WO2003014292A2 - Bibliotheque combinatoire de molecules bifonctionnelles - Google Patents

Bibliotheque combinatoire de molecules bifonctionnelles Download PDF

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WO2003014292A2
WO2003014292A2 PCT/IL2002/000642 IL0200642W WO03014292A2 WO 2003014292 A2 WO2003014292 A2 WO 2003014292A2 IL 0200642 W IL0200642 W IL 0200642W WO 03014292 A2 WO03014292 A2 WO 03014292A2
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dtc
moiety
molecule
combinatorial library
sod
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WO2003014292A3 (fr
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Jonathan Gressel
Ilana Rogachev
Ying Wang
Abraham Warshawsky
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Technion Research and Development Foundation Ltd
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Technion Research and Development Foundation Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C333/00—Derivatives of thiocarbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C333/14—Dithiocarbamic acids; Derivatives thereof
    • C07C333/16—Salts of dithiocarbamic acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

  • the present invention relates to chemical combinatorial libraries in general and to a combinatorial library of bifunctional molecules in particular.
  • bifunctional molecules usually describes two distinctly different chemical entities combined together.
  • An example for this type of bifunctionality are targeted molecules, where one entity selectively bines to a specific site while the other entity, e.g., a drug, has the desirable chemical or biological activity to be expressed at this particular site, h a typical library of bifunctional compounds, variation in one of the functional groups is meant to cause a chemical variation.
  • the present invention discloses a unique class of bifunctional compounds where two functions, different by nature are presented. The first is a specific chemical or biological activity which is kept unchanged, while the second has a sole physical function for tuning the physical properties of the molecule.
  • a general aspect of the present invention is a combinatorial library comprising of a plurality of bifunctional molecules wherein each member of the library comprises a first moiety being of a specific chemical function and a second moiety being of specific physical properties.
  • members in the library differ from each other by variations either in the first moiety, in the second moiety or in both. Variations among members cause changes in two vectors.
  • the chemical function is fixed, the physical function may be changed and vice versa, i.e., when the physical function is fixed the chemical function may by changed.
  • the second moiety optionally further imparts the molecule a second chemical function.
  • Another aspect of the present invention is a combinatorial library comprising of a plurality of bifunctional molecules of the structure CF - PF wherein, CF is a moiety of a first specific chemical or biological activity; and PF is a second moiety imparting the molecule certain physical properties and wherein structural modifications in the second moiety vary the physical properties of the bifunctional molecule without significantly modifying the first chemical or biological activity for obtaining optimized effect of the first chemical or biological activity at the environment where the chemical or biological activity is to be expressed.
  • the varied physical properties are preferably one or combination of the following: lipophilic solubility, hydrophilic solubility, lipophilic- hydrophilic partition, surface activity, transport properties and hydrolysis rate.
  • the first moiety can be any bioactive or agroactive group such as a drug, a cosmetic, a peptide, a hormone, a UV responsive molecule, a light responsive molecule, an ultrasound responsive molecule, a microwave responsive molecule, an NMR or EPR responsive molecules, an olfactory molecule, a taste-responsive molecule, an oligonucleotide, a nucleic acid, a protein etc.
  • the second moiety can be an amphiphilic group, more preferably an oligoether group.
  • the second moiety further imparts the molecule a second specific chemical or biological activity which is significantly unchanged when the physical properties of the bifunctional molecule are varied.
  • the first moiety is a metal chelator, more preferably the first moiety is a disubstituted dithiocarbamic acid or a salt thereof and the second moiety is an amphiphilic group, more preferably an oligoether group.
  • R 1 and R 2 are independently a straight or branched, substituted or known substituted alkyl of 1 to 20 carbons, more preferably R 1 is selected from the group consisting of ethyl and butyl and R 2 is an alkyl of more than 6 carbons, most preferably
  • R 2 is selected from the group consisting of hexyl, octyl, decyl and dodecyl
  • the second moiety is a herbicide molecule, more preferably a paraquat molecule and the first moiety is disubstiruted dithiocarbamic acid or a salt thereof.
  • a third aspect of the present invention is a bifunctional molecule comprising a first moiety and a second moiety, the first moiety imparts said molecule a first specific chemical activity and the second moiety imparts the molecule specific physical properties and optionally a second specific chemical activity, wherein structural modifications of said second moiety tune the physical properties of said bifunctional molecule without significantly modifying said first and optionally second chemical activities for obtaining optimized effect of said first and optionally second chemical activities at the environment where said chemical activities are to be expressed.
  • Figure 3 Formation of Cu(DTC) 2 complex upon addition of Cu ions to the Fe(DTC) 3 complex.
  • Spectra Fe(DTC) 3 (0.5 mM in 75% EtOH) - thin line; Cu(DTC) 2 (0.1 mM in 75% EtOH) - dotted line; the final mixture, containing both Cu(DTC) 2 and Fe(DTC) 3 complexes - thick line.
  • FIG. 5 Inhibition of the dismutation activity of CuZnSOD by amphiphilic dithiocarbamates (DTCs).
  • DTCs amphiphilic dithiocarbamates
  • Figure 7 Inhibition of peroxides activity of SOD by different DTCs.
  • FIG 11 Full substitution of calcein from the calcein-copper complex by DTC.
  • Figure 12 Scheme 3 is a schematic description of the experiment on the influence of
  • Figure 13 Detection of both calcein and calcein-copper complex in the inner volume of vesicles after the second gel filtration.
  • the fluorescent spectra of the liposomes before and after treatment with DTC, after the second gel filtration and after Triton X-100 and EDTA addition are shown.
  • the fluorescent signals 1 - after the first gel filtration; 2 - after the treatment with less than stoichiometric amount of DTC; 3 - after the second gel filtration (sample was diluted on the column); 4 - after the addition of EDTA and Triton X-100. Numbers on the fluorescent spectra correspond to the numbers of the steps of the experiment, presented in the Scheme 3
  • Figure 16 Scheme 5 general formula for inner salt structure
  • Figure 17 Scheme 6 structural formula of the monosubstituted MPQ-DTC (compound 7) is an inner salt
  • FIG. 18 UV absorption spectra of PQ-DTC, the precursor of PQ-DTC — compound 5, Et 2 DTC and the spectrum difference between PQ-DTC and compound 5.
  • Figure 19 Chelation of copper by PQ-DTC. Changes in the spectrum of 0.1 mM
  • PQ-DTC solution upon addition of different amounts of Cu ions (as CuSO 4 solution), formation of PQ-DTC band at 435 nm.
  • Figure 23 Inhibition of the dismutation activity of CuZnSOD by Et DTC, PQ-DTC and compound 5.
  • Figure 24 The weak herbicidal activity of PQ-DTC in vivo system.
  • bifunctional describes two distinctly different chemical entities.
  • variation in one of the functional groups is meant to cause a chemical variation, h the present case, we have two functions, different by nature. The first is a specific chemical or biological activity which is unchanged and has the sole chemical function of binding metal ions). The second has a sole physical function of trying to change the physical properties of the molecule.
  • CF was chosen to be a metal chelator of the dithiocarbamic acid (DTC) class, of known selectivity to Cu( ⁇ ) ions.
  • DTC dithiocarbamic acid
  • the PF was chosen as an oligoether group. Such a group, because it is an amphiphile, allows the change of solubility, surface activity, and transport properties without affecting other chemical properties, such as the property of the CF group.
  • the first library (part A in the following) comprises a plurality of metal chelator - amphiphile bifunctional molecules, more precisely of disubstituted diethyldithiocarbamat (DTC) - oligoeyher, wherem the chemical function (CF) is the chemical ability to bind specific metal ions.
  • the physical function (PF) is introduced in the form of an amphiphile which allows changing the physical properties of the molecule, in this case the micellar properties such as surface tension.
  • the second library (part II in the following) is of a herbicide - metal chelator bifunctional molecules, more precisely of paraquat - DTC. In this case the amphiphile group of the first library is replaced by paraquat, which is not only an amphophile but in addition imparts the bifunctional molecules a second chemical activity of the herbicide type.
  • amphiphile physical function can be combined with a chemical function (CF) of any bioactive group or agroactive group such as a drug, a cosmetic, a peptide, a hormone, UV or light responsive molecules, an ultrasound responsive molecule, a microwave responsive molecule, an NMR or EPR responsive molecules, an olfactory molecule a taste-responsive molecule, a oligonucleotide, a nucleic acid, a protein, etc.
  • Said groups can be defined as Interactive Functions (IF) having affinity interaction or long term interaction, such as host-guest interaction or agonist-antagonist interaction, with a specific site or molecule in the media where said molecules are to be expressed.
  • IF Interactive Functions
  • IF-amphiphile libraries By constructing such IF-amphiphile libraries, it will be possible to monitor the physical properties of large sections of bioactive molecules of great importance in human and animal care and in agricultural applications. Furthermore, instead of with amphophilic moiety, said groups can be combined with a moiety having a controlled hydrolysis, and in particular a DTC (see Table 3 below) in applications such as drug delivery, where said moiety of controlled hydrolysis serves for allowing slow and/or controlled release of the other moiety.
  • a controlled hydrolysis and in particular a DTC (see Table 3 below) in applications such as drug delivery, where said moiety of controlled hydrolysis serves for allowing slow and/or controlled release of the other moiety.
  • Oxidative stress resulting from the excessive production of reactive oxygen species (ROS) is an important phenomenon in many biological systems. 1 Aerobic organisms had to evolve an antioxidant defense system to exist. This system may be comprised of (a) low molecular weight compounds that scavenge oxidants and/or (b)
  • SOD Superoxide dismutase
  • herbicides This concept of combining herbicides and SOD inhibitors to reduce the load of herbicide use is the basis for the present work.
  • One way to inhibit CuZnSOD is to inactivate its copper cofactor with copper- specific chelators.
  • combinatorial libraries of bifunctional copper- specific metal chelators are proposed as inhibitors of superoxide dismutase and, hence, as augmentors (or synergists, as we propose to call them) to the action of herbicides.
  • Chelators such as diethyldithiocarbamate can remove copper from superoxide dismutase, inhibiting the
  • Part A describes a limited "sublibrary” comprising bifunctional metal chelators wherein the first chemical function is the metal chelator
  • DTCs disubstituted dithiocarbamates
  • R 1 hexyl, octyl, decyl or dodecyl
  • R 1 Et or Bu
  • the influence of DTCs on SOD superoxide dismutation activity was measured by the cytochrome C/xanthine/xanthine oxidase assay.
  • the SOD dismutation activity was recovered after incubation of inactivated SOD with copper.
  • Inhibition of peroxidase activity of SOD by different DTCs was determined using EPR spectra of the DMPO-'OH adduct formed in solutions containing H 2 O 2 and CuZnSOD and in the presence of the spin trap compound.
  • Part A also describes the preparation of phospholipid vesicles incorporating copper ion and a fluorescent chelator, as well as the use of a sensitive fluorescent method for determining the rate of transport of the metal chelating amphiphiles into the liposomes.
  • Structural attenuation leads to a balance in desired properties.
  • Library members with decyl and dodecyl groups are poor inhibitors of SOD dismutase activity.
  • Amphiphilic dithiocarbamates (hexyl and octyl substituted) reduce the peroxidase activity of SOD.
  • N-Alkyl-N-(2-(2-ethoxyethoxy)ethyl)dithiocarbamates or N-Alkyl-N-(2-(2- ethoxyethoxy)butyl)dithiocarbamates were synthesized according to the Scheme shown in Scheme 2 in Fig. 1.
  • Synthesis of glycol(alkyl)dithiocarbamates can be conventionally divided into two steps: step one is the synthesis of secondary amines; step two is the synthesis of dithiocarbamates.
  • 2,3-dioxypropylchloride with 8-10 times excess of alkylamine was adapted for the synthesis of alkyl(ethoxyethoxyalkyl)amines.
  • Relatively pure secondary amines in good yields were prepared from ethoxyethoxyethylchloride or ethoxyethoxybutylchloride using 10-fold excess of primary amines.
  • the main problem in this alkylation step was the formation of aminehydrochlorides, which hampered the distillation process, blocking the distillation apparatus and reducing the yield of secondary amines. This problem appeared only during the synthesis of more lipophilic amines with the long alkyl chains.
  • the synthesis of sodium dithiocarbamates 18 is performed by reaction of a secondary amine with CS 2 in the presence of ⁇ aOH.
  • Et-Oct-DTC and Bu-Oct-DTC were synthesized from the corresponding amines by the reaction with ⁇ aOH and carbon disulfide in water. These compounds were then purified by washing with cooled diethyl ether. More lipophilic dithiocarbamates (Bu-Dec-DTC and Bu-Dodec-DTC) were prepared from the reaction of the corresponding secondary amines in tetrahydrofuran with sodium hydride and an excess of carbon disulfide.
  • the increasing lipophilicity of the dithiocarbamic salts due to the increasing size of the aliphatic substituents on nitrogen, produced problems in the process of purification of the DTCs after synthesis. More lipophilic dithiocarbamates were less soluble in water and had a good solubility in Et 2 O. Therefore, these compounds were purified by several consecutive recrystallizations from cooled hexane solutions. All dithiocarbamates were characterized by H ⁇ MR. The dithiocarbamate salts were converted to the corresponding methyl esters by alkylation with methyl iodide prior to analytical characterization by TLC, NMR, mass spectra or elemental analysis.
  • the partition coefficient (P ow ) and the distribution ratio (D ow ) between octanol and water are the most commonly used expressions for the hydrophobicities of
  • the degree of octanol/water partitioning is useful in predicting the in vivo transport properties of any compound through hydrophobic barriers (cell membranes skin or leaf cuticles).
  • the hydrolytic stabilities of the various dithiocarbamic acids were determined to address the first possibility that of a catalytic enhancement effect.
  • the time of "half maximal" decrease of UN absorbance of dithiocarbamic acids was taken as the time of 50% decomposition of these compounds (t 50 ). Data on decomposition of dithiocarbamic acids are presented in Table 3.
  • the CS 2 spectrum overlaps with the absorbance spectrum of the dithiocarbamic ligand, rendering the UN absorbance measurements imprecise.
  • Another method e.g., iodometric titration
  • Table 3 show that chemical instability increases for long aliphatic substituents.
  • the rates of decomposition of amphiphilic dithiocarbamates are positively correlated with the lipophilicity of the compounds.
  • Et-Oct-DTC The stability of Et-Oct-DTC is independent of the cation present in the dithiocarbamic salt. Thus, a "crown ether effect" may not be ruled out for the DTCs with the short-chain oligoglycol substituent (up to two CH 2 CH 2 O groups) and requires synthesis of longer-chain oligoether substituents.
  • the comparative stability constants of Et-Et-DTC, Et-Bu-DTC, Et-Hex-DTC, and Bu 2 DTC were determined spectrophotometrically by the competition method described by Janssen. 23 ' 2 This method is based on the competition of dithiocarbamate ligands with 8-hydroxyquinoline ligands for copper. Its application to amphiphilic DTCs is described in detail in literature. 25
  • the competitional (Kc) values presented in this article show the relative competitive stabilities of Cu(DTC) 2 complexes over Cu(hydroxyquinoline) 2 (Table 5). The higher the K c values are, the stronger the copper- dithiocarbamate complex is.
  • the overall stability constant of Cu(DTC) 2 complex ⁇ 2 is equal to K c x ⁇ ' 2 , where ⁇ ' 2 is the stability constant of Cu(hydroxyquinoline) 2 complex.
  • ⁇ ' 2 (determined in the same solvent as K c ) is required for obtaining an overall stability constant of Cu(DTC) 2 .
  • Diethyldithiocarbamate has higher selectivity for copper over iron.
  • the comparative stabilities of Cu(Et-Hex-DTC) 2 complex over Fe(Et-Hex-DTC) 3 complex were determined to prove that the selectivity for copper over iron, typical to diethyldithiocarbamate, also holds true for all the amphiphilic DTCs in this work.
  • Fe(DTC) 3 complex was prepared, and the ability of Cu ion to substitute
  • Fe exchange (with the composition: Cu(DTC) 2 (0.1 mM), Fe(DTC) 3 (0.43 mM), and
  • Cu(DTC) 2 complexes are stable at acidic pH, as ascertained in separate experiments
  • the DTC/SOD molar ratio leading to 50% decrease of SOD activity during preincubation ranges from 3.4 for Et 2 DTC to 7.9 for Et-Oct-DTC and Bu-Oct-DTC.
  • the catalytic activity of SOD is pH independent in the range pH 5 to 9 and decreases at more alkaline values.
  • 32 We used alkaline solutions (pH 10.2) for preincubation of the dithiocarbamates with SOD to reduce the decomposition of DTCs. pH 7.8 was used for measuring the SOD activity. The high pH could lead to some decrease of SOD activity. It was checked and found that this decrease of activity is mainly reversible by lowering the pH. 32 Nevertheless, to correct for the influence of the changes in pH, all measured SOD activity after incubation with DTCs was normalized against the activity of native SOD kept at pH 10 buffer for the same 2.5 hour preincubation period.
  • DMPO DMPO- * OH adduct from the * OH generated by CuZnSOD and H 2 O 2 .
  • 33 ' 34 Superoxide dismutase was preincubated in Et 2 DTC or with amphiphilic dithiocarbamates for 2.5 hours at a 25:1 [DTC]/[SOD] molar ratio. This excessive amount of the chelators almost completely inhibited the SOD dismutation activity (see Figure 5).
  • the generation of DMPO-'OH was also drastically decreased by preincubation of superoxide dismutase with Et 2 DTC, Et-Hex-DTC, and Et- Oct-DTC ( Figure 7).
  • Bu-Oct-DTC was less effective than other dithiocarbamates, for reasons not clear at this time.
  • the Cu(DTC) 2 complexes did not produce paramagnetic DMPO adducts in the presence of H 2 O 2 (data not shown), implying that no interfering signals from the Cu- DTC complexes were formed during the experiment.
  • CMCs critical micellar concentration
  • the ability of free DTC ligands and their DTC-copper complexes to cause leakage in liposomes was studied and compared with the well-known diethyldithiocarbamate.
  • the liposomes were encapsulated with calcein at a self-quenching concentration, 41 and the fluorescence emanating upon addition of varying concentrations of DTCs was measured.
  • the test is based on the fact that the fluorescence of polar-fluorescent dyes (e.g., calcein) is quenched at high concentrations.
  • polar-fluorescent dyes e.g., calcein
  • Various processes resulting in a dilution of the dyes produce an increase in the fluorescence, thereby providing a facile method for monitoring these events.
  • Calcein entrapped in lipid vesicles at high concentrations displays very low fluorescence intensity because of self-quenching. If the liposomes lose integrity, the probe is released from the vesicles.
  • the local concentration of calcein decreases and the amplitude of the fluorescent signal increases due to the dilution of calcein in the bulk-surrounding phase.
  • Egg lecithin/cholesterol small unilamellar liposomes were prepared, containing calcein-copper complex in the internal volume. Compounds traverse the liposome membranes and substitute the copper ions from the calcein-copper complex inside the liposomes. When less than a stoichiometric amount of DTC ligand is used, the reaction of the ligand exchange inside the liposomes proceeds only partially. The vesicles then contain three components: DTC-copper complex formed; calcein-copper complex remained; and some free calcein was displaced by DTC from its copper complex.
  • the liposomes are returned to the gel filtration column (the second gel filtration procedure) to separate them from any released calcein or calcein-copper that would react with EDTA.
  • the DTC-copper complex is lipophilic enough to penetrate through the bilayer to the external solution. If this penetration process does not cause leakage (or the penetration does not happen) and the vesicles remain intact ⁇ two things happen.
  • the free calcein forms, and the calcein-copper complex remains in the internal volume of the liposomes after the second gel filtration (both calcein and calcein-copper do not traverse the liposome bilayer).
  • Scheme 3 in Fig. 2 shows the schematic representation of this experiment.
  • the presence of free calcein after the second gel filtration is shown in Figure 13 (spectrum 3 shows higher fluorescence than the spectrum 1). Nevertheless, spectrum 3 lies lower than spectrum 2 because the liposome sample was 1.7 times diluted on the column.
  • Figure 13 also demonstrates the presence of calcein-copper in the inner volume of liposomes after the second gel filtration.
  • the fluorescent signal increased as the result of the appearance of free calcein, due to the reaction of calcein-copper with EDTA (EDTA plays the same role as DTC, complexing
  • the crossing of a liposomal bilayer reduces the initial rates of the ligand exchange reaction only by a factor of 1.4-1.6 (Table 7).
  • the measured rates consist of the addition of two steps: the penetration of the DTC through the membrane bilayer and the following ligand exchange reaction inside the liposomes.
  • the close numbers for the penetration rates for the measured DTCs suggests that the liposome bilayer is an easy barrier for the dithiocarbamate ligands and that the ligand exchange reaction is the limiting step.
  • the stability experiments showed that neither amphiphilic DTC nor their copper complexes cause leakage of the phospholipid bilayer membranes.
  • Liposomes can accommodate charged molecules, sometimes to a large extent. 39
  • the difference between the two models of biological membranes is exemplified by the distribution of ionized pentachlorophenol, which is several hundred times greater for egg-PC membranes compared with octanol. 38 Conclusive Summary
  • a 50 are DTC/SOD molar ratios leading to 50% decrease of SOD activity after a 2.5 hours of preincubation of SOD with DTCs.
  • a 50 are DTC/SOD molar ratios leading to 50% decrease of SOD activity after a 2.5 hours of preincubation of SOD with DTCs.
  • D t 50 is the time of 50% decomposition of DTCs at pH 10.
  • Bu-Oct-DTC show similar rates of Cu( ⁇ ) abstraction from the enzyme, and the most bulky chelator (Bu-Dodec-DTC) is less active in Cu(lT) removal. Similarly, these three chelators inhibit the dismutation activity of SOD, and the more bulky DTCs (Bu-Dec-DTC and Bu-Dodec-DTC) are much less effective, even at
  • Phase A presents the effects concerning the transport of the DTCs.
  • the transport is affected by (a) rate of hydrolytic decomposition of the DTCs, which is a function of their structure and pH (b) rate of transport via the cell membrane and (c) solubilization in the membrane, as indicated by D ow -
  • Phase B presents the events taking place following the transport step, inside the cell, with a very complex equilibrium among the ligand DTC, the transition metal ions, Cu( ⁇ ) and Fe(flj, and the enzyme SOD (other metaloenzymes are ignored at this time). More complications arise from the side reactions, such as Fenton reaction, in which H 2 O 2 is producing OH * radicals. As known, the Fenton reaction is effected by the DTCs.
  • Phase C discusses the aspects of enzyme inhibition. The conclusion is that the less bulky DTCs totally inhibit the enzyme. The effective inhibition of SOD by Et 2 DTC, Et-Hex-DTC, Et-Oct-DTC, and Bu-Oct-DTC required at least 10-50 molar excess of these compounds. Then the DTCs are free to interact with free Fe(fl) in the cell and produce OH * radicals. In this way, they should synergies the herbicide. Finally, at Phase D (infinitesimal time too varying from one DTC to another) the DTCs are fully decomposed, and a system D, free of DTC, is restored.
  • thionylchloride E. Merck, for synthesisoctylamine (Fluka, AG); decylamine (Fluka, AG); dodecylamine (Fluka, AG); dibutylamine (Fluka, AG); potassium hydroxide (E. Merck, GR, for analysis); sodium hydroxide (E. Merck, GR, for analysis); sodium hydroxide volumetric standard (1.018 N solution in water, Aldrich); sodium carbonate (E.
  • Proton NMR spectra were measured on a Bruker WH-270, a Bruker DPX-250, or a Bruker AMX-400 NMR spectrometer. Either all chemical shifts are reported in ⁇ units downfield from tetramethylsilane as an internal standard, or the H 2 O signal was used as a reference. The following abbreviations are used: s-singlet, d-doublet, t-triplet, q- quartet, and m-multiple. Deuterated CDC1 3 (Aldrich) and D 2 O (E. Merck) used for NMR were of 99.8% isotope purity.
  • Flash column chromatography separations were performed on silica gel Merck 60 (230-400 mesh ASTM). UV/VIS spectra were measured on a Hewlett-Packard 8450A diode array spectrophotometer. TLC was performed on E. Merck Kieselgel 60 F254 plates. Staining of TLC plates was done by (a) basic aqueous 1% KMnO 4 and (b) 0.3% ninhydrin in EtOHabs- Tetrahydrofuran was distilled under LiAlH4 and passed through an Al 2 O 3 column. High Resolution mass spectra (Dl, EI- MS) were measured on PGS-70B Finnigan-Mat instrument at the Chemical Faculty of the Israel Institute of Technology (Technion), Haifa. Syntheses and analyses
  • Dibutyl dithiocarbamic acid was synthesized from dibutylamine in water according to reference. 19 Synthesis of 2-(2-ethoxyethoxy)ethyl chloride was described in literature. 47 2-(2-Ethoxyethoxy)butyl chloride
  • n-Octyl (2-(2-ethoxyethoxy)butyl)amine n-Octylamine (65 ml, 50.6 g, 0.39 mole, 10 times excess) was placed in a 250 ml three-necked flask, fitted with a magnetic stirrer, dropping funnel, and reflux condenser with the nitrogen outlet.
  • 2-(2-Ethoxyethoxy)butyl chloride (7.08 g, 39 mmole) was added dropwise at room temperature. The reaction mixture was heated in an oil bath at
  • n-Octyl (2-(2-ethoxyethoxy)ethyl)amine Synthesis was as described for «-octyl (2-(2-ethoxyethoxy)butyl)amine, except that 2-(2-ethoxyethoxy)ethyl chloride was used instead of 2-(2-ethoxyethoxy)butyl chloride.
  • TLC Rf - 0.82 (CHC1 3 : MeOH : NH 3 (25% aq) 9 : 1 : 0.1 staining with ninhydrin).
  • Me 4 Si 0.85-0.94 (6H, m, two CH 3 CH 2 ), 1.2-1.4 (16H, m, CH 3 (CH 2 ) 7 and C ⁇ 3 CH 2 C ⁇ 2 C ⁇ 2 O), 1.4-1.63 (4H, m, CH 2 CH 2 CH 2 N and CH 3 CH 2 CH 2 CH 2 O), 1.79 (IH, br s, NH), 2.59 (2H, t, CH 2 CH 2 CH 2 N), 2.78 (2 ⁇ , t, OCH 2 CH 2 N), 3.46 (2 ⁇ , t,
  • n-Octyl (2-(2-ethoxyethoxy)butyl)dithiocarbamic acid, sodium salt (compound Bu-Oct- DTC) Synthesis was as described for compound Et-Oct-DTC, except that w-octyl (2-(2- ethoxyethoxy)butyl)amine was used instead of w-octyl (2-(2-ethoxyethoxy)ethyl)amine.
  • n-Dodecyl (2-(2-ethoxyethoxy)butyl)dithiocarbamic acid, sodium salt (compound Bu- Dodec-DTC) n-Dodecyl (2-(2-ethoxyethoxy)butyl)amine (2.93 g, 8.89 mmole), 20 ml of THF and 80% NaH (0.259 g, 8.63 mmole of pure compound) were placed in a 50 ml round- bottom flask, equipped with a magnetic stirrer and ice bath, and connected to the Ar system. CS 2 (1.5 ml, 24.9 mmole) was added to the rapidly stirred, cooled mixture.
  • n-Octyl (2-(2-ethoxyethoxy)butyl)dithiocarbamic acid, methyl ester n-Octyl (2-(2-ethoxyethoxy)butyl)amine sodium salt (0.292 g, 0.79 mmole) and 5 ml of absolute ethanol were placed in a 25 ml round-bottom flask equipped with a magnetic stirrer and an ice bath. C ⁇ 3 I (49 ⁇ l, 0.79 mmole) was added to the rapidly stirred cooled mixture. The ice bath was then removed, and the mixture was stirred overnight at room temperature. The ethanol was removed under vacuum.
  • H ⁇ MR ⁇ (CHC1 3 , 400 MHz; Me 4 Si): 4.2-3.8 (6H, m, OCH 2 CH 2 ⁇ CH 2 ); 3.61-3.56 (4 ⁇ , m, OCH 2 CH 2 0); 3.46 (2H, t, CH 3 CH 2 CH 2 CH 2 O); 2.63 (3 ⁇ , s, SCH 3 ); 1.71 (2 ⁇ , br m, CH 2 CH 2 CH 2 N); 1.59-1.55 (2H, m, CH 3 CH 2 CH 2 CH 2 O); 1.39-1.35 (2H, m, CH 3 CH 2 CH 2 O); 1.31 (10H, m, CH 3 (CH 2 ) 5 ); 0.94-0.86 (6 ⁇ , m, CH 3 CH 2 CH 2 CH 2 O and GH 3 (CH 2 ) 5 ).
  • Synthesis was as described for methyl ester of n-octyl (2-(2- ethoxyethoxy)butyl)dithiocarbamic acid, except that the sodium salt of n-octyl (2-(2- ethoxyethoxy)ethyl)amine was used instead of the sodium salt of n-octyl (2-(2- ethoxyethoxy)butyl)amine, and the preparative TLC was made with chloroform.
  • Iron (m) sulfate pentahydrate, copper (II) sulfate pentahydrate, and iron (III) chloride hexahydrate were purchased from Aldrich. Analytical ethanol (Frutarom) was used.
  • the superoxide dismutation activity of SOD was measured by monitoring the inhibition of the reduction of ferricytochrome C by superoxide generated by a xanthine/xanthine oxidase reaction, as described in (27). All DTCs were preincubated with 1 mM SOD at pH 10.2 for 2.5 hours and room temperature in 50 mM carbonate buffer (pH 10.2) at molar ratios of DTC/SOD 2:1 to 50:1. Enzyme-dithiocarbamate solutions were diluted after the preincubation with 50 mM potassium phosphate buffer (pH 7.8). The SOD activity reaction mixture contained 3 nM enzyme. SOD, preincubated for 2.5 hours at pH 10.2 without a DTC, was used as a control.
  • the copper-deficient SOD was reconstituted to an active form by the addition of
  • the Cu-DTC complexes were first formed by incubation of 1.5 mM SOD with 10 mM DTC in buffer (pH 10.2) for 24 hours. The complexes were then 10-fold diluted with the same buffer and sedimented from the protein by centrifugation at 39,000xg- for
  • Cu(Et 2 DTC) 2 and Cu(Et-Hex-DTC) 2 (10 ⁇ M) in 10% DMSO/phosphate buffer (pH 7.8) with 12.5 times excess of dithiocarbamate ligands were prepared as follows: CuSO 4 (20 ⁇ l of 10 mM), 2 ml of DMSO, and then 50 ⁇ l of 50 mM solution of dithiocarbamate ligand in buffer (pH 10.2) were put into the 20 ml volumetric flask. Volume was adjusted with 50 mM phosphate buffer (pH 7.8).
  • the peroxidase activity of superoxide dismutase refers to the "bound" hydroxyl radical: * OH produced by SOD and H 2 O 2 remains bound to the copper atom at the active site.
  • the peroxidase activity was measured by spin-trapping technique and electron paramagnetic resonance (EPR) spectroscopy. ' The spin-trapping technique converts transient free radicals to stable free radicals. A relatively long-lived spin adduct
  • DMPO/'OH is fo ⁇ ried from SOD, hydrogen peroxide, and a diamagnetic spin trap DMPO.
  • the identity of the free radical was determined on the basis of hyperfine coupling (hfc) constants of the spin adduct by EPR spectroscopy.
  • EPR solutions for measurements of the influence of Cu(DTC) 2 complexes on the peroxidase activity of SOD in the presence of H 2 O 2 contained 45 mM DMPO, 5 ⁇ M Cu(DTC) 2j and 40 mM H 2 O 2 in 50 mM phosphate buffer (pH 7.8).
  • the solutions of SOD inactivated by DTCs contained 45 mM DMPO, 2.5 ⁇ M SOD (and 62.5 ⁇ M DTC for spectra B, C, D and E ( Figure 7) and 30 mM H 2 O 2 in 50 mM phosphate buffer at pH 7.8.
  • the EPR signals were measured in 100- ⁇ l flat cell at room temperature.
  • Spectral acquisition began 3 minutes after initiation of the reaction by the addition of hydrogen peroxide.
  • the spectrometer settings were as follows: receiver gain, 4xl0 5 ; modulation amplitude, 1 G; time constant, 1.25 sec; sweep time, 200 sec; center field, 3500 G; sweep width, 100 G; microwave power, 20 mW.
  • Regression curves were constructed on the basis of average values (the same values that were used for the construction of the curves: SOD activity (% of control) versus [DTC]/[SOD] molar ratio, Figure6), as the duplicates were dependent values.
  • the residual plots showed that the underlying assumptions for the regressions were met, and therefore no further transformations were needed. 95% confidence intervals were calculated for the slopes, and overlapping of these confidence intervals means that the corresponding slopes are not significantly different.
  • CMCs of amphiphilic dithiocarbamates were determined from the Wilhelmy plate surface tension measurements.
  • the Wilhemy plate experiments were performed with a KrDss digital instrument K10T using small volume (15 ml) glass measurement cells with a glass side arm incorporated in their walls.
  • the surface of the buffer solution was swept with blotting silk paper and its surface tension value ( ⁇ o) was recorded. Then, keeping in contact the roughened platinum Wilhemy plate in contact with the buffer solution, a DTC solution was injected into the buffer subphase through the side arm of the measurement cell.
  • Calcein-copper complex (Cal-Cu) was prepared as follows: stock Cu solution
  • the resulting calcein-Cu 2+ complex solution contained 60 ⁇ M Cal-Cu and 5 ⁇ M of free Cu ions (totally 65 ⁇ M of
  • Small unilamellar vesicles were prepared by sonication of multilamellar vesicles using a probe sonicator. The following procedure was used (100): egg phosphatidylcholine (PC) and cholesterol (in 1:1 molar ratio) was dissolved in chloroform/methanol mixture (2:1 v/v) to obtain 10 mg/ml solution of phospholipid. The solvents were then evaporated under a stream of nitrogen to yield a dry thin lipid film on the wall of a glass vial (5 mg lipid / 2 cm vial). Then, the film was dried under high vacuum for 2-3 hours.
  • PC egg phosphatidylcholine
  • cholesterol in 1:1 molar ratio
  • SUV containing calcein-copper complex were prepared as follows: a solution of calcein-copper complex (60 ⁇ M in tris-saline buffer, 1 ml) were added to the vial with the thin film of lipid to obtain 5 mg/ml PC solution, and the mixture was shaken on a vortex for 10 minutes. To form unilamellar liposomes, the multilamellar vesicles thus formed were sonicated until the solution was transparent.
  • SUV containing the self-quenching concentration of calcein were prepared the same way as the SUV containing calcein-copper complex, but the stock solution of calcein (60 mM) was added instead of calcein-copper.
  • a fluorescent technique 5 was used for checking the leakage and fusion of the liposomes in the presence of DTC.
  • the liposomes were encapsulated with calcein at self-quenching concentration (60 mM in tris-saline buffer) to obtain 5 mg/ml PC.
  • the fraction of SUV after gel filtration (fraction with the higher absorbance at 340 nm) was then diluted more than 100 times to keep absorbance at 496 nm not higher than 0.2.
  • the fluorescence of this liposome solution and the fluorescence upon addition of varying concentrations of DTCs was measured (excitation wavelength was 495 nm and emission wavelength - 515 nm).
  • Triton X-100 (final concentration 0.5%) was added at the end of each experiment to destroy the vesicles and to get the full value of fluorescence . . b) Leakage caused by the formation of DTC-Cu complex Calcein-copper containing liposomes collected after the gel filtration column were first checked for their integrity (a calcein-Co - EDTA assay for monitoring vesicle fusion was used as a basis). EDTA ligand liberates calcein from calcein-copper complex and does not penetrate the liposome bilayer.
  • EDTA solution 60 mM final concentration was added to the liposomes after the gel filtration with subsequent breakage of liposomes with Triton X-100 (0.5%) to determine the assay end-point.
  • DTC (0.3 ⁇ M final concentration) was added to the Sephadex purified liposomes and after 5 minutes the fluorescence was measured (excitation 484 nm, emission range 500-550 nm). Then, the sample was returned to the same gel filtration column (second gel filtration). EDTA (67 mM final concentration) and Triton X-100 (0.3 % final concentration) were added to the liposomes after the second gel filtration, and after 5 minutes the fluorescence was measured. Rates of penetration through liposome bilayer
  • the fluorescence was measured under constant mixing. The measurements were monitored each 0.5 seconds for a few seconds before adding DTC (baseline) and until 6 minutes after adding it to the vesicle mixture (2 mM final concentration of DTC). Triton X-100 (0.1% final concentration) was added at the end of experiments containing liposomes to break the vesicles and get the full value of fluorescence.
  • Diquaternary salts of 2,2' and 4,4'-b ⁇ py ⁇ dyls are well known herbicides.
  • the mode of action of bipyridylium herbicides, 49"51 is related to one-electron metabolic reduction of paraquat (PQ 2+ ) by replacing naturally occurring ferridoxin to form the corresponding radical PQ ' and instantaneous reoxidation to produce very reactive oxygen species (ROS).
  • PQ 2+ paraquat
  • ROS very reactive oxygen species
  • the reduction is due to photoinduced electron transport from chlorophyll through the photosystems and it is generally believed that the phyto toxic activity of paraquat is due to membrane hpoxidation , causing membrane structure disintegration with water loss, leading to wilting.
  • paraquat exerts its phytotoxic effect by accepting (iron-sulfur centers, replacing ferredoxin of photosystem I (PSI). 54
  • the electrons are transferced to oxygen (O 2 ) to produce different and very reactive toxic oxygen species, such as superoxide radical (O 2 " ), hydrogen peroxide (H 2 O ), and hydroxyl radical (OH).
  • Transition metals such as iron and copper, are able to accelerate the conversion of H O 2 to hydroxyl radical (OH), 83, 84 ' 65 ⁇ and hence transition-metal specific chelators especially iron chelators can reduce or prevent biological deleterious effects of paraquat.
  • Toxicity investigations of combined paraquat with diethyldithiocarbamate (Et 2 DTC) are unequivocal in in vitro systems 86 .
  • Te 2 DTC diethyldithiocarbamate
  • Part A of the present application shows that amphiphilic dithiocarbamates (DTCs) strongly inhibit SOD, with a pronounced increased ability to cross lipophylic membrane barriers compared to DTC salts.
  • DTCs amphiphilic dithiocarbamates
  • the next step was to consider a bifunctional herbicide and DTC combination, so that quickly penetrating paraquat will carry the covalently linked DTC unit which will act as a strong copper-specific chelator and inhibit SOD increasing activity. Therefore, we designed and synthesized a bifunctional molecule (PQ- DTC), incorporating the well-known paraquat and metal chelator, presupposed to aid in the production of H 2 O 2 from 02 " ' by inhibiting SOD.
  • PQ- DTC bifunctional molecule
  • Phosphate buffer 50 mM pH 7.8 of was prepared from potassium dihydrogen phosphate and disodium hydrogen phosphate anhydrous (both from E. Merck, analysis grade).
  • Carbonate buffer 50 mM pH 9.5
  • was prepared from sodium carbonate anhydrous E. Merck, for analysis
  • sodium hydrogen carbonate Aldrich, A.C.S.
  • UV/VIS spectra were recorded from a Hewlett-Packard 8450A diode array spectrophotometer.
  • Proton NMR spectra were measured on a Bruker WH-270, or Bruker DPX-250 NMR spectrometers.
  • TLC was performed on Kieselgel 60 F254 , E. Merck plates.
  • Electrospray Ionization mass spectra (ESI-MS) were detected on Micromass
  • Table 9 lists strutures and 1H NMR data ( ⁇ , ppm) for all synthesized compounds.
  • PQ-DTC-Cu 2+ complex Formation of PQ-DTC-Cu 2+ complex (see Figure 19) PQ-DTC (0.1 mM) was mixed with various concentrations of Cu 2+ (as CuSO 4 ) in
  • Herbicidal activities of PQ-DTC and paraquat were determined in vivo on Spirodella oligorrhiza sp. by the bleaching of chlorophyll 72 h after the addition of various concentrations PQ-DTC and paraquat, and incubation at 25°C at a light intensity -1 -2 of 500 ⁇ Em s m .
  • the quantification of chlorophyll was made by extraction of chlorophyll into 80% acetone. The absorption value was recorded at 645 nm and 663 nm, respectively, and chlorophyll contented calculated according to Arnon.
  • PQ-DTC in DMF exhibits the strongest absorption at 577 nm and the fastest rate to reach a constant radical concentration compared to the other three solvents.
  • UV spectroscopy is the basis for most of the evidence of PQ radical-cation dimerization or association and there are many examples and references cited in [47 - 1 . Therefore, the spectral variation of PQ-DTC could be related to the form of association of the cation radical [PQ-DTC] +' With either mono-cation dimers or di-cation dimers in different solvents.
  • the precursor of PQ-DTC (compound 5) was spectrally analyzed in the same solvents to try again and to understand the production of such stable radical from PQ- DTC,.
  • [PQ-DTC] +' radical may be attributed to dimerization and to an intrinsic factor related to delocalization caused by electronegative atoms.
  • PQ-DTC decomposes into compound 5 and CS .
  • the percent decomposition of PQ-DTC can be roughly calculated.
  • Fig. 22 Decomposition increases with time and depends on pH. The decomposition rate increases rapidly at a more acidic pH. However, the decomposition of PQ-DTC is much slower than that of the family of amphiphilic dithiocarbamates (see Part A).
  • the PQ unit in PQ-DTC bearing two quaternary nitrogens at one end, is capable of forming inner salts with the ⁇ CS 2 " group.
  • MPQ-DTC monosubstituted MPQ-DTC
  • compound 7 is an inner salt as shown in Scheme 6 ( Figure 17).
  • the formation of inner salts could be one of the reasons why PQ-DTC (compound 6) is hydrolyticly more stable than the family of amphiphilic dithiocarbamates.
  • PQ-DTC Activity of PQ-DTC in vitro and in vivo systems hi order to analyze the possible modes of action of a bifunctional compound carrying a herbicidal active group, e.g., paraquat, and a metal binding group, e.g., DTC, the activity of each function should be examined separately.
  • a herbicidal active group e.g., paraquat
  • a metal binding group e.g., DTC
  • Compound 5 is totally inactive, while PQ-DTC(contains 4% of its precursor compound 5) has some ability to inhibit SOD, but it is lower than the activity of Et 2 DTC at all DTC/SOD ratios. Herbicidal activity of PQ-DTC in vivo
  • the PQ-DTC herbicidal activity in vivo was evaluated by the loss of chlorophyll from the Spirodella oligorrhiza and was compared with the herbicidal activity of paraquat. (Fig.24).
  • the PQ-DTC has (about two orders of magnitude) less herbicidal activity than to paraquat on a molar equivalence basis.
  • PQ-DTC The great reduction in herbicidal activity of PQ-DTC can be interpreted as stemming from the close interaction between the PQ unit and the DTC unit, through the formation of ionic associations (shown in Schemes 5 and 6, in Figures 16 and 17). Structural changes, such as removing the secondary amine position along the aliphatic chain may not affect such interactions and increases the herbicidal activity of paraquat. The only solution would be to change the DTC chelator to a Cu chelator with less ionic properties.
  • the extension of this work to other PQ-DTC compounds with variable spacer groups, and to other paraquat chelators (PQ-Che) of attaching some synergistic entity to any rapidly penetrating herbicide is of great interest.
  • the synergistic entity may be an inhibition of the degradation of the herbicide in the target plant (Gressel) It may allow designing herbicides with controllable properties (toxicity, herbicidal activity). This may also lead to new directions towards drug-like molecules with controlled activity and controlled degradation of themselves or of the toxic metabolites they generate.
  • Paraquat is a potent oxygen radical generating compound in aerobic systems, especiall; plants but including all aerobic types. Its effects are often partially offset by Cu/Zn superoxide dismutase (SOD), which detoxifies superoxide. Cu/Zn SOD can be inhibited by copper specific chelators.
  • SOD superoxide dismutase
  • Cu/Zn SOD can be inhibited by copper specific chelators.
  • This is a first example of a bifunctional-herbicide chelator (paraquat), and a dithiocarbamate (DTC)-copper chelator, that was constructed into one molecule (PQ-DTC).
  • N-Methyl-N'-(5-bromopentyl)- 4,4 '-bipyridinium bromide iodide (4) N-methyl-4-(4-pyridyl)pyridinium iodide (60 mg, 0.2 mmol) was dissolved into 25 ml acetonitrile . A solution of 1,5-dibromopentane (1.4 ml) in 5 ml of acetonitrile was added dropwise with stirring. The mixture was refluxed for 12 hours.

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Abstract

L'invention concerne une nouvelle bibliothèque combinatoire comprenant une pluralité de molécules bifonctionnelles de formule CF - PF, dans laquelle CF est une fraction présentant une activité chimique ou biologique spécifique et PF est une seconde fraction conférant des propriétés physiques régulables à ces molécules. Les modifications structurales de la seconde fraction font varier les propriétés physiques de la molécule bifonctionnelle sans modification significative de son activité chimique ou biologique, d'où l'obtention d'un effet optimisé de l'activité chimique ou biologique dans l'environnement où cette activité chimique ou biologique doit être exercée. La seconde fraction peut éventuellement conférer à la molécule une seconde activité chimique ou biologique.
PCT/IL2002/000642 2001-08-06 2002-08-06 Bibliotheque combinatoire de molecules bifonctionnelles Ceased WO2003014292A2 (fr)

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Publication number Priority date Publication date Assignee Title
CN108570093A (zh) * 2018-05-08 2018-09-25 中国石油大学(华东) 一种螯合铜离子的多肽及其用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MACIAS ET AL.: 'Dithiocarbamates derived from naturally occurring amino acids and their complexes with nickel and copper' JOURNAL OF COORDINATION CHEMISTRY vol. 46, no. 1, 1998, pages 71 - 77, XP002960196 *
ROGACHEV ET AL.: 'Synthesis, properties and use of copper-chelating amphiphilic dithiocarbamates as synergists of oxidant-generating herbicides' PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY vol. 60, no. 3, 1998, pages 133 - 145, XP002960195 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108570093A (zh) * 2018-05-08 2018-09-25 中国石油大学(华东) 一种螯合铜离子的多肽及其用途
CN108570093B (zh) * 2018-05-08 2021-10-29 中国石油大学(华东) 一种螯合铜离子的多肽及其用途

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