH.sub.2).sub.nR.sup.8, R.sup.7 is H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkyl-aryl, or C.sub.1-C.sub.6 alkyl-heteroaryl, R.sup.8 is C(O)NH.sub.2, aryl, heteroaryl, SH, or SC.sub.1-C.sub.6alkyl, and n is 0, 1, 2 or 3.
9. The compound according to claim 1, wherein X is ##STR00127##
10. The compound according to claim 1, responding to the following formula (I-A): ##STR00128##
11. The compound according to claim 1, being selected from the following compounds: ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134## and the pharmaceutically acceptable salts and/or solvates thereof.
12. A pharmaceutical composition comprising at least one compound of formula (I) as defined in claim 1 and at least one pharmaceutically acceptable excipient.
13. (canceled)
14. A method for treating a VASH peptidase activity associated disorder comprising the administration to a person in need thereof of an effective dose of a compound according to claim 1 or a pharmaceutically acceptable salt and/or solvate thereof.
15. The method according to claim 14, wherein the VASH peptidase activity associated disorder is a disorder involving altered microtubule detyrosination and/or polyglutamylation.
16. A conjugate comprising a fragment of a compound of formula (I) as defined in claim 1 linked to a biomolecule.
17. (canceled)
18. Use of compound according to claim 1 or a conjugate according to claim 16 as research tool for research and development activities.
19. The method according to claim 15, wherein the VASH peptidase activity associated disorder is selected from neurodegenerative diseases, glaucoma, psychiatric disorders, neuronal disorders, cancers, muscular dystrophies, infertility, retinal degeneration, Purkinje cell sicknesses, infantile onset degeneration, male infertilities, and ciliopathies.
20. The conjugate according to claim 16, wherein the biomolecule is selected from a peptide, a protein, a biomarker, an affinity probe, or a E3 ubiquitin ligase recruiter.
21. The use of claim 18, wherein research and development activities are selected in the group consisting of: in vitro and/or in cellulo screening assays for identifying new VASH inhibitors and/or quantifying their inhibitor efficiency, in vitro method for studying the role of tubulin detyrosination, in vitro diagnostic methods for identifying or monitoring a VASH peptidase activity associated disorder involving altered microtubule detyrosination and/or polyglutamylation, and related kits for performing said screening assays and methods.
Description
DESCRIPTION OF THE FIGURES
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EXAMPLES
1) Synthesis
Abbreviations:
[0278] Aad: -aminoadipic acid [0279] All: Allyl group [0280] BOP: benzotriazol-1-yloxytris(dimethylamino)phosphonium haxefluorophosphate [0281] DCM: Dichloromethane [0282] DIEA: N,N-Diisopropylethylamine [0283] DMF: dimethylformamide [0284] DMSO: dimethylsulfoxide [0285] HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate [0286] NMP: N-methyl-2-pyrrolidone [0287] PyAOP: 7-Azabenzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate [0288] RM: reaction mixture [0289] RT: room temperature (18 C.-25 C.) [0290] SPPS: solid phase peptide synthesis [0291] TES: Trans epoxy succinate [0292] TFA: trifluoroacetic acid [0293] THF: tetrahydrofuran [0294] TiS: triisopropylsilane [0295] Tyr: Tyrosine
1.1) Material
[0296] All of the Fluorenylmethyloxycarbonyl (Fmoc) protected amino acids and 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU) were provided by Iris Biotech GmbH. Piperidine, N,N-Diisopropylethylamine (DIEA), TFA, Triisopropylsilane (TIS), dichloromethane (DCM), 1,2-Dichloroethane (DCE), N,N-Dimethylformamide (DMF), N-methylpyrrolidinone (NMP) and ethyl ether (Et.sub.2O) were provided by Sigma Aldrich. AmphiSpheres 40 RAM 0.38 mmol/g 75-150 m resin was purchased from Agilent Technologies. Solvents used for HPLC and LC/MS were of HPLC grade.
SPPS Procedure
[0297] Peptide synthesis was performed using a standard SPPS protocol. Each synthesis was performed using Fmoc-Rink amide AmphiSpheres 40 resin (0.37 mmol/g). The Fmoc protected amino acids (4 eq), HATU (4 eq) and DIEA (6 eq) were added to the syringe reactor and the mixture was stirred for 1 hour at room temperature. After each coupling reaction, the peptide-resin was submitted to two 5 min deprotection cycles with DMF/piperidine 80/20 v/v solution.
Compounds Purification
[0298] All crude compounds were purified by preparative HPLC (Waters 4000 apparatus) on a C18 reversed-phase column (C18 Deltapak column, 100 mm40 mm, 15 m, 100 ) at a flow rate of 50 mL/min of a H2O+0.1% TFA and CH3CN+0.1% TFA mixture in gradient mode with UV detection at 214 nm. Fractions containing the pure product were collected and lyophilized.
LC/MS Analyses
[0299] Samples were prepared in an acetonitrile/water (50/50 v/v) mixture containing 0.1% TFA. The LC/MS system consisted of a Waters Alliance 2690 HPLC coupled to a Micromass (Manchester, UK) ZQ spectrometer (electrospray ionization mode, ESI+). All analyses were carried out using a C18 Chromolith Flash 254.6 mm column. A flow rate of 3 mL/min and a gradient of 0-100% Acetonitrile over 5 min of a H2O+0.1% HCOOH and CH3CN+0.1% HCOOH mixture in gradient mode with UV detection at 214 nm. Positive-ion electrospray mass spectra were acquired at a solvent flow rate of 100-200 l/min. Nitrogen was used for both the nebulizing and drying gas. The data were obtained in a scan mode ranging from 200 to 1700 m/z in 0.1 s intervals; ten scans were summed up to obtain the final spectrum. Retention times are given in minutes. Solvents used for HPLC and LC/MS were of HPLC grade.
1.2) Synthesis and Characterization
[0300] 1.2.1) Synthesis of compounds of formula (I) according to the present invention
Synthesis of Compound 1
[0301] ##STR00078##
[0302] Boc-Tyr(Bn)-OH (2 g, 5.4 mmol, FLUKA AG) was dissolved in 3 ml of anhydrous DMF followed by Cs.sub.2CO.sub.3 (1.8 g, 5.4 mmol). The RM was stirred at RT during 5 min before allyl bromide (696.6 l, 8.1 mmol) was added. After a 2-hour-long reaction at RT, RM was diluted in brine and EtOAc; then extracted 2 times. The regrouped organic phases were then washed 2 times with 1M KHSO.sub.4 solution, 2 times with NaHCO.sub.3 saturated solution and 2 times with brine; then organic phase was dried on MgSO.sub.4, filtered, and concentrated to afford Boc-Tyr(Bn)-OAll (2.45 g, yield: 110%, t.sub.R=2.19, MS (ESI+): m/z=412.3 [M+H].sup.+). The product was used as it is without further purification for the next step.
[0303] Boc-Tyr(Bn)-OAll (2.22 g, 5.4 mmol) was dissolved in 15 ml of DCM, followed by 15 ml of TFA/TIS/H.sub.2O 95/2.5/2.5 v/v/v. The RM was then stirred for 1 h10 at RT. The RM was concentrated dry, and the remaining oil was precipitated with cold ether. The precipitate was filtered and dried over reduced pressure to afford compound 1 as a white solid. The product was used as it is without further purification. [0304] Expected mass: 2.37 g [0305] Obtained mass: 2.83 g [0306] Yield: quantative with the presence of some impurities [0307] t.sub.R=1.38, MS (ESI+): m/z=312.2 [M+H].sup.+
Synthesis of Compound 2
[0308] ##STR00079##
[0309] (2,3S)-Oxirane-2,3-dicarboxylic acid (66 mg, 0.5 mmol) was dissolved in 3 ml of NMP, followed by compound 1 (212 mg, 0.5 mmol) and DIEA (374 l, 2 mmol). The RM was stirred and 260 mg (0.5 mmol) of PyAOP were added. The RM was stirred at room temperature for 20 min, and then poured in 50 ml of cold water and kept in ice until precipitation appeared. The precipitate was removed by filtration and the filtrate pH was adjusted to 3 with 1M HCl solution. The filtrate was then extracted 3 times with EtOAc and the organic phase was washed 3 times with 1M HCl solution, one time with brine, dried over MgSO.sub.4 and evaporated dry to afford 100 mg of compound 2 as transparent oil. The product was used as it is without further purification. [0310] Expected mass: 212.5 mg [0311] Obtained mass: 100 mg [0312] Yield: 47%
[0313] HPLC purity=95%, t.sub.R=3.33, MS(ESI+): m/z=426.2 [M+H].sup.+, (expected m/z=426.16)
Synthesis of Compound 3
[0314] ##STR00080##
[0315] Fmoc-Aad(OtBu)-OH (1 g, 2.28 mmol) was dissolved in 5 ml of NMP followed by the 2-phenylethan-1-amine (316 l, 2.5 mmol) and the DIEA (1.175 ml, 6.83 mmol). The RM was stirred and the PyAOP was added (1.18 g, 2.28 mmol). The RM was stirred for 15 min at RT. It was then poured in 50 ml of EtOAc, washed 1 time with water/brine 50/50 v/v mixture, 3 times NaHCO.sub.3 saturated solution, 3 times 1M HCl solution, 1 time with brine, dried over MgSO.sub.4 and concentrated dry to afford 1.24 g of compound 3 as transparent oil. The compound was used without further purification. [0316] Expected mass: 1.235 g [0317] Obtained mass: 1.24 g [0318] Yield: quantitative [0319] HPLC purity=95%, t.sub.R=4.28, MS (ESI+): m/z=543.3 [M+H].sup.+
Synthesis of Compound 4
[0320] ##STR00081##
[0321] Compound 3 (542 mg, 1 mmol) was dissolved in 5 ml of NMP followed by the addition of the diethylamine (208 l, 2 mmol). The RM was then stirred at RT for one hour. Next, the RM was poured in 50 ml of water and the pH was adjusted to 11 by 1M NaOH solution. The aqueous phase was then extracted 3 times with EtOAc, the regrouped organic phases were washed with brine, dried over Na2.sub.SO.sub.4 and concentrated dry to afford 294 mg of compound 4 as transparent oil. The product was used without any further purification. [0322] Expected mass: 320 mg [0323] Obtained mass: 294 mg [0324] Yield: 92% [0325] tR=2.28, MS (ESI+): m/z=321.2 [M+H]+
Intermediate A Synthesis
[0326] ##STR00082##
[0327] Compound 4 tert-butyl(S)-5-amino-6-oxo-6-(phenethylamino) hexanoate (131 mg, 0.41 mmol) and compound 2 HO-TES-Tyr(OBn)-OAll (174 mg, 0.41 mmol) were solubilized in DMF followed by the addition of DIEA (139 l, 0.82 mmol) and BOP (173 mg, 0.41 mmol). After 2 hours, the product was isolated by extraction. The obtained white powder was then dried over night at 0.2 mbar to afford 260 mg of intermediate A as white solid. [0328] Expected mass: 260 mg/Obtained mass: 300 mg/Crude Yield: 86% [0329] t.sub.R=2.18, MS (ESI+): m/z=728.4 [M+H].sup.+
Synthesis of Compound 5
[0330] ##STR00083##
[0331] (+,)-trans-Oxirane-2,3-dicarboxylic acid (200 mg, 1.52 mmol, TCI chemicals) was dissolved in 5 ml of NMP, followed by the HCl.Math.H-Tyr (OtBu)-OtBu (500 mg, 1.52 mmol, Iris Biotech GmbH) and the DIEA (1.340 ml, 8 mmol). The PyAOP was then added (792 mg, 1.52 mmol) and the reaction mixture (RM) was stirred for 80 min at room temperature (RT). The RM was then poured in 180 ml of cold water. The obtained precipitate was removed by filtration. The pH of the filtrate was then adjusted to 3 with 1M KHSO.sub.4 solution and extracted 3 times with EtOAc. The organic phases were regrouped, washed with brine, dried over Na.sub.2SO.sub.4 and concentrated dry to afford 433 mg of compound 5 as transparent oil. [0332] Expected mass: 618 mg [0333] Obtained mass: 433 mg [0334] Yield: 70% [0335] t.sub.R=3.4, MS (ESI+): m/z=408.4 [M+H].sup.+
Synthesis of LV-80
[0336] ##STR00084##
[0337] Intermediate A (100 mg, 0.137 mmol) was dissolved in 15 ml of DCM, followed 15 ml of TFA/TIS/H.sub.2O. The RM was then stirred for 30 min at RT. The RM was concentrated dry and the remaining oil was precipitated with cold EtOEt. The precipitate was filtered and dried over reduced pressure. Next, the precipitate was dissolved in 1 ml of dry DMF and the allyl ester was removed by treatment with Pd.sup.0 Tetrakis (5.15 mg, 0.0045 mmol) and PhSiH.sub.3 (37 l, 0.3 mmol). After 20 min the RM was dissolved in 20 ml EtOAc that was washed 1 time with 1M HCl solution, 1 time with brine and concentrated dry. The obtained precipitate was then purified by preparative HPLC to obtain 41 mg of LV-80 as white solid. [0338] Expected mass: 91 mg/Obtained mass: 41 mg/Crude Yield: 45% [0339] t.sub.R=3.10, MS (ESI+): m/z=632.4 [M+H].sup.+
Synthesis of LV-86
[0340] ##STR00085##
[0341] Intermediate A (100 mg, 0.137 mmol) was dissolved in 15 ml of DCM, followed 15 ml of TFA/TiS/H.sub.2O. The RM was then stirred for 30 min at RT. The RM was concentrated dry and the remaining oil was precipitated with cold Et.sub.2O. The precipitate was filtered and dried over reduced pressure to obtain Intermediate B.
[0342] Intermediate B (15 mg, 0.022 mmol) was dissolved in NMP, followed by the addition of cesium carbonate (7.15 mg, 0.022 mmol) and iodoethane (2.34 l, 0.029 mmol). The RM was then stirred at RT for 1 hour. Next, the RM was poured in 30 ml of water. The aqueous phase was extracted 3 times with EtOAc. The regrouped organic phases were washed 3 times with saturated NaHCO.sub.3, 3 times with KHSO.sub.4 1M then washed with brine, dried over Na.sub.2SO.sub.4 and evaporated dry to afford 17 mg white solid. Next, the white solid was dissolved in 1 ml of dry DMF and the allyl ester was removed by treatment with Pd.sup.0 Tetrakis (1 mg, 0.00087 mmol), PhSiH.sub.3 (3 l, 0.024 mmol). After 20 min the RM was dissolved in 20 ml EtOAc that was washed 1 time with 1M KHSO.sub.4 solution, 1 time with brine and concentrated dry. The obtained precipitate was then purified by preparative HPLC to obtain 8.1 mg of LV-86 as white solid. [0343] Expected mass: 15.8 mg/Obtained mass: 8.1 mg/Crude Yield: 51% [0344] t.sub.R=3.49, MS (ESI+): m/z=660 [M+H].sup.+
[0345] .sup.1H NMR (500 MHZ, DMSO) 8.70 (s, 1H), 8.60 (s, 1H), 8.18 (t, J=5.5, 1H), 7.44 (d, J=7.5, 2H), 7.38 (t, J=7.4, 2H), 7.32 (t, J=7.3, 1H), 7.27 (t, J=7.5, 2H), 7.21-7.16 (m, 2H), 7.14 (d, J=8.3, 2H), 6.93 (d, J=8.2, 2H), 5.05 (s, 2H), 4.38 (d, J=4.6, 1H), 4.23 (dd, J=13.6, 7.6, 1H), 4.04 (q, J=7.1, 2H), 3.57 (d, J=5.1, 2H), 3.22 (td, J=13.0, 6.9, 2H), 3.02 (dd, J=13.8, 4.4, 1H), 2.84 (dd, J=13.8, 9.4, 1H), 2.69 (t, J=7.2, 2H), 2.24 (t, J=7.2, 2H), 1.57 (m, J=6.7, 1H), 1.50 (m, 1H), 1.45 (m, J=16.4, 8.0, 3H), 1.17 (t, J=7.1, 3H) 13C NMR (126 MHz, DMSO) 172.63, 172.50, 170.67, 165.65, 165.49, 157.11, 139.33, 137.20, 130.23, 129.57, 128.73, 128.46, 128.32, 127.85, 127.79, 126.15, 114.52, 69.15, 59.80, 53.95, 52.58, 52.46, 52.35, 50.63, 35.77, 35.04, 33.09, 31.52, 20.80, 14.17.
Synthesis of LV-87
[0346] ##STR00086##
[0347] Intermediate B (10 mg, 0.015 mmol) was dissolved in anhydrous THF. N-Methylmorpholine (3.82 L, 0.030 mmol) was added to the solution. The reaction mixture was then cooled down to 15 C. and stirred for 5 min. Isobutylchloroformate (1.94 L, 0.015 mmol) was added, and the mixture was stirred for 10 min at 15 C. O-Tritylhydroxylamine (8.25 mg, 0.030 mmol) was added and the stirring continued for 15 min at 15 C. and 30 min at room temperature. Afterward, the mixture was poured into EtOAc (20 mL) washed 1M KSHO.sub.4, saturated solution of NaHCO.sub.3, dried over MgSO.sub.4 and concentrated dry to afford 14 mg of intermediate C as a white powder [0348] Expected mass: 14 mg/Obtained mass: 18 mg/Crude Yield: quantitative
[0349] Intermediate C (13.92 mg, 0.015 mmol) was dissolved in 10 ml of DCM, followed 10 ml of TFA/TiS/H.sub.2O. The RM was then stirred for 30 min at RT. The RM was concentrated dry and the remaining oil was precipitated with cold EtOEt. The precipitate was filtered and dried over reduced pressure. Next, the precipitate was dissolved in 0.5 ml of dry DMF and the allyl ester was removed by treatment with Pd.sup.0 Tetrakis (1 mg, 0.00087 mmol), PhSiH.sub.3 (2.2 l, 0.0175 mmol). After 20 min the RM was dissolved in 20 ml EtOAc that was washed 1 time with 1M HCl solution, 1 time with brine and concentrated dry. The obtained precipitate was then purified by preparative HPLC to obtain 2 mg of LV-87 as white solid. [0350] Expected mass: 11 mg/Obtained mass: 2 mg/Crude Yield: 17% [0351] t.sub.R=2.93, MS (ESI+): m/z=647.3 [M+H].sup.+
[0352] .sup.1H NMR (500 MHZ, DMSO) 8.80 (d, J=8.0, 1H), 8.70 (s, 1H), 8.61 (d, J=8.1, 1H), 8.17 (t, J=5.6, 1H), 7.44 (d, J=7.2, 2), 7.38 (d, J=14.8, 2H), 7.32 (t, J=7.3, 1H), 7.29-7.26 (m, 2H), 7.20-7.17 (m, 3H), 7.15 (d, J=8.6, 2H), 6.94 (d, J=8.7, 2H), 5.06 (s, 2H), 4.44-4.38 (m, 1H), 4.26-4.19 (m, 1H), 3.58 (s, 2H), 3.26-3.20 (m, 2H), 3.02 (dd, J=13.8, 4.6, 1H), 2.84 (dd, J=13.9, 9.4, 1H), 2.72-2.67 (m, 2H), 1.90 (dd, J=13.0, 6.6, 2H), 1.60-1.53 (m, 1H), 1.49-1.43 (m, 2H), 1.42-1.37 (m, 1H).
[0353] .sup.13C NMR (500 MHZ, DMSO) 172.50, 168.75, 165.75, 165.45, 157.14, 139.32, 137.18, 130.20, 128.71, 128.45, 128.35, 127.84, 127.78, 126.15, 114.43, 69.14, 53.82, 52.53, 52.37, 48.50, 35.07, 31.98, 31.79, 30.14, 21.63.
Synthesis of LV-81
[0354] ##STR00087##
[0355] The H-Aad(tBu)Phe-sequence was synthetized via SPPS on cystamine-Trt resin (CA) charged 0.5 mmol/g. Next, the solid supported sequence was attached via an amide bond to compound HO-TES-Tyr(OBn)-OAll (95 mg, 0.225 mmol) via PyAOP (95 mg, 0.225 mmol) and DIEA (77 l, 0.450 mmol) activation, overnight at RT. After 2 hours, Pd Tetrakis (8.66 mg, 0.008 mmol) and PhSiH.sub.3 were added, leading to the deprotection of the OAll ester. The thiolated version of LV-80 was cleaved from the resin by a 2 hours long treatment with TFA/DCM/H2O/TiS 50/50/2.5/2.5. The filtrate was then concentrated dry to provide a brown oil that was precipitated in cold Et.sub.2O to obtain a white solid that was isolated by filtration. The compound was then purified by preparative HPLC to obtain 32 mg of LV-81 as white solid. [0356] Expected mass: 55 mg/Obtained mass: 32 mg/Crude Yield: 58%
Intermediate D Synthesis
[0357] ##STR00088##
[0358] HCl.Math.H-Tyr-OEt (100 mg, 0.407 mmol) and (2S,3S)-trans-Oxirane-2,3-dicarboxylic acid (80.8 mg, 0.611 mmol) were dissolved in 1.5 mL of NMP, followed by the addition of DIEA (1,628 mmol, 374 L). The mixture was stirred and HATU (309.5 mg, 0,814 mmol) was added. The mixture was stirred at room temperature for 20 min. The reaction mixture was poured in a Na.sub.2CO.sub.3 1 M solution and extracted 3 times with 30 ml of diethyl ether. The aqueous phase was acidified with KHSO4 1M and the resulting solution was extracted 3 times with 30 mL of ethyl acetate. The organic layers were combined, washed with brine then dried over MgSO.sub.4, filtered and concentrated under reduced pressure to afford intermediate D as a yellow oil. The product was used as it is without further purification. [0359] Expected mass: 131 mg/Obtained mass: 104 mg/Yield: 79.3% [0360] HPLC purity=98%, t.sub.R=1.2 min, MS (ESI+): m/z=324.3 [M+H]+, (expected m/z=324.31)
Synthesis of LV-111
[0361] ##STR00089##
[0362] Cs.sub.2CO.sub.3 (314 mg, 0.966 mmol) was added to Intermediate D (104 mg, 0.321 mmol) dissolved in anhydrous DMF and the reaction was stirred for 15 min. Iodoethane (77.6 L, 0.966 mmol) was added to the solution, and the reaction was stirred at room temperature overnight and monitored by HPLC. The solution was poured in 30 ml of water and extracted 3 times with 30 ml of ethyl acetate then washed 3 times with a solution of NaHCO.sub.3 1 M and 3 times with a solution of KHSO.sub.4 1 M. The organic layer was dried over MgSO.sub.4, filtered, and concentrated under reduced pressure to afford the desired product as white crystals. The product was then purified by preparative chromatography to obtain 51.5 mg of LV-111 as white solid. [0363] Expected mass: 121.66 mg/Obtained mass: 51.5 mg/Yield: 42% [0364] HPLC purity=98%, t.sub.R=2.1 min, MS (ESI+): m/z=380.2 [M+H].sup.+, (expected m/z=380.16)
[0365] .sup.1H NMR (500 MHZ, DMSO) 8.78 (d, J=7.9, 1H), 7.07 (d, J=8.3, 2H), 6.78 (d, J=8.2, 2H), 4.42 (dd, J=14.2, 8.4, 1H), 4.17-3.89 (m, 6H), 3.61 (s, 1H), 3.40 (s, 1H), 2.93 (dd, J=13.8, 5.7, 1H), 2.83 (dd, J=13.7, 9.2, 1H), 1.29-1.05 (m, 9H).
[0366] .sup.13C NMR (126 MHz, DMSO) 171.40, 167.43, 165.49, 157.81, 130.66, 128.90, 114.60, 63.32, 62.06, 61.24, 54.20, 53.06, 51.69, 36.17, 15.14, 14.34.
[0367] HRMS (micrOTOF-Q): calculated for C.sub.19H.sub.25NO.sub.7 [M+H].sup.+: 380.1631, found: 380.1704.
Synthesis of LV-101
[0368] ##STR00090##
[0369] HCl.Math.H-Tyr-OEt (100 mg, 0.407 mmol) and (2S,3S)-trans-Oxirane-2,3-dicarboxylic acid (27 mg, 0.204 mmol) were dissolved in 1.5 ml of NMP, followed by DIEA (1,628 mmol, 374 L). The mixture was stirred for 5 min and HATU (154.7 mg, 0,407 mmol) was added. The mixture was stirred at room temperature for 20 min. The solution was extracted 3 times with ethyl acetate. The organic layers were washed 3 times with KHSO.sub.4 1M and NaHCO.sub.3 1 M, then one time with brine and dried over MgSO.sub.4, filtered and concentrated under reduced pressure to afford the desired product as a white powder. [0370] HPLC purity=98%, t.sub.R=1.42 min, MS (ESI+): m/z=515.20 [M+H].sup.+, (expected m/z=515.20)
Synthesis of LV-104
[0371] ##STR00091##
[0372] Cs.sub.2CO.sub.3 (126.8 mg, 0.389 mmol) was added to LV-101 (100 mg, 0.195 mmol) dissolved in anhydrous DMF and the reaction was stirred for 15 min. Iodoethane (47.0 L, 0.585 mmol) was added to the solution, and the reaction was stirred at room temperature overnight and monitored by HPLC. The solution was poured in 30 ml of water and extracted 3 times with 30 mL of ethyl acetate then washed 3 times with a solution of NaHCO.sub.3 1 M and 3 times with a solution of KHSO.sub.4 1 M. The organic layer was dried over MgSO.sub.4, filtered, and concentrated under reduced pressure to afford the desired product as a white powder. The product was then purified by preparative chromatography to obtain 75.6 mg of LV-104 as white powder. [0373] Expected mass: 111.15 mg/Obtained mass: 75.6 mg/Yield: 68% [0374] HPLC purity=99%, tR=3.81 min, MS (ESI+): m/z=571.3 [M+H]+, (expected m/z=571.26)
[0375] .sup.1H NMR (500 MHZ, DMSO) 8.87 (d, J=7.7, 2H), 7.07 (d, J=8.2, 4H), 6.78 (d, J=8.1, 4H), 4.38 (dd, J=14.2, 8.3, 2H), 4.02 (q, J=7.1, 4H), 3.93 (q, J=6.9, 4H), 3.45 (s, 2H), 2.92 (dd, J=13.8, 5.7, 2H), 2.82 (dd, J=13.8, 9.2, 2H), 1.25 (t, J=6.9, 6H), 1.08 (t, J=7.1, 6H).
[0376] .sup.13C NMR (126 MHz, DMSO) 170.97, 165.50, 157.29, 130.25, 128.36, 114.14, 62.70, 60.62, 53.79, 52.17, 35.51, 14.62, 13.91.
[0377] HRMS (micrOTOF-Q): calculated for C.sub.30H.sub.38N.sub.2O.sub.9 [M+H]+: 571.6390, found: 571.2650
Synthesis of Intermediate E
[0378] Carbonyldiimidazole CDl (113.6 mg; 1.2 eq) was dissolved in a minimum of DMF and the RM was placed at 10 C. Boc-NHNH.sub.2 (92.5 mg; 1.2 eq) was also dissolved in a minimum of DMF and added to the RM which was then stirred during 30 min at 10 C. After this, TFA.Math.HTyr (OEt) OEt (203.6 mg; 1 eq) was also dissolved in a minimum of DMF and was added to the RM followed by Et.sub.3N (168 L). The RM was then stirred at room temperature during 2 h50. For the work up step, distilled water and AcOEt were added in the RM and placed in a separation funnel. The organic phase was then washed with HCl 1M (2), saturated NaHCO.sub.3 (2) and brine (2). The solution obtained was then dried with MgSO.sub.4, filtered and concentrated to give an oil (154.3 mg; Yield: 56%). This obtained oil was solubilized in a TFA/TIS/H.sub.2O (95/2.5/2.5) solution and stirred at room temperature during 30 min and then concentrated dry to give Intermediate E as crude oil. The compound was used without any further purification.
Synthesis of LV-119
[0379] ##STR00092##
[0380] CDl (63.3 mg; 1.2 eq) was dissolved in a minimum of DMF and the RM was placed at 10 C. Intermediate E (159.7 mg; 1.2 eq) was also dissolved in a minimum of DMF and added to the RM which was then stirred during 30 min at 10 C. The pH was adjusted to 8 with few drops of Et.sub.3N. After this, TFA.Math.HTyr (OEt) OEt (115.9 mg; 1 eq) was also dissolved in a minimum of DMF and was added to the RM followed by Et.sub.3N (102.2 L; 2.2 eq). The RM was then stirred at room temperature during 3 h. For the work up step, distilled water and AcOEt were added in the RM and placed in a separation funnel. The organic phase was then washed with HCl 1M (2), saturated NaHCO.sub.3 (2) and brine (2). The solution obtained was then dried with MgSO.sub.4, filtered and concentrated to give an oil.
[0381] The raw product was then purified on HPLC and lyophilized to give very light and white crystals (43.9 mg; Yield: 24%; Purity: >96%).
[0382] .sup.1H NMR (500 MHz, CDCl.sub.3) 6.99 (d, J=8.5, 4H), 6.75 (d, J=8.6, 4H), 6.50 (s, 2H), 6.05 (d, J=8.1, 2H), 4.60 (dd, J=14.3, 6.6, 2H), 4.16-4.04 (m, 4H), 3.92 (q, J=7.0, 4H), 3.03-2.89 (m, 4H), 1.38-1.28 (m, 6H), 1.18 (t, J=7.1, 6H).
Synthesis of Intermediate F
[0383] ##STR00093##
[0384] Boc-5-Ava-OH (1 g, 4.6 mmol) was dissolved in anhydrous DMF (30 ml) and EtOH (1.3 ml, 23 mmol) was added. Then was added in this order: DMAP (56.4 mg, 0.46 mmol), NMM (605 l, 5.5 mmol), OxymaPure (653.7 mg, 4.6 mmol) and EDCi (1.1 g, 5.98 mmol). The RM was stirred at RT during 2h05. Three AcOEt extractions were proceeded. The organic layer was then washed with NAHCO.sub.3 (sat.), KHSO.sub.4 (1M), and brine three times each. Organic layer was then dried over MgSO.sub.4, filtered, and concentrated dry. [0385] Expected mass: 1.13 g; Obtained mass: 966.3 mg; Yield: 86%
Synthesis of Intermediate G
[0386] ##STR00094##
[0387] Intermediate F (100 mg, 0.41 mmol) was dissolved in DCM (700 l), followed by TFA/TIS/H.sub.2O 95/2.5/2.5 v/v/v (663 l). The RM was then stirred for 1 h at RT and concentrated dry. [0388] Expected mass: 106.2 mg; Obtained mass: 286.02 mg; Crude Yield: 269%
Synthesis of IBMT23
[0389] ##STR00095##
[0390] Intermediate G (106.2 mg, 0.41 mmol) and IBMT7 (49.2 mg, 0.14 mmol) were dissolved in anhydrous DMF (911 l). Then was added in this order: DMAP (1.2 mg, 0.01 mmol), NMM (127.6 l, 1.16 mmol), OxymaPure (19.9 mg, 0.14 mmol) and EDCi (34.5 mg, 0.18 mmol). The RM was stirred at RT overnight. Three AcOEt extractions were proceeded. The organic layer was then washed with NAHCO.sub.3 (sat.), KHSO.sub.4 (1M), and brine three times each. Organic layer was then dried over MgSO.sub.4, filtered, and concentrated dry to afford 47.03 mg of desired product. The crude product was purified on inverse phase preparative HPLC. [0391] Expected mass: 66.9 mg; Obtained mass: 33.4 mg; Yield: 50%; Purity: 100% (MS (ESI+): m/z=479.0 [M+H].sup.+; time=3.12 min) (expected m/z=479.55)
Synthesis of Intermediate H
[0392] ##STR00096##
[0393] Boc-Phe(4-NH.sub.2)OH (100 mg, 0.36 mmol) was dissolved in NMP (691 l) and DBU (54 l, 0.36 mmol) was added. Iodoethane (29 l, 0.36 mmol) was previously put at 0 C. and then added to the reaction mixture dropwise. After a 30 min stirring at RT, H.sub.2O and AcOEt were added and then poured in a separation funnel. Three AcOEt extractions were proceeded, and the organic layer was then washed with KHSO.sub.4 (1M) and brine three times each. Organic layer was then dried over MgSO.sub.4, filtered, and concentrated dry. [0394] Expected mass: 110.9 mg; Obtained mass: 67.5 mg; Crude Yield: 61%
Synthesis of Intermediate I
[0395] ##STR00097##
[0396] Intermediate H (47.9 mg, 0.16 mmol) was dissolved in anhydrous DMF (139 l) and Pyridine (78 l, 0.96 mmol) was added. Ms-Cl (37 l, 0.48 mmol) was then added and the RM was stirred during 1 h at RT. H.sub.2O and AcOEt were added and then poured in a separation funnel. Three AcOEt extractions were proceeded. The organic layer was then washed with NAHCO.sub.3 (sat.), KHSO.sub.4 (1M), and brine three times each. Organic layer was then dried over MgSO.sub.4, filtered, and concentrated dry. [0397] Expected mass: 61.8 mg; Obtained mass: 19.8 mg; Crude Yield: 32%
Synthesis of Intermediate J
[0398] ##STR00098##
[0399] Intermediate I (19.8 mg, 0.05 mmol) was dissolved in DCM (200 l), followed by TFA/TIS/H.sub.2O 95/2.5/2.5 v/v/v (122 l). The RM was then stirred for 1 h at RT and concentrated dry. [0400] Expected mass: 20 mg; Obtained mass: 24.4 mg; Crude Yield: 122%
Synthesis of IBMT35
[0401] ##STR00099##
[0402] Intermediate J (20 mg, 0.05 mmol) was dissolved in NMP (240 l), followed by (2S,3S)-trans-Oxirane-2,3-dicarboxylic acid (6.6 mg, 0.05 mmol) and DIEA (61.2 l, 0.35 mmol). The RM was stirred and HATU (19.1 mg, 0.05 mmol) was added. The RM was stirred at room temperature for 3 h. The RM was then extracted 3 times with EtOAc and the organic phase was washed 3 times with HCl (1M), one time with brine, dried over MgSO.sub.4, filtered, and evaporated dry to obtain the 18 mg mixture of 1 and 2. The crude product was purified on inverse phase preparative HPLC.
[0403] (IBMT35) Expected mass: 16.7 mg; Obtained mass: 3.2 mg; Yield: 19%; Purity: 98% (MS (ESI+): m/z=669.0 [M+H].sup.+; time=2.64 min) (expected m/z=668.7)
Synthesis of Intermediate K
[0404] ##STR00100##
[0405] Boc-Phe(4-NH.sub.2)OH (50 mg, 0.18 mmol) was suspended in a solution of NaOH (1M) and the RM was placed at 0 C. Then was added Ts-Cl and the RM was stirred during 2h at 0 C. Acidification was proceed with HCl (1M) until pH 2-3 and the RM was extracted three times with AcOEt.
[0406] Then organic layer was washed three times with KHSO.sub.4 (1M) and two times with brine, dried over MgSO.sub.4, filtered, and concentrated dry. [0407] Expected mass: 78.1 mg; Obtained mass: 76.9 mg; Yield: 98%
Synthesis of Intermediate L
[0408] ##STR00101##
[0409] Intermediate K (78.1 mg, 0.18 mmol) was dissolved in anhydrous DMF (1.2 ml) and EtOH (62.6 l, 1.08 mmol) was added. Then was added in this order: DMAP (2.5 mg, 0.02 mmol), NMM (24.2 l, 0.22 mmol), OxymaPure (25.6 mg, 0.18 mmol) and EDCi (44 mg, 0.23 mmol). The RM was stirred at RT during 40 min. Three AcOEt extractions were proceeded. The organic layer was then washed with NAHCO.sub.3 (sat.), KHSO.sub.4 (1M), and brine three times each. Organic layer was then dried over MgSO.sub.4, filtered, and concentrated dry. [0410] Expected mass: 83.3 mg; Obtained mass: 69.4 mg; Yield: 83%
Synthesis of Intermediate M
[0411] ##STR00102##
[0412] Intermediate L (69.4 mg, 0.15 mmol) was dissolved in DCM (400 l), followed by TFA/TIS/H2O 95/2.5/2.5 v/v/v (365 l). The RM was then stirred for 1 h at RT, concentrated dry, and precipitated in diethyl ether. [0413] Expected mass: 71.5 mg; Obtained mass: 69.8 mg; Crude Yield: 98%
Synthesis of IBMT38
[0414] ##STR00103##
[0415] Intermediate M (69.8 mg, 0.15 mmol) was dissolved in anhydrous DMF (385 l), followed by (2S,3S)-trans-Oxirane-2,3-dicarboxylic acid (13.2 mg, 0.1 mmol) and DIEA (63.8 l, 0.38 mmol).
[0416] The RM was stirred and HATU (57 mg, 0.15 mmol) was added. The RM was stirred at room temperature for 2h20. The RM was then directly purified on inverse phase preparative HPLC. [0417] (IBMT38) Expected mass: 61.5 mg; Obtained mass: 8.3 mg; Yield: 13%; Purity: 91% (MS (ESI+): m/z=821.0 [M+H].sup.+; time=3.64 min) (expected m/z=820.9) 1.2.2) Synthesis of conjugate of formula (I) according to the present invention
Synthesis of LV-82
[0418] ##STR00104##
[0419] Compound 81 (7.35 mg, 0.01 mmol) was dissolved in 800 l of DMF followed by the addition of biotin-maleimide (4.5 mg, 0.01 mmol) and 400 l PBS solution. The mixture was lightly heated in order to solubilize both reagents and then stirred at RT for 1 h. The RM was then directly purified by preparative HPLC to afford 7 mg of LV-82 as white solid. [0420] Expected mass: 11.85 mg/Obtained mass: 7 mg/Yield: 59% [0421] t.sub.R=2.89, MS (ESI+): m/z=1186.2 [M+H].sup.+, 593.7 [M+2H].sup.2+
1.2.3) Synthesis of Comparative Example LV-43
[0422] ##STR00105##
[0423] Fmoc-Aad(OtBu)-OH (88 mg, 0.216 mmol) was attached via a standard SPPS HATU (82 mg, 0.216 mmol) DIEA (50 l, 0.288 mmol) activation. The RM was stirred with 200 mg of rink amide (RA) resin charged at 0.37 mmol/g in solid phase synthesis reactor for 2 h. After standard washings, the Fmoc protection was removed by two times 10 min DMF/Pip 80/20 v/v treatment. As a next step compound 5 (88 mg, 0.216 mmol) was attached to the liberated amine via a PyAOP (113 mg, 0.216 mmol), DIEA (545 l, 0.63 mmol) activation. After overnight reaction, the resin was washed 3 times DMF, 3 times DCM and the inhibitor was cleaved by a 20 ml TFA treatment for 50 min. The product containing TFA filtrate was then evaporated dry, and the peptide was precipitated in EtOEt, filtered, purified by preparative HPLC and lyophilized to obtain 10.7 mg of LV-43 as a white solid. [0424] Expected mass: 31.4 mg [0425] Obtained mass: 10.7 mg [0426] Yield: 34% [0427] HPLC purity=100%, tR=0.84, MS (ESI+): m/z=438.20 [M+H].sup.+ (expected m/z=438.15)
2) Biological Results
2.1) Experimental Model
Cell Culture
[0428] Human CHL-1 cells (melanoma derived cell line, Nieuwenhuis J. et al. Science, 2017, 358 (6369): 1453-1456) were routinely cultured in DMEM containing 10% (v/v) fetal bovine serum (FBS), 50 U/ml penicillin and 50 g/ml streptomycin (pen/strep) and incubated at 37 C. Prior testing, the CHL-1 cells were seeded in adequate plates (96, 48, 24, 12 or 6 wells) at 30% confluency. Next day cells were treated and collected for further analysis (immunoblotting, qPCR, immunofluorescence . . . ).
[0429] Cortical cell cultures were seeded either on glass coverslips or plastic plates coated with 0.1 mg/ml poly-L-lysine (Sigma) at 10.sup.5 cells per cm.sup.2 or 210.sup.4 cells per cm.sup.2 for low-density cultures. Neurons were plated in DMEM containing 10% (v/v) fetal bovine serum (FBS), 50 U/ml penicillin and 50 g/ml streptomycin (pen/strep), and 1-2 h later the medium was replaced with Neurobasal medium supplemented with 2% B27, pen/strep, 0.6% glucose and 1% Glutamax (all reagents from Life Technologies).
Drosophila Melanogaster
[0430] Drosophila were raised at 25 C. yw flies were used as control for yw; Tub84B3 mutant. Total males were disrupted in laemli loading buffer, boiled, sonicated and loaded for immunoblotting.
2.2) Method Details
Protein Expression and Purification
[0431] We used the methods as disclosed in the application WO2020/012002.
[0432] Human VASH1 (hVASH1) was cloned in the expression vector with a poly-histidine tag. Bacteria were transformed and induced with Isopropyl -d-1-thiogalactopyranoside (IPTG) overnight or for 4 hours. Bacteria were collected and disrupted using a HTU-DIGI-F press (Heinemann). Recombinant proteins were purified using nickel-based affinity chromatography (IMAC) according to the manufacturer's protocol (GE Healthcare).
In Vitro Detyrosination Assays
[0433] Spodoptera frugiperda Sf9 cells were grown, lysed and used for tubulin purification by affinity chromatography. Microtubules were obtained with taxol and stored until use. In vitro analysis of detyrosination activities was performed using the recombinant hVASH1. Detyrosination assays were performed in the presence of 0.5 M microtubules. Reactions were stopped by addition of the denaturing loading buffer followed by 5 min incubation at 95 C., and samples were loaded on SDS PAGE for immunoblot analysis.
Standardized In Vitro Detyrosination ELISA Based Assays
[0434] Standardized primary ELISA-based in vitro detyrosination assay in a 96-wells format has been developed according to the method disclosed in WO2020/012002. Briefly, the substrate of the VASH enzyme has been produced and 100 L of the enzymatic mix (enzyme and inhibitor) is added to each well, then the plate is incubated 5 minutes at 37 C. Primary (rabbit anti-detyrosinated tubulin) is incubated for 1 hour and after washes, secondary (anti-rabbit) antibody is added to each well. The plate is incubated 1 h at room temperature. Development consists of addition of TMB (3,3,5,5-Tetramethylbenzidine) to each well, incubation 30 minute at room temperature. The colour development is stopped by addition of 0.5 M sulfuric acid. OD is measured at 450 nm.
Deglutamylation Assays and Tubulin Purification
[0435] Deglutamylation assays have been performed as previously described (Rogowski et al., Cell, 2010, 143 (4): 564-78).
Transfection of Human Cells
[0436] Plasmid transfections were performed and cells were collected 24 hours after transfection for immunoblotting and immunofluorescence analysis.
Quantitative PCR
[0437] Total RNA was isolated with TRIzol (Invitrogen). Reverse transcription was carried out with random hexanucleotides. Quantitative PCR assays were performed using the Lightcycler SYBR Green Master mix on a Lightcycler apparatus (Roche). All used primers were intron-spanning. The relative amount of target cDNA was obtained by normalization by geometric averaging of multiple internal control genes.
Immunofluorescence Labeling
[0438] Methanol-fixed CHL-1 cells and mouse cortical neurons were analyzed using a Zeiss Axioimager Apotome microscope after standard immunofluorescence experiments. Briefly, samples were incubated with primary antibodies overnight, washed three times in PBS before 1 h incubation with the following secondary antibodies: Alexa Fluor 488-conjugated goat anti-rat, goat anti-mouse and Alexa Fluor 555-conjugated goat anti-rabbit (Invitrogen). After incubation, samples were washed three times in PBS, stained with DAPI, and mounted.
Microscopy
[0439] Microscopy image acquisition was performed at the Montpellier RIO Imaging facility and images were processed using OMERO.
Bioanalysis of the Penetration and the Conversion of the Compounds by LC-MS on Human Cells
[0440] Peptide stability in serum Human serum reaction sample contained 250 l of heat inactivated Human Serum (Sigma Aldrich) and 750 L of RPMI Medium 1640 (Sigma Aldrich). The reactions were initiated by addition of 50 l compound (in mM stock solution in DMSO). Assays were performed in a shaking water bath at 37 C. 100 l samples were collected at known time intervals and added to 200 l of acetonitrile 1% % TFA to precipitate serum proteins. Cloudy sample was cooled to 4 C. for 15 min and then submitted to centrifugation for 10 min at 12,000 rpm to pellet the precipitated serum proteins. 150 l of the clear supernatants were collected and peptides were finally analyzed by RPHPLC and LC-MS.
2.3) Results
2.3.1 in Cellulo VASH-Mediated Tubulin Detyrosination Assay
[0441] We used human CHL-1 cells in which tubulin detyrosination is catalyzed exclusively by VASHs. In these cells, detyrosination is predominantly VASH-dependent (Nieuwenhuis J. et al. Science, 2017, 358 (6369): 1453-1456). Cells were pre-treated with taxol before incubation with Epo-Y. Samples were immunoblotted using the indicated antibodies.
[0442] By using CHL-1 cells, we analyzed the use of Epo-Y to reduced taxol induced detyrosination. Cells were routinely cultured in a standard humidified tissue culture incubator at 37 C. in presence of 5% CO2 and plated in a 6-wells culture dish. The cells were treated for 2 hours with Taxol in absence or presence of Epo-Y.
[0443] The cells were collected in a RIPA buffer (of 50 mM Tris HCl, 150 mM NaCl, 1.0% (v/v) NP-40, 0.5% (w/v) Sodium Deoxycholate, 1.0 mM EDTA, at a pH of 7.4), and quantitation of total protein performed using BCA kit (Thermo Fisher Scientific). A 20 g protein sample of a total cell extract was run on 10% SDS-PAGE, transferred to nitrocellulose, and probed with each antibody. Western blot analysis showed a striking decrease of taxol treated (2 hours) and consequent tubulin detyrosination in CHL-1 cells. As illustrated in
[0444] So this in cellulo screening assay with CHL-1 cells and taxol is a relevant screening assay for identification of putative inhibitor of VASH-mediated tubulin detyrosination.
2.3.2 Putative Inhibitor Parthenolide does not Inhibit VASH-Mediated Tubulin Detyrosination
[0445] To study the inhibitory effects of parthenolide (PTL) on VASH-mediated tubulin detyrosination, we used the previously developed in vitro detyrosination assay disclosed in WO2020/012002. Reactions were incubated with inhibitor, stopped and analysed by immunoblotting with the indicated antibodies. As illustrated in
2.3.3 Newly Developed Inhibitors Target VASH1 and VASH2 and Peptidase Activity
[0446]
2.3.4 Cell Penetrant Potent Inhibitors of VASH Peptidase Activity
[0447] A small, highly reactive epoxide-based molecule such as Epo-Y might interact with free thiols present on proteins other than the VASHs or with nucleophilic lysine residues in the active sites of tyrosine kinases. Therefore, through iterative chemical variations and analysis with our standardized in vitro assay, we identified a compound, which we termed LV-43 (
2.3.5 Complete Inhibition of VASH-Mediated Tubulin Detyrosination in Cells
[0448] To test the LV-80 potency on the endogenous detyrosination activity, we compared wild type CHL-1 cells incubated with LV-80 to double knockout CHL-1 cells (2KOs) that lack both VASH1 and VASH2. Cells were collected and analysed by immunoblotting with the indicated antibodies. After 24-hour incubation with LV-80, the level of detyrosination was comparable in treated CHL-1 and CHL-1 2KOs cells (
2.3.6 Advantageous Safety Profile of VASH Enzyme Inhibitors
[0449] We next choose a widely used MTT-based colorimetric assay to assess the potential metabolic toxicity and cell viability alterations in the presence of LV-80. The MTT-based assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) was performed 48 hours after incubation with the indicated compounds to monitor cell viability.
[0450] Incubation for 24 hours with 0.1 M taxol and 10 M PTL resulted in a significant reduction of cell viability. Conversely, incubation with a much higher concentration of LV-80 (100 M) had no significant effect on cell viability, which was similar to that of cells incubated with vehicle (
2.3.7 Newly Developed VASH Inhibitors are Specific to VASH Enzymatic Activity
[0451] Next, we tested LV-80 inhibitory activity towards cytosolic carboxypeptidases (CCPs), another family of proteases that target tubulin C-terminal tails and act as tubulin deglutamylases. The inhibitor did not affect CCP1 and CCP5 activities. Samples were immunoblotted using the indicated antibodies. When polyglutamylated brain tubulin was incubated in the presence of protein lysates from HEK293 cells transfected with GFP-CCP1, we observed that CCP1-dependent deglutamylation activity towards long glutamate chains was not affected by addition of LV-80 (
2.3.8 Biotinylated Version of Newly Developed Inhibitor as Research Tool
[0452] Since detyrosination levels are particularly high in brain, we tested whether LV-80 fused to biotin could be used to pull down VASH1 from human brain extracts. Cells were pre-treated with taxol before incubation with biotinylated LV-80. Samples were immunoblotted using the indicated antibodies First, we confirmed that biotinylated LV-80 retained its inhibitory properties towards VASH1- and VASH2-mediated detyrosination in vitro (
2.3.9 Strong Reduction of Tubulin Detyrosination in Primary Cortical Neurons Treated with VASH Inhibitor
[0453] We decided to assess the functional role of tubulin detyrosination in differentiating primary cortical neurons purified from cerebral cortical tissue of mouse embryos. After dissociation and filtration, we differentiated cells in the presence or absence of LV-80 for 7 days. Cells were collected and analysed by immunoblotting with the indicated antibodies. Samples were analysed by immunoblotting and the relative optical density measured in three independent assays (n=3 and error bars represent SEM). P-values were calculated with the multiple t-test (n=3, *<0.05, **<0.01, ***<0.001, ****<0.0001). Analysis of the level of tubulin posttranslational modifications using specific antibodies (
2.3.10 Detyrosination Renders Tubulin Permissive to Glutamylation
[0454] Samples were analysed by immunoblotting and the relative optical density measured in three independent assays (n=3 and error bars represent SEM). P-values were calculated with the multiple t-test (n=3, *<0.05, **<0.01, ***<0.001, ****<0.0001).
[0455] Strikingly, while acetylation levels remained unaffected in treated neurons (
2.3.11 Tubulin Detyrosination Impacts Tau Protein Binding in Primary Cortical Neurons
[0456] To study the role of tubulin detyrosination in regulating the interaction of MTs with MT associated proteins (MAPs), we first completed differentiation of the cortical neurons for 5 days in absence of VASH inhibitor and followed by a treatment of 3 days. This approach allows for monitoring the cell distribution of tau protein independently of cell differentiation. Cells were collected and analysed by immunoblotting with the indicated antibodies. Incubation of cortical neurons with LV-80 for 3 days efficiently reduced tubulin detyrosination level and significantly increased tyrosinated tubulin level (
2.3.12. Newly Designed Low nM in Cellulo VASH Inhibitors
[0457] We designed a set of highly effective prodrugs efficient at low nM IC50 in cellulo (including examples as LV-104 and LV-111) and are cell penetrant, as illustrated in
[0458] The compounds were added prior Taxol-treatment and abolished VASH-mediated detyrosination in low nM ranges in CHL-1 cells. Quantification shows LV-104 and LV-111 (IC50 of 8 nM and 20 nM, respectively) are much more efficient on cells than LV-80 (IC50 of 500 nM). Two examples of prodrug compounds (LV-104 and LV-111) and a direct VASH inhibitor. Both prodrugs did not have any effect on VASH1 and VASH2 in the context of in vitro assays.
[0459] Considering all these results, we propose that targeting VASH peptidase activity is a safe and well tolerated mechanism for treating neurodegenerative diseases like Alzheimer's disease but also cancer, muscular dystrophy and ciliopathies. Interestingly these compounds have strong effect on tubulin glutamylation and may also be used for reducing TTLL-dependent modifications of tubulin in e.g. infantile onset neurodegeneration or glaucoma.
[0460] We showed that parthenolide, a molecule extensively used as an inhibitor of detyrosination, does not inhibit VASH activity neither in vitro nor in cellulo. This is in agreement with a recently published study, which demonstrated that the effect of parthenolide on detyrosination is indirect and most likely results from changes in microtubule dynamics caused by its covalent binding to tubulin (Hotta et al. Curr Biol. 2021, Volume 31, Issue 4, Pages 900-907). More importantly, we described medicinal chemistry-based optimization of the Epo-Y compound, which led to the development of a new highly specific and cell-penetrant VASH inhibitor. We showed that our newly developed compounds (active ingredients) have the ability to completely inhibit VASH activity and, in contrast to parthenolide, shows no detectable toxicity. Furthermore, by applying the VASH inhibitor to primary neuronal cultures, we have discovered previously unknown crosstalk between tubulin detyrosination and another important tubulin modification called polyglutamylation. The newly identified link between these two modifications has far-reaching consequences for the establishment of neuronal polarity and the localization of the main neuronal microtubule associated protein called tau. Overall these compounds hold strong potential for the use in pharmaceutical development and for research purposes.
2.3.13 In Vitro and in Cellulo Detyrosination Assay of IBMT11
[0461] The in vitro inhibition activity of IBMT11 is tested by adding the compound to the reaction medium as described above (see part 2.2) and then detyrosination was analysed by immunoblotting with the indicated antibodies.
[0462] While LV-104 did not inhibit VASH in vitro, we found that IBMT11 inhibited VASH1-mediated tubulin detyrosination more potently than Epo-Y, as indicated by their in vitro IC50 (0.6 M for IBMT-11 versus 10 M for Epo-Y) (
[0463] When tested in a cell-based assay involving taxol treated CHL-1 cells, IBMT11 was found to have a weak inhibition activity (
2.3.14. Conversion of LV104 into IBMT11 Inside Human Cells
[0464] The bioanalysis of the penetration and the conversion of the compound LV-104 has been achieved using the above described protocol on human cells (see part 2.2). The results are illustrated on
[0465] They confirm the total conversion of the prodrug LV-104 into IBMT11 inside human cells. Some conversion occurs in cell's culture medium (supernatant), however LV104 was undetectable inside cells (Cells). These data confirm our hypothesis according to which the presence of less polar groups, such as esters, in particular on the position corresponding to R.sup.3, increase significantly their cell penetration. Once inside the cell, the compound is hydrolysed thus converting esters to carboxylic acids for example and provides a potent VASH inhibitor.
[0466] Thus, IBMT11 is the corresponding drug of the prodrug LV-104. Indeed, groups corresponding to R.sup.3 and R.sup.6 are ethyl esters in LV-104 whereas they are carboxylic acid in IBMT11. That is why LV-104 cannot disclose an in vitro activity and IBMT-11 has a poor in cellulo activity; And, in the other way round, IBMT-11 is very efficient in vitro, while LV-104 is potent VASH inhibitor in cellulo.
2.3.15. In Vitro and in Cellulo Assays of Compounds According to the Invention
a) IBMT23
[0467] IBMT23 is found to be inactive when tested in an in vitro detyrosination assay (using the protocol as described above) (
b) IBMT34
[0468] IBMT34 is the sodium salt of LV80. It is found to have same activity in vitro and in cellulo as the corresponding base LV-80 but it possesses a dramatically increased solubility in water or PBS solution (see
c) IBMT28 and IBMT28hydro
[0469] IBMT28 is found to be inactive when tested in an in vitro detyrosination assay (using the protocol as described above) (
d) IBMT38hydro
[0470] IBMT38hydro has been tested in an in vitro detyrosination assay, as described above. It is found to have an in vitro activity as good as LV80 (see
2.3.16. Calculated in Vitro IC.SUB.50
[0471] The following compounds have been tested in in vitro detyrosination assays as described above and their IC.sub.50 have been calculated and compared to the IC.sub.50 of the compound LV-80. The column fold change corresponds to the ratio IC.sub.50(compound)/IC.sub.50(LV80) for each compound.
TABLE-US-00001 IC50 Fold change/ Compound Formula (m) LV-80 LV-37
2.3.17. Comparative Example with SD-139
##STR00118##
[0472] SD139 was synthesized as previously described by Aillaud, C. et al. Science, 2017, 358, p. 1448-1453.
[0473] SD139hydro was obtained after incubation with 150 mM NaOH for 5 min, then neutralization with 150 mM HCl.
[0474] SD-139 and SD-139hydro (also named SD-139sapo) were tested in in vitro detyrosination assays and compared to the activities of LV-80 and EPO-Y (see