Cyclic tetrapeptides and their metal complexes.

Abstract

The present invention provides cyclic tetrapeptides consisting of alternating α- and β-amino acids and metal complexes thereof. The cyclic tetrapeptides are particularly useful for coordinating metals selected from Pb, Cd, Hg and As. The present invention further encompasses the use of the cyclic tetrapeptides in treating diseases, particularly metal poisoning, and in the remediation of contaminated water and soil. Methods for detecting the above metals in various substrates are also provided.

Description

[Technical Field]

[0001] The present invention relates to cyclic tetrapeptides and their metal complexes. The cyclic tetrapeptides are suitable for coordinating metals such as Pb, As, Cd, and Hg. The present invention further relates to the use of cyclic tetrapeptides in the treatment and diagnosis of diseases, particularly metal poisoning. Also provided are methods for removing or detecting these metals by applying the cyclic tetrapeptides to contaminated soil, water, and other materials. [Background technology]

[0002] Toxic metals, such as lead (Pb), arsenic (As), mercury (Hg), and cadmium (Cd), can be found in contaminated soil or water, posing a risk to the ecosystem and health of living organisms. For example, toxic metals can enter the human body through contaminated drinking water. Furthermore, metals can accumulate in crops and animals in the food chain and thus be ingested by humans.

[0003] Lead (Pb) is a non-essential element and a toxic metal considered to be the most harmful to human health. Pb poisoning causes up to one million deaths annually worldwide. Alarmingly, one in three children suffers from Pb poisoning, and even in the United States, more than 3% of children are found to have unsafe Pb blood levels.

[0004] The molecular mechanisms of Pb toxicity are diverse and include interference with both cellular processes and organ function. Under physiological conditions, Pb interacts with thiols of various proteins, mainly cysteine ​​(Cys) and aspartic acid (Asp) or glutamic acid (Glu) residues. 2+ This tight metal binding alters the enzyme's conformation and reduces its function. 2+ It also replaces some essential metal ions, mainly calcium (Ca) and zinc (Zn) ions, in metalloproteins, causing protein dysfunction.

[0005] After uptake, Pb 2+ is distributed in soft tissues, with the highest accumulation levels in the liver and kidney. 2+ Due to the similar ionic radius, Pb 2+ can cross the blood-brain barrier, resulting in accumulation in the brain. Finally, a significant fraction of Pb is stored in calcified tissues and released into the blood during pregnancy, providing a source of exposure to the fetus during placental crossing.

[0006] Chelation therapy is the latest treatment for Pb poisoning. It is based on the administration of drugs called chelating agents (CAs), which ideally should have several essential characteristics: (a) low toxicity of the CA and the complexes formed, (b) selectivity for individual metal ions, (c) water solubility, (d) formation of removable complexes, and (e) the ability to penetrate cells and tissues. CAs used for Pb poisoning are preferentially ethylenediaminetetraacetic acid (EDTA) and dimercaptosuccinic acid (DMSA; Figure 1).

[0007] These small molecule drugs achieve some of the requirements mentioned above. However, despite being a temporary treatment for Pb poisoning, they have significant drawbacks, mainly low metal selectivity, which results in essential metals being depleted from the body during treatment, which increases drug toxicity. Furthermore, EDTA cannot cross the cell membrane, limiting its use to extracellular targets. 2+ They are also suspected of redistributing ions to the brain. As a result, these chelators are only approved for medical use in cases of proven extremely high toxic metal levels. Critically, however, they are not approved for use in pregnant women and only in rare pediatric cases, even though these segments are among the most affected populations.

[0008] Based on the above-mentioned state of the art, the object of the present invention is to provide means and methods for the treatment and diagnosis of metal poisoning, as well as means and methods for detecting and removing metals from substrates such as contaminated water or soil. This object is achieved by the features of the independent claims of the present application and in further advantageous aspects as explained in the dependent claims, examples, figures and general description of the present specification. Summary of the Invention

[0009] A first aspect of the present invention is a compound of formula 1 , especially the formula 1 A: [ka] (In the formula, each R, independently of any other R, is independently selected from -CH and -H; R A1 and R A2 are independent of each other, C 1-4 -alkyl or phenyl, where C 1-4 -Alkyl or phenyl is -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -substituted by one or more substituents independently selected from alkyl, -SO3H, -COOH, -NH2, -CONH2, -NHC(=NH)(NH2), a 5-10 membered heterocyclic ring, a cyclic hydrocarbon moiety containing 3 to 10, particularly 3 to 6, carbon atoms, wherein the 5-10 membered heterocyclic ring or the cyclic hydrocarbon moiety is C 1-4 -Alkyl, -SH, (=S), -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 - optionally substituted with one or more substituents selected from alkyl, -SO3H, (=O), -COOH, -NH2, -CONH2; R B1 and R B2 are independent of each other. -H, or -OH, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4-Alkyl, -COOH, -NH2, -NH-C 1-4 -Alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, 5-10 membered heterocycle or a group containing 1-12 C atoms Mutan a moiety selected from the group consisting of -OH, (=O), -SH, (=S), -SC, -H, -S ... 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 optionally substituted with one or more substituents independently selected from alkyl, -NH-C(=NH)(NH), -CONH, -SOH and a 5- to 10-membered heterocycle; a linker suitable for binding to a detectable marker or a solid support, a detectable marker, optionally linked by a linker, or Linkers attached to solid supports is) The present invention relates to the compound

[0010] The compound of Formula 1 is a cyclic tetrapeptide consisting of two α-amino acids and two β-amino acids. The amino acids form a head-to-tail cyclization and can alternatively be represented by cyclic-[Xaa-βXaa-Xaa-βXaa] (SEQ ID NO: 012), where Xaa represents the α-amino acid and βXaa represents the β-amino acid.

[0011] Cyclic tetrapeptides are suitable for binding metals. Pb 2+ The distinction between toxic metals such as cadmium and other ions that are essential for humans is based on the cavities formed and the R A and R B This is achieved by a combination of the number and selection of metal binding groups in the

[0012] R for α-amino acids A1 and R A2 contributes to metallic bonding. In particular, for Pb bonding, R A1 and R A2Each of the formulas (I) and (II) includes a soft or intermediate linking moiety. Non-limiting examples of such moieties are thiol or carboxylic acid moieties, such as the thiol moiety of cysteine ​​or the β-carboxylic acid moiety of aspartic acid.

[0013] R for β-amino acids B1 and R B2 can fulfill various functions such as contributing to metal binding, mediating water solubility, facilitating cyclization in synthesis, and stabilizing the ring structure and metal complex.

[0014] β-alanine is used as βXaa, i.e., R B When is H, intramolecular cyclization during synthesis is promoted and the stability of the ring structure of the cyclic tetrapeptide is enhanced.

[0015] The water solubility of cyclic tetrapeptides is enhanced by the addition of a moiety R containing a functional group such as an alcohol, amide, carboxylic acid, or primary amine. B can be increased by using

[0016] Enhancement of metal binding affinity can be achieved by additional coordination sites or by the addition of suitable R B Selectivity can also be improved by stereocontrol. For example, R B1 and / or R B2 The aliphatic or aromatic residues in allow complexation with smaller metal ions such as Hg.

[0017] Further functionalization of the cyclic tetrapeptides can be achieved by R B1 and / or R B2 This can be achieved by a linker, a linker attached to a solid support, or a detectable marker, such as in. Such cyclic tetrapeptides can be used in the diagnosis of metal poisoning, determining the degree of contamination of substrates such as water or soil, or in the remediation of metal-contaminated soil or water.

[0018] A second aspect of the present invention relates to a metal complex comprising a ligand and a metal, wherein the ligand is a compound according to the first aspect of the present invention.

[0019] As described above, the compound according to the first aspect of the present invention is R A and R B For example, thiol and / or carboxylic acid moieties can be attached to Pb in their deprotonated form. 2+ The metal complexes may contain only one ligand (monomeric complexes) or two ligands (dimeric complexes).

[0020] A third aspect of the invention relates to the use of a compound according to the first aspect of the invention in the treatment of disease.

[0021] A fourth aspect of the present invention relates to the use of compounds according to the first aspect of the present invention in the treatment of metal poisoning.

[0022] In another embodiment, the present invention relates to a pharmaceutical composition comprising at least one compound of the present invention or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.

[0023] A fifth aspect of the present invention is a method for determining whether a patient has or is at risk of developing metal poisoning, comprising the steps of: a. determining the level of a metal in an ex vivo blood, plasma or serum sample taken from a patient using a compound according to the first aspect of the invention; and b. Demonstrate the statistical significance of metal concentrations The present invention relates to a method comprising:

[0024] In particular, R B The compound according to the first aspect of the invention comprising a detectable marker, optionally attached by a linker, is suitable for determining the amount of a metal in a sample.

[0025] A sixth aspect of the present invention relates to a method for removing metals from a substrate, comprising using a compound according to the first aspect of the present invention.

[0026] As mentioned above, there is a constant need to remediate soil and water contaminated with metals such as Pb.

[0027] A seventh aspect of the present invention relates to a method for detecting a metal in a substrate, comprising using a compound according to the first aspect of the present invention.

[0028] In particular, R B The compounds according to the first aspect of the invention comprising a detectable marker, optionally attached by a linker, are suitable for the detection of metals, such as Pb, in substrates such as contaminated water or soil.

[0029] Terms and Definitions For the purposes of interpreting this specification, the following definitions shall apply, and where appropriate, terms used in the singular shall include the plural and vice versa. In the event that any definition set out below conflicts with any document incorporated herein by reference, the definition set out below shall control.

[0030] The terms "comprising," "having," "containing," and "including," and other similar forms and their grammatical equivalents, when used herein, are intended to be equivalent in meaning and to be open-ended in that the item or items preceded by these words are not meant to be a closed listing of such item or items, nor are they meant to be limited to only the listed item or items. For example, an item "comprising" components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C, but also one or more other components. Thus, "comprising" and similar forms and their grammatical equivalents are intended and understood to encompass disclosures of "consisting essentially of" or "consisting of" embodiments.

[0031] Where a range of values ​​is given, unless the context clearly dictates otherwise, it is understood that each value between the upper and lower limit of that range and any other stated or intervening value, to the tenth of the unit of the lower limit, is encompassed within the disclosure, subject to any limit specifically excluded in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the disclosure.

[0032] Reference herein to "about" a value or parameter encompasses (and describes) variations on that value or parameter itself. For example, a description that refers to "about X" includes a description of "X."

[0033] "A," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art (for example, in cell culture, molecular genetics, nucleic acid chemistry, hybridization technology and biochemistry).Standard techniques are used for molecular, genetic and biochemical techniques (usually see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th ed., John Wiley & Sons, Inc.) and scientific procedures.

[0035] The term "tetrapeptide" in the present context refers to a molecule consisting of four amino acids that form a linear chain in which the amino acids are linked by peptide bonds. A tetrapeptide contains two α-amino acids and two β-amino acids.

[0036] The term "cyclic tetrapeptide" refers to a tetrapeptide as described above, in which the amino acids form a head-to-tail ring as shown in Formula 1.

[0037] Amino acid residue sequences are given from the amino to the carboxyl terminus. Capital letters refer to L-amino acids in the single-letter code for sequence positions (Stryer, Biochemistry, 3 rded. p. 21). A lowercase letter for an amino acid sequence position or a "D" preceding an amino acid name or amino acid code refers to the corresponding D- or (2R)-amino acid. Sequences are written from left to right in the amino- to carboxyl-terminal direction. Following standard nomenclature, α-amino acid residue sequences are designated by either three-letter or one-letter codes, as shown below: Alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V). Three-letter or one-letter codes are also used after the Greek letter "β" for β-amino acids that contain a residue at the same β-carbon as the corresponding α-amino acid, e.g., "β-Ala" or "βAla" refer to the β-amino acid β-alanine. Homologs of α- or β-amino acids that differ by an additional methylene bridge (-CH2-) in the side chain are referred to as "homo" amino acids, e.g., homocysteine. "Homo" is also abbreviated as "h," e.g., hCys refers to the α-amino acid homocysteine, and "βhGlu" refers to β-homoglutamic acid.

[0038] In the context of the present invention, the term "5-10 membered heterocycle " refers to compounds consisting of 5 to 10 carbon atoms, one or more of which are replaced by heteroatoms N, S or O, especially N. Similarly, "5- to 6-membered heterocycle " consists of 5 to 6 carbon atoms, one or more of which are replaced by heteroatoms N, S or O, especially N. The carbon atoms and one or more of the heteroatoms are linked by single and / or double bonds to form a ring structure. The ring structure may be monocyclic or bicyclic.

[0039] The term "hydrocarbon moiety containing 3 to 10 carbon atoms" refers to a hydrocarbon moiety containing carbon-carbon single, double and / or triple bonds, particularly carbon-carbon single and / or carbon-carbon double bonds. The carbon atoms may form a linear, branched or cyclic structure or a combination thereof.

[0040] The term alkyl refers to a straight or branched chain hydrocarbon moiety. 1-4 -Alkyl refers to a saturated straight or branched chain hydrocarbon having 1, 2, 3 or 4 carbon atoms. 1-3 -Alkyl refers to a straight or branched chain hydrocarbon having up to 3 carbon atoms. 1-4 Non-limiting examples of -alkyl include methyl, ethyl, propyl, n-butyl, 2-methylpropyl, and tert-butyl. 1-4 -Alkyl refers to methyl (Me), ethyl (Et), propyl (Pr), isopropyl (iPr), n-butyl (Bu) and tert-butyl (tBu).

[0041] The term "cyclic hydrocarbon moiety" refers to a monocyclic or polycyclic hydrocarbon moiety containing a carbon-carbon single bond, a double bond, and / or a triple bond, particularly a carbon-carbon single bond and / or a carbon-carbon double bond. The ring structure of a polycyclic hydrocarbon moiety can be bridged, fused, or spirocyclic. Non-limiting examples of cyclic hydrocarbon moieties are aryl, e.g., phenyl, and cycloalkyl, e.g., cyclohexyl.

[0042] In the context of this specification, the term C 5-6 -Cycloalkyl relates to a saturated hydrocarbon ring having 5 or 6 carbon atoms.

[0043] In the present context, the term fluorescent dye relates to small molecules capable of fluorescing in the visible or near infrared spectrum. [Brief explanation of the drawings]

[0044] [Figure 1]FIG. 1 shows DMSA and EDTA as benchmark drugs against Pb poisoning. [Figure 2a] Figure 1 shows the detoxification potential of test peptides compared to benchmark drugs and glutathione (GSH) at the highest dose concentration in vivo in DH5α cells (10 equivalents) at 120 mM. Values ​​are the mean + SD of more than three replicates, each performed in triplicate. [Figure 2b] Figure 1 shows the detoxification potential of test peptides compared to benchmark drugs and glutathione (GSH) at the highest dose concentration in vitro in HT-29 cells (5 equiv.) at 10 mM. Values ​​are the mean + SD of more than three replicates, each performed in triplicate. [Figure 2c] Figure 1 shows the concentration-dependent detoxification ability of 8 and 2 drugs in HT-29 cells. Values ​​are the mean + SD of more than three repeats, each performed in triplicate. [Figure 2d] Figure 1 shows the concentration-dependent detoxification capacity of EDTA and 8 as Ca vs. Na salts in HT-29 cells. Values ​​are the mean + SD of more than three repeats, each performed in triplicate. [Figure 2e] Figure 1 shows the toxicity of 8 and 2 drugs in HT-29 cells. Values ​​are the mean + SD of more than three repeats, each performed in triplicate. [Figure 3a] FIG. 1 shows a metal complex consisting of Pb and cyclic [Cys-βAla-Asp-βAla] as a monomeric ligand. [Figure 3b] FIG. 1 shows a metal complex consisting of Pb and cyclic [Cys-βAla-Asp-βAla] as a dimeric ligand. [Figure 4] FIG. 1 shows the dose-dependent recovery of HT-29 cells treated with Pb(NO) (2 mM), followed by Na, CaNaEDTA, and NaDMSA (1 h after addition of Pb ions; values ​​were calculated relative to cells intoxicated with Pb ions as a negative control). [Figure 5a] FIG. 1 shows the mean blood lead levels (BLL) of eight mice per group, collected at the end of the study (day 18) and analyzed by ICP-MS. [Figure 5b] FIG. 1 shows urinary Pb from 8 mice per group (only 34 of 40 mice) collected on the final day of the study (day 18) and analyzed by ICP-MS. [Figure 6] FIG. 1 shows peptides 1f (R=SH) and 8f (R=COOH) coupled to polystyrene tentagel resin. [Figure 7] Figure 1 shows the Pb concentrations detected by ICP-MS and calculated relative to the original solution between the negative control 0f and the two immobilized peptides 1f and 8f after two filtration rounds (dark grey) and one regeneration round with EDTA (light grey). [Figure 8] Figure 1 shows the Pb concentrations detected by ICP-MS and calculated relative to the original solution of equimolar ZnCl2 + Pb(NO3)2 and CaCl2 + Pb(NO3)2 solutions and Pb(NO3)2-spiked human serum for 0f (black bars), 1f (light grey bars), and 8f (medium grey bars) (all salt solutions are 25 mM). DETAILED DESCRIPTION OF THE INVENTION

[0045] A first aspect of the present invention is a compound of formula 1 , especially the formula 1 A: [ka] (In the formula, each R, independently of any other R, is independently selected from -CH and -H; R A1 and R A2 are independent of each other, C 1-4 -alkyl or phenyl, where C 1-4 -Alkyl or phenyl is -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4-substituted by one or more substituents independently selected from alkyl, -SO3H, -COOH, -NH2, -CONH2, -NHC(=NH)(NH2), a 5-10 membered heterocyclic ring, a cyclic hydrocarbon moiety containing 3 to 10, particularly 3 to 6, carbon atoms, wherein the 5-10 membered heterocyclic ring or the cyclic hydrocarbon moiety is 、C 1-4 -Alkyl, -SH, (=S), -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -SO3H, (=O), -COOH, -NH2, -CONH2, especially -C 1-4 -Alkyl, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 - optionally substituted with one or more substituents selected from alkyl, -SO3H, -COOH, -NH2, -CONH2; R B1 and R B2 are independent of each other. -H, or -OH, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 - a moiety selected from alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, a 5- to 10-membered heterocycle or a hydrocarbon moiety containing 1 to 12 C atoms, wherein the 5- to 10-membered heterocycle or The charcoal The hydrogen moiety is -OH, (=O), -SH, (=S), -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 optionally substituted with one or more substituents independently selected from alkyl, -NH-C(=NH)(NH), -CONH, -SOH and a 5- to 10-membered heterocycle; a linker suitable for binding to a detectable marker or a solid support, a detectable marker, optionally linked by a linker, or Linkers attached to solid supports is) The present invention relates to the compound

[0046] In some embodiments, at least one R is H and another R is -CH3.

[0047] In some embodiments, at least two R are H and another R is -CH3.

[0048] In some embodiments, at least three R are H and another R is -CH3.

[0049] In some embodiments, the moiety R A1 and R A2 At least one of R contains a heteroatom S, N or O, especially S. When the compound of formula 1 is used to bind a metal, R A The heteroatom of forms bonds with metals such as Pb, Hg, As, and Cd, especially Pb. Binding of Pb, Hg, As, and Cd, especially Pb, may not be achieved by a hydroxyl moiety such as in the side chain of serine. Therefore, α-serine is a suitable R A However, β-serine is still a suitable amino acid for providing the moiety R which enhances the water solubility of the cyclic tetrapeptide. B may be used to provide

[0050] In some embodiments, the compound is of formula 2, 3, 4, 5, 6 or 7, particularly a compound of formula 2a, 3a, 4a, 5a, 6a or 7a. [ka] TIFF0007784745000004.tif187143 TIFF0007784745000005.tif58139

[0051] Cyclic tetrapeptides can be formed from L- or D-amino acids or mixtures thereof. For economic reasons, L-amino acids in particular are used as they are usually cheaper than the corresponding D-amino acids.

[0052] To form a stable metal complex, the metal-binding moiety R A1 and R A2 In particular, Pb 2+ should be facing in the same direction to capture

[0053] In some embodiments, the α-amino acids of the cyclic tetrapeptide are both L-amino acids or both D-amino acids, particularly both L-amino acids. A1 and R A2 are both bonded to the α-carbon atom by an up-wedge bond, or R A1 and R A2 are both connected to the α-carbon atom by down-wedge bonds.

[0054] In some embodiments, the compound is of formula 2, 5, 6 or 7, particularly a compound of formula 2a, 5a, 6a or 7a.

[0055] In some embodiments, the compound is of formula 2 or 5, particularly formula 2a or 5a.

[0056] In some embodiments, the compound is of Formula 2, particularly Formula 2a.

[0057] In some embodiments, R A1 and R A2 are, independently of each other, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4C substituted by one or more, in particular one or two, substituents independently selected from alkyl, -SO3H, -COOH, -NH2, -CONH2, a 5-10 membered heterocycle, a cyclic hydrocarbon moiety containing 3-6 carbon atoms; 1-4 -Alkyl, especially C 1-3 Alkyl, more particularly C 1-2 alkyl, wherein the 5- to 10-membered heterocycle or the cyclic hydrocarbon moiety is C 1-4 -Alkyl, -SH, (=S), -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -SO3H, (=O), -COOH, -NH2, -CONH 2、 Especially C 1-4 -Alkyl, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 It may be optionally substituted with one or more, in particular one, substituent selected from -alkyl, -SO3H, -COOH, -NH2, -CONH2.

[0058] In some embodiments, R A1 and R A2 are, independently of each other, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 C substituted by one or more, in particular one or two, substituents independently selected from alkyl, -SO3H, -COOH, -NH2, -CONH2, a 5-10 membered heterocycle, a cyclic hydrocarbon moiety containing 3-6 carbon atoms; 1-4 -Alkyl, especially C 1-3 Alkyl, more particularly C 1-2 alkyl, wherein the cyclic hydrocarbon moiety is C 1-4 -Alkyl, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 It may be optionally substituted with one or more, in particular one, substituent selected from -alkyl, -SO3H, -COOH, -NH2, -CONH2.

[0059] In some embodiments, R A1 and R A2is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, imidazolyl, mercaptoimidazolyl, thiofuranyl, oxazolonyl, indolyl, mercaptopurinyl, benzothiophenyl, especially imidazolyl, mercaptoimidazolyl, thiofuranyl, indolyl, more especially mercaptoimidazolyl.

[0060] Thiofuran is also called thiophene.

[0061] Benzothiophene is also known as benzothiofuran.

[0062] In some embodiments, R A1 and R A2 in heterocycle is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, imidazolyl, mercaptoimidazolyl, thiofuranyl, oxazolonyl.

[0063] In some embodiments, R A1 and R A2 in heterocycle is selected from pyrrolyl, pyrazolyl, imidazolyl, mercaptoimidazolyl, thiofuranyl, oxazolonyl.

[0064] In some embodiments, R A1 and R A2 in heterocycle is selected from imidazolyl, mercaptoimidazolyl, and thiofuranyl.

[0065] In some embodiments, R A1 and R A2 in heterocycle is selected from pyrrolyl, pyrazolyl, and imidazolyl.

[0066] In some embodiments, R A1 and R A2 in heterocycleis selected from imidazolyl, indolyl.

[0067] In some embodiments, R A1 and R A2 in heterocycle is selected from imidazolyl.

[0068] In some embodiments, imidazolyl is 1H-imidazol-4-yl. For example, R A is 1H-imidazol-4-yl when histidine is used as the α-amino acid.

[0069] In some embodiments, the indolyl is 1H-indol-3-yl. For example, R A is 1H-indol-3-yl when tryptophan is used as the α-amino acid.

[0070] In some embodiments, R A1 and R A2 The cyclic hydrocarbon moiety in is selected from cyclopentyl, cyclohexyl and phenyl.

[0071] In some embodiments, R A1 and R A2 The cyclic hydrocarbon moiety in is phenyl.

[0072] In some embodiments, R A1 and R A2 is substituted by one or two substituents independently selected from -SH, -S-CH3, -SeH, -Se-CH3, -SO3H, -COOH, -NH2, -CONH2, imidazolyl, indolyl and phenyl; 1-3 Alkyl, especially C 1-2 alkyl, wherein said phenyl may be optionally substituted with one or more, especially one, substituent selected from -SH and -SeH, especially -SH.

[0073] In some embodiments, R A1 and RA2 is substituted by one or two substituents independently selected from -SH, -S-CH3, -SeH, -Se-CH3, -SO3H, -COOH, -NH2, -CONH2, 1-3 Alkyl, especially C 1-2 It is alkyl.

[0074] In some embodiments, R A1 and R A2 is substituted by one or two substituents independently selected from -SH, -S-CH3, -SeH, -Se-CH3, -SO3H, -COOH, -NH2, -CONH2, imidazolyl, indolyl and phenyl; 1-3 Alkyl, especially C 1-2 alkyl, wherein said phenyl may be optionally substituted with one or more, especially one, substituent selected from -SH and -SeH, especially -SH.

[0075] In some embodiments, R A1 and R A2 are independently selected from -CH-SH, -(CH)-SH, -CH-S-CH, -(CH)-S-CH, -CH(SH)(-CH-SH), -CH-CH(SH)(-CH-SH), -CH(SH)(-COOH), -CH(SH)-CH-COOH, -CH-CH(SH)(-COOH), -phenyl-SH, -CH-SOH, -(CH)-SOH-CH-COOH, -(CH)-COOH, -CH-NH, -(CH)-NH, -CH-CONH, -(CH)-CONH, -CH-imidazolyl, -CH-mercaptoimidazolyl, and -CH-phenyl.

[0076] In some embodiments, R A1 and R A2are independently selected from -CH-SH, -(CH)-SH, -CH-S-CH, -(CH)-S-CH, -CH(SH)(-CH-SH), -CH-CH(SH)(-CH-SH), -CH(SH)(-COOH), -CH(SH)-CH-COOH, -CH-CH(SH)(-COOH), -phenyl-SH, -CH-SOH, -(CH)-SOH-CH-COOH, -(CH)-COOH, -CH-NH, -(CH)-NH, -CH-CONH, -(CH)-CONH.

[0077] In some embodiments, R A1 and R A2 are independently selected from -CH-SH, -(CH)-SH, -(CH)-S-CH, -CH-CH(SH)(-CH-SH), -CH(SH)(-COOH), -phenyl-SH, -CH-SOH, -CH-COOH, -CH-NH, -CH-CONH, -CH-imidazolyl, and -CH-phenyl.

[0078] In some embodiments, R A1 and R A2 are independently selected from —CH2—SH, —(CH2)2—S—CH3, and —CH2—COOH.

[0079] In one embodiment, -R A1 and R A2 are identical and -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 C substituted by one or more, in particular one or two, substituents independently selected from alkyl, -SO3H, -COOH, -NH2, -CONH2, a 5-10 membered heterocycle, a cyclic hydrocarbon moiety containing 3-6 carbon atoms; 1-4 -Alkyl, especially C 1-3 Alkyl, more particularly C 1-2 alkyl, Here, the cyclic hydrocarbon moiety is -SH, -SC 1-4 -Alkyl, -SeH, -Se-C1-4 -substituted with one or more, in particular one, substituent selected from alkyl, -SO3H, -COOH, -NH2, -CONH2, and Here, the 5- to 10-membered heterocycle is C 1-4 -Alkyl, -SH, (=S), -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -SO3H, (=O), -COOH, -NH2, -CONH 2、 Especially C 1-4 -Alkyl, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 optionally substituted with one or more, in particular one, substituent selected from: -alkyl, -SO3H, -COOH, -NH2, -CONH2; and / or -R A1 -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, and -COOH, especially -SH, -SC 1-4 C substituted by one or two substituents selected from -alkyl and -COOH 1-4 -Alkyl, especially C 1-3 Alkyl, more particularly C 1-2 alkyl, and R A2 -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 C substituted by one or more, in particular one or two, substituents independently selected from alkyl, -SO3H, -COOH, -NH2, -CONH2, a 5-10 membered heterocycle, a cyclic hydrocarbon moiety containing 3-6 carbon atoms; 1-4 -Alkyl, especially C 1-3 Alkyl, more particularly C 1-2 alkyl, wherein the 5- to 10-membered heterocyclic ring or the cyclic hydrocarbon moiety is C 1-4 -Alkyl, -SH, (=S), -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -SO3H, (=O), -COOH, -NH2, -CONH2、 Especially C 1-4 -Alkyl, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -alkyl, -SO3H, -COOH, -NH2, -CONH2, In particular, R A2 -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 C substituted by one or two, especially one, substituent independently selected from alkyl, -COOH, -NH2, -CONH2, a 5- or 6-membered heterocycle, especially imidazolyl, mercaptoimidazolyl or thiofuranyl, phenyl, especially unsubstituted phenyl; 1-4 -Alkyl, especially C 1-3 Alkyl, more particularly C 1-2 alkyl, wherein the phenyl is -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 - optionally substituted by one or more, in particular one, substituent selected from alkyl, R A2 is R A1 is selected to be different from

[0080] In one embodiment, -R A1 and R A2 are identical and substituted by one or two substituents independently selected from -SH, -S-CH3, -SeH, -Se-CH3, -SO3H, -COOH, -NH2, -CONH2, imidazolyl, mercaptoimidazolyl, thiofuranyl, indolyl, and phenyl; 1-3 Alkyl, especially C 1-2 alkyl, where phenyl is substituted with one or more, in particular one, substituent selected from -SH and -SeH, in particular -SH; and / or -R A1 -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4-Alkyl and -COOH, especially -SH, -SC 1-4 C substituted by one or two substituents selected from -alkyl and -COOH 1-3 Alkyl, especially C 1-2 alkyl, and R A2 is substituted by one or two substituents independently selected from -SH, -S-CH3, -SeH, -Se-CH3, -SO3H, -COOH, -NH2, -CONH2, imidazolyl, mercaptoimidazolyl, thiofuranyl, indolyl, and phenyl; 1-3 Alkyl, especially C 1-2 alkyl, wherein phenyl is optionally substituted with one or more, in particular one, substituent selected from -SH and -SeH, in particular -SH; In particular, R A2 is substituted by one or two substituents, in particular one substituent, independently selected from -SH, -S-CH3, -SeH, -Se-CH3, -COOH, -NH2, -CONH2, imidazolyl and phenyl, in particular unsubstituted, phenyl or imidazolyl, C 1-3 Alkyl, especially C 1-2 alkyl, wherein said phenyl is optionally substituted with one or more substituents, in particular one substituent, selected from -SH and -SeH, in particular -SH; R A2 is R A1 is selected to be different from

[0081] In one embodiment, -R A1 and R A2 are identical and selected from -CH2-SH, -(CH2)2-SH, -(CH2)2-S-CH3, -CH2-CH(SH)(-CH2-SH), -CH(SH)(-COOH), -phenyl-SH, -CH2-SO3H, -CH2-COOH and -CH2-imidazolyl, and / or -R A1is selected from -CH-SH and -CH(SH)(-COOH), and R A2 is selected from -CH2-SH, -(CH2)2-SH, -(CH2)2-S-CH3, -CH2-COOH, -CH2-NH2, -CH2-CONH2, -CH2-imidazolyl and -CH2-phenyl; R A2 is R A1 is selected to be different from

[0082] In some embodiments, R A1 and R A2 are identical.

[0083] In some embodiments of any of the aspects of the invention, R A1 No or R A2 The alkyl portion of is not substituted with a 5- to 6-membered heterocyclic or cyclic hydrocarbon moiety.

[0084] In some embodiments of any of the aspects of the invention, R A1 No or R A2 The alkyl portion of is not substituted with a cyclic hydrocarbon moiety.

[0085] In some embodiments, R B1 and R B2 teeth, -H, or -OH, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 - a moiety selected from alkyl, -NH-C(=NH)(NH), -CONH, -SOH, a 5- to 10-membered heterocycle or a hydrocarbon moiety containing 1 to 12 C atoms, Here, the 5- to 10-membered heterocyclic ring or the hydrocarbon moiety is -OH, (=O), -SH, (=S), -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4- a moiety optionally substituted with one or more substituents independently selected from alkyl, -NH-C(=NH)(NH), -CONH, -SOH, and a 5- to 10-membered heterocycle; are independent of each other.

[0086] In some embodiments, R B1 and R B2 teeth, -H, or -OH, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 - a moiety selected from alkyl, -NH-C(=NH)(NH), -CONH, -SOH, a 5- to 10-membered heterocycle or a hydrocarbon moiety containing 1 to 12 C atoms, where: The charcoal The hydrogen moiety is -OH, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 - a moiety optionally substituted with one or more substituents independently selected from alkyl, -NH-C(=NH)(NH), -CONH, -SOH, and a 5- to 10-membered heterocycle; are independently selected from

[0087] To enhance the water solubility of the cyclic tetrapeptides of the present invention, one or both moieties R B1 and R B2 may contain a hydrophilic moiety. In some embodiments, at least one R B1 and R B2 are independently selected from -OH, -COOH, -NH2, -CONH2, -SO3H, a 5-10 membered heterocycle or a hydrocarbon moiety containing 1-12 C atoms, optionally substituted with one or more substituents independently selected from -OH, -COOH, -NH2, -CONH2, -SO3H and a 5-10 membered heterocycle.

[0088] To enhance metal binding affinity, R B1 and R B2 may include moieties that provide additional coordination sites and / or a second coordination sphere. In some embodiments, R B1 and R B2 are, independently of each other, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 -alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, a 5-10 membered heterocycle or a hydrocarbon moiety containing 1 to 12 C atoms, wherein the 5-10 membered heterocycle or hydrocarbon moiety is (=O), -SH, (=S), -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 -alkyl, -NH-C(=NH)(NH), -CONH, -SOH, and a 5- to 10-membered heterocycle. B1 and R B2 At least one of the following is independently -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 -alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, a 5-10 membered heterocycle or a hydrocarbon moiety containing 1 to 12 C atoms, wherein the hydrocarbon moiety is selected from -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 -Optionally substituted with one or more substituents independently selected from alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H and 5-10 membered heterocycle.

[0089] In some embodiments, R B1 and R B2 teeth, -H, or -OH, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 -Alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, 5-10 membered heterocycle, cyclopentyl, cyclohexyl, phenyl or C 1-8 Alkyl, especially C 1-4 alkyl, where cyclopentyl, cyclohexyl, phenyl or C 1-8 Alkyl, especially C 1-4 Alkyl is -OH, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 -optionally substituted with one or more substituents independently selected from alkyl, -NH-C(=NH)(NH), -CONH, -SOH and a 5- to 10-membered heterocycle; and 5-10 membered heterocycles include -OH, (=O), -SH, (=S), and -SC. 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 - a moiety optionally substituted with one or more substituents independently selected from alkyl, -NH-C(=NH)(NH), -CONH, -SOH are independently selected from

[0090] In some embodiments, R B The cyclopentyl, cyclohexyl or phenyl is unsubstituted.

[0091] In some embodiments, R B1 and R B2 of heterocycleis selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, imidazolyl, in particular imidazolyl, mercaptoimidazolyl, thiofuranyl, oxazolonyl, indolyl, mercaptopurinyl, benzothiophenyl, benzimidazolyl, quinolyl, isoquinolyl, diazanaphthalenyl.

[0092] In some embodiments, R B1 and R B2 of heterocycle is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, imidazolyl, in particular imidazolyl, indolyl.

[0093] In some embodiments, R B1 and / or R B2 of heterocycle is R A1 and R A2 is defined as for R A1 and R A2 Reference is made to specific embodiments relating to the above.

[0094] In some embodiments, R B1 and R B2 -H, -C 3-6 Alkyl, in particular -CH2-CH(CH3)2, -CH2-phenyl, -SH, -(CH2) m -SH, -(CH2) m -COOH and -(CH2) r -CONH2; and m and r are 0, 1, 2, or 3.

[0095] In some embodiments, R B1 and R B2 are H, -SH, -(CH2) m -SH, -(CH2) m -COOH and -(CH2) r -CONH2; and m and r are 0, 1, 2, or 3.

[0096] In some embodiments, R B1 and R B2 is H, -(CH2) m -COOH and -(CH2) r -CONH2; and m and r are 0, 1, 2, or 3.

[0097] In some embodiments, R B1 and R B2 is H, -(CH2) m and m is independently selected from —COOH and —CONH 2 ; and m is 1, 2, or 3.

[0098] In some embodiments, m is 1, 2, or 3.

[0099] In some embodiments, r is 0 or 1, particularly 1.

[0100] In some embodiments, R B1 and R B2 is -H.

[0101] In some embodiments, R B1 and R B2 are identical.

[0102] To facilitate detection by the cyclic tetrapeptides of the present invention and / or metal complexes comprising the cyclic tetrapeptides of the present invention, the cyclic tetrapeptides may comprise a detectable marker.

[0103] In some embodiments, the detectable marker is selected from a moiety comprising a dye, an affinity tag, a magnetic bead, and a radioisotope.

[0104] Suitable dyes are for example fluorescent dyes known to those skilled in the art.

[0105] For detection of the cyclic tetrapeptide by affinity tag, commonly known tags can be used, non-limiting examples of which include strep-tag, glutathione-S-transferase (GST) tag, and poly(His) tag.

[0106] In one embodiment, the linker is a hydrocarbon moiety containing up to 50 C atoms, in particular up to 20 C atoms, in which one or more C atoms may optionally be replaced by O, S or N.

[0107] In some embodiments, the solid support is a resin, a bead, an electrode surface or the bottom / wall of a reaction vessel, particularly an electrode surface, a resin or a bead, more particularly a resin or a bead.

[0108] The compounds according to the first aspect of the invention may be attached via a linker to a reaction vessel such as a 96-well plate or to a flow-through device, facilitating their use in diagnostic / detection methods and in the remediation of contaminated water and soil, respectively.

[0109] In one embodiment, the compound according to the first aspect of the present invention is a compound of formula X1 to X22, particularly formula X1 to 11 or X14 to 22. [ka] TIFF0007784745000007.tif127155

[0110] In certain embodiments of any of the aspects of the invention described herein, R A1 and R A2 is not -CH2-imidazolyl and R A1 and R A2 is not -CH2-phenyl.

[0111] In certain embodiments of any of the aspects of the invention described herein, R A1and R A2 is not -CH2-imidazolyl.

[0112] In certain embodiments of any of the aspects of the invention described herein, R A1 and R A2 is not -CH2-phenyl.

[0113] In some embodiments of any of the aspects of the invention described herein, the compound of Formula 1 is not a compound of Formula D1 or D2. [ka]

[0114] A second aspect of the present invention relates to a metal complex comprising a ligand and a metal, wherein the ligand is a compound according to the first aspect of the present invention.

[0115] As described above, the compound according to the first aspect of the present invention is R A and R B The metal can be bound via a suitable moiety in

[0116] In certain embodiments, the binding moiety of the compound according to the first aspect of the invention binds to a metal in its deprotonated form, for example, a thiol and / or carboxylic acid moiety in its deprotonated form binds to a metal, as shown below: 2+ (See also Figure 3). [ka]

[0117] In some embodiments, the ligand is an anion.

[0118] Typically, the ratio of metal to peptide is 1:1 or 1:2, ie, the complex is monomeric or dimeric.

[0119] In some embodiments, the complex is a dimer, particularly a homodimer.

[0120] In some embodiments, the metal is selected from Pb, As, Cd and Hg, and in particular the metal is Pb.

[0121] For the ligand, reference is made to the embodiment of the first aspect of the invention.

[0122] A third aspect of the invention relates to the use of a compound according to the first aspect of the invention in the treatment of disease.

[0123] In one embodiment, the compounds according to the first aspect of the invention are for use in the treatment of a disease.

[0124] For the compounds, reference is made to the embodiments of the first aspect of the invention.

[0125] A fourth aspect of the present invention relates to the use of compounds according to the first aspect of the present invention in the treatment of metal poisoning.

[0126] In one embodiment, the compounds according to the first aspect of the invention are for use in the treatment of metal poisoning.

[0127] In some embodiments, the metal poisoning is selected from Pb poisoning, As poisoning, Cd poisoning, and Hg poisoning.

[0128] In some embodiments, the metal poisoning is Pb poisoning.

[0129] In the medical context, the compounds according to the first aspect of the invention may be applied by standard methods as described in Sears, ME (2003).

[0130] For the compounds, reference is made to the embodiments of the first aspect of the invention.

[0131] A fifth aspect of the present invention provides a method for determining whether a patient has or is at risk of developing metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning, comprising the steps of: a. Determining the level of metals, in particular Pb, As, Cd and / or Hg, in an ex vivo blood, plasma or serum sample taken from a patient using a compound according to the first aspect of the invention; and b. Demonstrate the statistical significance of metal concentrations The present invention relates to a method comprising:

[0132] In particular, R B The compound according to the first aspect of the invention comprising a detectable marker, optionally attached by a linker, is suitable for determining the amount of a metal in a sample.

[0133] Statistical significance can be established by determining the ratio of free ligand, i.e., a compound according to the first aspect of the present invention, to the metal complex. The signal obtained when the marker is detected can be compared to a standard.

[0134] With respect to the compounds, reference is made to embodiments of the first aspect of the invention.

[0135] The present invention further encompasses the use of a compound according to the first aspect of the invention for use in the manufacture of a kit for detecting the onset of metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning.

[0136] Wherever alternatives for single separable features are described herein as "embodiments," it is to be understood that such alternatives can be freely combined to form separate embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for the detectable label can be combined with any of the alternative embodiments of the ligand / compound according to the first aspect of the invention, and these combinations can be combined with any pharmaceutical application or diagnostic method described herein.

[0137] A sixth aspect of the present invention relates to a method for removing metals, in particular metals selected from Pb, As, Cd and Hg, more in particular Pb, from a substrate, in particular soil or an aqueous solution or suspension, which method comprises using a compound according to the first aspect of the present invention.

[0138] In particular, compounds according to the first aspect of the invention which contain a detectable marker, such as an affinity tag, or which are attached to a solid support via a linker, are suitable for this method.

[0139] For the compounds, reference is made to the embodiments of the first aspect of the invention.

[0140] A seventh aspect of the present invention relates to a method for detecting a metal, in particular a metal selected from Pb, As, Cd and Hg, more in particular Pb, in a substrate, in particular soil or an aqueous solution or suspension, comprising using a compound according to the first aspect of the present invention.

[0141] In particular, R B The compound according to the first aspect of the invention comprising a detectable marker, optionally attached by a linker, is suitable for determining the amount of a metal in a sample.

[0142] For the compounds, reference is made to the embodiments of the first aspect of the invention.

[0143] Another aspect of the present invention relates to the preparation of a compound according to the first aspect of the present invention, which preparation comprises the steps of: - providing a tetrapeptide consisting of two α-amino acids Xaa and two β-amino acids βXaa, characterized from the N-terminus to the C-terminus by the sequence βXaa-Xaa-βXaa-Xaa (SEQ ID NO: 013) or Xaa-βXaa-Xaa-βXaa (SEQ ID NO: 014), in particular βXaa-Xaa-βXaa-Xaa (SEQ ID NO: 013), - adding a coupling reagent and a base to obtain a reaction mixture; - in a dilution step, diluting the reaction mixture in an organic solvent, in particular CH2Cl2 or DMF, more in particular CH2Cl2 Includes.

[0144] In some embodiments, the coupling agent is selected from the group consisting of PyBOP, HATU (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, CAS number 148893-10-1), HCTU (O-(1H-6-chlorobenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, CAS No. 330645-87-9), HOBt / DIC (benzotriazol-1-ol, CAS No. 2592-95-2) and N,N'-di(propan-2-yl)methanediimine, CAS No. 693-13-0), DCC (N,N'-dicyclohexylmethanediimine, CAS No. 538-75-0), DPPA (diphenylphosphoryl azide, CAS No. 26386-88-9).

[0145] In some embodiments, the coupling agent is PyBOP. The term "PyBOP" refers to benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (CAS number 128625-52-5).

[0146] In one embodiment, 1 to 2 molar equivalents of coupling agent are used relative to the molar amount of tetrapeptide.

[0147] In one embodiment, 1.5 molar equivalents are used relative to the molar amount of the tetrapeptide.

[0148] In certain embodiments, the base is Hunig's base. The term "Hunig's base" relates to N-ethyl-N-(propan-2-yl)propan-2-amine (CAS number 7087-68-5).

[0149] In one embodiment, 2 to 6 molar equivalents of base are used relative to the molar amount of tetrapeptide.

[0150] In one embodiment, 3 molar equivalents of base are used relative to the molar amount of tetrapeptide.

[0151] In one embodiment, the concentration of the tetrapeptide in the dilution step is 0.01 mM to 10 mM, particularly 0.05 mM to 2 mM.

[0152] In one embodiment, the concentration of the tetrapeptide in the dilution step is 0.1 mM.

[0153] In one embodiment, the dilution step is carried out for 12 to 72 hours, particularly 16 to 48 hours.

[0154] In some embodiments, the dilution step is followed by an evaporation step. To allow for fast evaporation, a low boiling point solvent, such as CH2Cl2, can be used. If the boiling point of the solvent, for example, DMF, is higher, evaporation can be cumbersome.

[0155] In one embodiment, the process is carried out at a temperature in the range of 15° C. to 40° C., particularly in the range of 20° C. to 25° C. The process may be carried out at ambient temperature. Heating or cooling of the reaction mixture is not required.

[0156] The tetrapeptides may contain protecting groups. Suitable protecting groups as well as methods of deprotection are known to those skilled in the art.

[0157] Medical Treatments, Dosage Forms and Salts Similarly, within the scope of the present invention is a method for treating metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning, in a patient in need thereof, which method comprises administering to the patient a compound according to the first aspect of the present invention.

[0158] Similarly, there is provided a dosage form for the prevention or treatment of metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning, comprising a compound according to any of the above aspects or embodiments of the invention.

[0159] Those skilled in the art will understand that any specific drug compound mentioned herein can exist as the pharmaceutically acceptable salt of the drug.Pharmaceutically acceptable salts comprise a counterion that bears the opposite charge to the ionized drug.Non-limiting examples of pharmaceutically acceptable anionic salts include acetate, benzoate, besylate, acid tartrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate, triethiodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine, and zinc.

[0160] The dosage form may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or inhalation form, or suppository. Alternatively, parenteral administration may be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection form. Optionally, pharmaceutically acceptable carriers and / or excipients may be present.

[0161] Topical administration is also within the scope of beneficial use of the present invention. Those skilled in the art will appreciate that topical administration is well within the scope of the present invention, as described in Benson and Watkinson (Eds.), Topical and Transdermal Drug Delivery: Principles and Practice (1st Edition, Wiley 2011, ISBN-13: 978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (2 nd Ed., CRC Press 2002, ISBN-13: 978-0824708610);Osborne and Amann(Eds.):Topical Drug Delivery Formulations(1 st The wide range of possible recipes for providing topical formulations is well known, as exemplified by the contents of The Journal of Clinical Chemistry, Vol. 1, No. 1, pp. 1997-2002, 1998. Ed. CRC Press 1989; ISBN-13: 978-0824781835.

[0162] Pharmaceutical Compositions and Administration Another aspect of the present invention relates to a pharmaceutical composition comprising a compound of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein.

[0163] In certain embodiments of the present invention, the compounds of the present invention are typically formulated into pharmaceutical dosage forms to provide easily controllable dosing of the drug and to present the patient with a smooth and easily handleable product.

[0164] In embodiments of the invention relating to topical use of the compounds of the invention, the pharmaceutical composition is formulated in a manner suitable for topical administration, such as an aqueous solution, suspension, ointment, cream, gel, or sprayable formulation, e.g., for delivery by aerosol, and comprises the active agent together with one or more solubilizers, stabilizers, tonicity enhancers, buffers, and preservatives known to those skilled in the art.

[0165] The pharmaceutical compositions can be formulated for enteral administration, particularly oral or rectal administration. In addition, the pharmaceutical compositions of the present invention can be made into a solid form (including, but not limited to, capsules, tablets, pills, granules, powders, or suppositories) or a liquid form (including, but not limited to, solutions, suspensions, or emulsions).

[0166] The pharmaceutical composition can be formulated for parenteral administration, for example, by iv infusion, intradermal, subcutaneous or intramuscular administration.

[0167] The dosing regimen for the compounds of the present invention will vary depending on known factors such as the pharmacodynamic characteristics of the particular drug and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and severity of the condition; the type of concomitant therapy; the frequency of treatment; the route of administration; the patient's renal and hepatic function; and the desired effect. In some embodiments, the compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses two, three, or four times daily.

[0168] In one embodiment, the pharmaceutical composition or combination of the present invention is administered in a unit dosage of about 1 to 1000 mg of active ingredient for a subject weighing about 50 to 70 kg. The therapeutically effective dosage of the compound, pharmaceutical composition, or combination thereof will depend on the subject's species, weight, age, and personal condition, and the disorder or disease or its severity being treated. A physician, clinician, or veterinarian skilled in the art can readily determine the effective amount of each active ingredient required to prevent, treat, or inhibit the progression of the disorder or disease.

[0169] The pharmaceutical compositions of the present invention can be subjected to conventional pharmaceutical processes such as sterilization, and / or can contain conventional inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers. They can be manufactured by standard processes, such as conventional mixing, granulation, dissolution, or lyophilization processes. Procedures and methods for manufacturing many such pharmaceutical compositions are known in the art, see, for example, L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Edition, 2013 (ISBN 8123922892).

[0170] Manufacturing methods and treatment methods according to the present invention The present invention further encompasses, as a further aspect, the use of a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, as defined in detail above, in a method for the manufacture of a medicament for the treatment or prevention of metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning.

[0171] Similarly, the present invention encompasses a method of treating a patient diagnosed with a disease associated with metal poisoning, particularly Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more particularly Pb poisoning, which method involves administering to the patient a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, as defined in detail herein.

[0172] The present invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be derived. These examples are intended to illustrate the invention and are not intended to limit its scope. [Example]

[0173] Example 1: Synthesis of cyclic tetrapeptides For the compounds described in this example, a scaffold composed of the sequence cyclic-[Xaa-βAla-Xaa-βAla] (SEQ ID NO: 015) (Xaa depicts any α-AA; Scheme 1) was chosen, as in addition to enhancing stability, the βAla was expected to facilitate the difficult intramolecular cyclization of the tetrapeptide. [ka]

[0174] Here, the inventors of the present invention 2+ We present a family of cyclic tetrapeptides designed to detoxify ions. The peptides were examined for their ability to restore Pb-exposed bacteria and human cells, where one particular peptide (8) significantly outperformed a benchmark chelator (CA). Mechanistic studies of the successful peptides shed light on their biological consequences and medical potential.

[0175] We began our research by synthesizing nine side-chain protected linear peptides (Table 1, 1-9). Typically, head-to-tail cyclization occurs in dimethylformamide (DMF) as the solvent, which is extremely rare for peptides shorter than pentamer lengths (White et al., 2011). We aimed to cyclize tetrapeptides in the absence of high-boiling solvents such as DMF. Through condition screening, we found ideal conditions: PyBOP and Hunig's base (1.5 and 3.0 equivalents, respectively) as the coupling reagent and base, respectively, and ultra-high dilution of peptide (0.1 mM) in CHCl for 16-48 hours until complete conversion was achieved. The cyclic peptides were then side-chain deprotected and purified without the need for HPLC, achieving >95% purity in 62-87% yields over the two steps (cyclization and deprotection). HR-ESI-MS and 1 H and 13 C NMR indicated exclusive intramolecular cyclization to form the desired tetramer.

[0176] [Table 1]

[0177] Example 2: In vivo and in vitro detoxification The desalted peptides were then evaluated for their ability to detoxify Pb (Figure 2a-d). We designed two assays for rapid and reliable screening of potential CAs both in vivo in bacteria and subsequently in vitro in human cells. Briefly, DH5α or HT-29 cells were first exposed to Pb(NO3)2 at slightly subminimal inhibitory concentrations and then treated with various concentrations ranging from 0.1 to 10 equivalents of the investigated CA. Cell viability was determined by colony counting or crystal violet staining (Feoktistova et al., 2016) for bacteria and human cells, respectively, and compared with intoxicated cells not treated with any CA as a negative control. Performing both assays proved highly valuable, primarily because the in vivo assay tests CAs on solidified media, eliminating limitations stemming from the compounds' low solubility. On the other hand, testing compounds in human cells, which cannot be performed with insoluble compounds, is more relevant for medical purposes.

[0178] Of the nine peptides, four performed exceptionally well in detoxifying intoxicated E. coli compared to benchmark compounds (Fig. 2a). All four of them contained at least one Cys and Pb. 2+ Peptides 1, 2, 6, and 8 contain additional residues capable of binding to Pb; Cys, DCys, Met, and Asp, respectively. Treatment with 1 increased recovery by more than eight-fold, while substitution of one LCys with DCys reduced the detoxification capacity of peptide 2. This is due to its preferred, unique, one-sided orientation. 2+This indicates the requirement that the binding moieties capturing the Pb should face in the same direction. Surprisingly, homo-functionalized peptides 3–5 exhibited poor activity, poor metal selectivity, or low Pb affinity. Notably, linear analogs of peptides 1–8 were also tested in vivo and in all cases demonstrated little detoxification ability. We conclude that in addition to the expected enhanced proteolytic stability, cyclization also fosters preorganization of the ligand, which confers its metal affinity by improving its coordination properties.

[0179] Despite their high in vivo activity, peptides 1, 2, and 6 exhibited poor water solubility, reducing their effectiveness as potential CAs. Attempts to dissolve them, such as various pH conditions, formulation with PEG, or cosolvent systems with DMSO, failed. Therefore, two analogs of 1 were synthesized in which βAla was replaced with βAsp or βhGlu to form peptides 1a and 1b, respectively. Although these peptides showed high solubility as Na or Ca salts, their detoxification ability in bacteria was unsatisfactory (Figure 2a). Their poor activity was due to (a) Pb 2+ This may be related to either (a) competition by the two carboxylates for coordination with the cation, which destabilizes the complex formation, or (b) a decrease in their metal selectivity and coordination with alkali or alkaline earth metal ions.

[0180] Nevertheless, we tested 1a and other soluble peptides in vitro for their ability to rescue intoxicated human cells (Fig. 2b). Among all compounds, peptide 8 showed a significantly greater degree of Pb recovery, with a recovery rate of 334 ± 42%, compared with 110 ± 10% for DMSA and 95 ± 16% for Na2CaEDTA. 2+ This peptide was dramatically superior at high concentrations compared to the benchmark drug and glutathione (GSH) as a natural reference peptide (Fig. 2b, 2c). 2+We also observed similar patterns for most compounds between the two assays, suggesting that the efficacy and chelation mechanism are similar in both systems.

[0181] Administration of EDTA as a Ca 2+ To reduce the unwanted depletion of ions, its Na salt was changed to Na2CaEDTA. Therefore, in the case of 8, it was tested whether the counter cation also affects its activity (Fig. 2d). Unlike EDTA, which shows high activity as a Ca-salt, 8 is hardly affected by the counter cation (Fig. 2d). These differences are due to the Pb content of 8. 2+ Unlike EDTA, the ability to bind to Ca is 2+ The lower activity of Ca₈ at high concentrations is associated with the slightly lower solubility of this salt compared to Na₂₈.

[0182] To conclude the efficacy of 8, we evaluated its in vitro toxicity (Fig. 2e), which was dramatically lower than that of DMSA and Na2CaEDTA, inhibiting the viability of only 15 ± 5% of the population.

[0183] material and method The peptides described herein are synthesized according to the reactions shown in Scheme 2. R represents the side chain of an α- or β-amino acid. The side chain can be protected with an appropriate protecting group (R'). Tetrapeptides are obtained by standard solid-phase peptide synthesis (SPPS) using a standard Fmoc-based protocol on chlorotrityl chloride resin. Cleavage (1% TFA) is achieved using TFA in CHCl for five rounds of 1 min each. Cyclization is achieved by reacting the side-chain-protected peptide with PyBOP (as a coupling reagent) and Hunig's base (DIPEA; as a base) in a ratio of 1.5 equivalents of PyBOP to 3 equivalents of base relative to the peptide. The peptide is highly diluted (0.1 mM) to avoid dimerization, and the solvent is CHCl alone. The reaction mixture is incubated overnight (16-48 hours). The side chains are deprotected with a TFA cocktail adjusted for the individual amino acid composition. Typically, a mixture of TFA:TIPS:EDT:HO (87.5:2.5:7.5:2.5) is applied for 1 h. Finally, the cyclic tetrapeptide is purified by precipitation in aqueous solution without the need for HPLC. Purity of over 95% and yields ranging from 62% to 87% (after purification) are achieved. In the final step, the peptide is purified by Cl precipitation because TFA is toxic. - The ions are reacted with HCl to replace the TFA anions. Complete removal of TFA is achieved by 19 Monitored by F NMR.

[0184] [ka]

[0185] The following cyclic tetrapeptides were synthesized as described above. Cys-βAla-Cys-βAla (SEQ ID NO: 001) HRMS(ESI)m / z:C 12 H 21 N4O4S2 + [M+H] + Calculated value: 349.09987; Measured value: 349.09946 Cys-βAla-Met-βAla (SEQ ID NO: 006) HRMS(ESI)m / z:C 14 H 25 N4O4S2 + [M+H] + Calculated value for: 377.13117; Measured value: 377.13120 His-βAla-His-βAla (SEQ ID NO: 004) HRMS(ESI)m / z:C 18 H 26 N8O4 2+ [M+2H] 2+ Calculated value: 209.10330; Measured value: 209.10341 Cys-βAla-His-βAla (SEQ ID NO: 007) HRMS(ESI)m / z:C 15 H 23 N6O4S + [M+H] + Calculated value for: 383.14960; Measured value: 383.14971 Asp-βAla-Asp-βAla (SEQ ID NO: 005) HRMS(ESI)m / z:C 14 H 19 N4O8 - [MH] - Calculated value: 371.12084; Measured value: 371.12065 Cys-βAla-Asp-βAla (SEQ ID NO: 008) HRMS(ESI)m / z:C 13 H 21 N4O6S + [M+H] + Calculated value for: 361.11763; Measured value: 361.11771 Cys-βAla-DCys-βAla (SEQ ID NO: 002) HRMS(ESI)m / z:C 12 H 21 N4O4S2 + [M+H] + Calculated value for: 349.09987; Measured value: 349.09978 Cys-βAsp-Cys-βAsp (SEQ ID NO: 010) HRMS(ESI)m / z:C 14 H 19 N4O8S2 - [MH] - Calculated value for: 435.06498; Measured value: 435.06564 Cys-βAla-Phe-βAla (SEQ ID NO: 009) HRMS(ESI)m / z:C 18 H 25 N4O4S + [M+H] + Calculated value for: 393.15910; Measured value: 393.15888 Met-βAla-Met-βAla (SEQ ID NO: 003) HRMS(ESI)m / z:C 16 H 28 N4O4SNa + [M+Na] + Calculated value for: 427.14442; Measured value: 427.14447

[0186] In vivo recovery test A single colony of DH5α E. coli WT cells was grown overnight in Tris minimal medium without antibiotics (TMM, pH 6.0; 5 mL) at 37°C and 220 rpm. The culture was then diluted to OD with additional TMM up to a total volume of 5 mL. 600 Dilute to 0.03 and measure the OD 600 The cell density was monitored. When the cell density reached 0.25, 1 mL of the culture was transferred to a cell culture tube and labeled as a positive control. To an additional 3 mL of the culture, 36 μL of Pb(NO3)2 1M was added (final concentration 12 mM). Both cultures were shaken at 37 °C and 220 rpm for an additional 5 hours.

[0187] Aqueous stock solutions of each CA were plated onto freshly prepared agar LB plates so that the final concentration of each compound was equal to 0.5, 1, 2, 5, and 10 equivalents relative to the amount of Pb(NO3)2 in 50 μL of pre-toxic medium. Stock solutions were prepared so that plating 30 μL of each solution and uniform spreading provided the desired amount of CA. To two additional plates, 30 μL of HO was added.

[0188] 50 μL of pre-toxic culture was evenly spread onto each CA-containing plate 5 hours before adding the metal to the culture. A Pb-containing culture was also placed on one of the two HO-containing plates, designated as a negative control. Finally, 50 μL of untoxicated culture was placed on the second HO-containing plate, designated as a positive control. All plates (positive and negative controls and five plates for each test compound) were then incubated overnight at 37°C. The plates were then photographed, and colonies were counted (using a Vilber Quantum Visualization System). Recovery for each concentration of CA was calculated according to Equation 1:

number

[0189] Each experiment was performed on three independent occasions, and values ​​are the mean ± SD of more than three replicates each performed in triplicate.

[0190] In vitro recovery test HT-29 cells (purchased from ATCC) were grown in 25 mM HEPES RPMI-1640 medium supplemented with 1% L-glutamine, 1% penicillin / streptomycin, and 10% fetal calf serum (FCS) Super (standard) at 37°C and 5% CO2. A 96-well plate was prepared so that each well contained 10,000 cells in 100 μL of medium, and the cells were allowed to adhere overnight. All wells except the positive control received 10 μL of 22 mM Pb(NO3)2 (final concentration 2 mM). 10 μL of HO was added to the positive control well. Sixty minutes after metal addition, 10 μL of each solution of test CA (2.4, 6, 12, 24, 48, and 120 mM) was added to reach final concentrations of 0.2, 0.5, 1, 2, 4, and 10 mM (0.1, 0.25, 0.5, 1, 2, and 5 equivalents, respectively). 10 μL of HO was added to positive control wells without metal and to negative control wells containing metal but no CA. Each condition was performed in triplicate. The plates were incubated at 37°C and 5% CO for an additional 23 hours, after which the medium was removed, and each well was washed with fresh medium and 50 μL of crystal violet solution (0.5% crystal violet powder in 20 mL MeOH and 80 mL HO). The plates were then gently shaken (60 rpm) for 20 minutes. The plates were then washed with HO until no unbound dye was observed and allowed to dry overnight. 200 μL of MeOH was added to each well, and the plates were gently shaken (60 rpm) for 20 minutes, after which their absorbance at 560 nm was read using a plate reader. The recovery rate of each concentration of CA was calculated according to Equation 2.

number

[0191] Each experiment was performed on three independent occasions, and values ​​are the mean ± SD of more than three replicates each performed in triplicate.

[0192] In vitro toxicity testing HT-29 cells (purchased from ATCC) were grown at 37°C and 5% CO2 in 25 mM HEPES RPMI-1640 medium supplemented with 1% L-glutamine, 1% penicillin / streptomycin, and 10% fetal calf serum (FCS) Super (standard). A 96-well plate was prepared so that each well contained 10,000 cells in 100 μL of medium, and the cells were allowed to adhere overnight. To all wells except the positive control, 10 μL of each test CA solution (2.4, 6, 12, 24, 48, and 120 mM) was added to reach final concentrations of 0.2, 0.5, 1, 2, 4, and 10 mM. 10 μL of HO was added to the positive control well. Each condition was performed in triplicate. The plates were incubated at 37°C and 5% CO2 for 24 hours, after which the medium was removed, and each well was washed with fresh medium and 50 μL of crystal violet solution (0.5% crystal violet powder in 20 mL MeOH and 80 mL H2O). The plates were then gently shaken (60 rpm) for 20 minutes. The plates were then washed with H2O until no unbound dye was observed and dried overnight. 200 μL of MeOH was added to each well, and the plates were gently shaken (60 rpm) for 20 minutes, after which their absorbance at 560 nm was read using a plate reader. The toxicity of each concentration of CA was calculated according to Equation 3.

number

[0193] Each experiment was performed on three independent occasions, and values ​​are the mean ± SD of more than three replicates each performed in triplicate.

[0194] The in vitro and in vivo assay settings for the determination of Pb detoxification capacity are shown in Table 2.

[0195] [Table 2]

[0196] Example 3: Peptide 8a In vitro and in vivo detoxification results The peptide that emerged as the best among all investigated peptides and also beat the standard of care (SOC) (Figure 4) has the sequence cyclic [SAsp-βAla-Asp-βAla] (8a; SEQ ID NO: 16). [ka]

[0197] Peptide 8a was then tested in mice. Forty male mice (C57BL / 6), 6–8 weeks old, were given 20 mM Pb(OAc)2 solution as their sole water supply for 7 days (days 1–7). This intoxication route mimics chronic exposure in humans. Two days after returning to clean water (day 9), they were randomly divided into five groups of eight mice each. They received 30 mg kg 2 Pb(OAc)2 once daily for 7 days, except for group 1, which served as a negative control. -1 were treated with either CaNa2EDTA, DMSA, or 8a at concentrations of 0.05 (Table 3).

[0198] Blood samples (100 μL) were collected from mice on days 10–15 before dosing and on day 18, 2 days after the final dose, when the experiment was terminated. Urine was also collected from 34 mice on day 18 and kept frozen until analysis.

[0199] [Table 3]

[0200] Blood samples taken on the final day clearly show that 8a is more effective than the two SOCs when administered both orally and IV (Table 3, Figure 5A). Specifically, when administered IV, 8a reduced mean blood lead levels (BLLs) by 2.1-fold compared to untreated and 1.6-fold compared to CaNa2EDTA (administered IV). When administered orally, the peptide reduced BLLs by 1.9-fold compared to untreated and 1.3-fold compared to DMSA.

[0201] The Pb content in the urine of 34 (of 40) mice collected on the final day of the experiment indicates that the mechanism of action of 8a is through chelation and excretion of toxic metals via urine. The higher Pb levels in the urine of groups 4 and 5 are consistent with the decreased BLL in these groups compared with groups 1–3. Comparing IV and oral administration of the peptide, Pb was excreted 2.9- and 2.8-fold higher than in the untreated group, respectively. The peptide also allowed for increased Pb removal compared with SOC, ranging from 1.3- to 2.2-fold.

[0202] Water remediation with immobilized peptides Tightly and selectively Pb 2+ Two peptides expected to bind ions were immobilized to a solid support with a long, flexible linker ((PEG2)2) and a photocleavable moiety (Figure 6).

[0203] Additionally, a negative control (0f) was synthesized in which the second PEG2 was acetylated. All three devices were then subjected to Pb(NO3)2 from a 25 mM Pb(NO3)2 solution. 2+ Their ability to trap ions was tested. One hour after adding the metal solutions to the device, the solutions were filtered and the Pb concentration in each was quantified by ICP-MS. The effectiveness was calculated by dividing the concentration of each solution by the concentration found in the original solution at 100% Pb content (Figure 7).

[0204] While 0f was unable to reduce the Pb concentration in the contaminated solution, 1f and 8f reduced the Pb concentration by 62±4% and 36±7%, respectively (Fig. 7, left dark gray bars), and removed Pb from the aqueous solution. 2+ Their effectiveness in removing ions was demonstrated.

[0205] The resins were then treated with 100 mM Na2EDTA solution for 10 min, and their Pb concentrations were quantified to demonstrate effective resin regeneration (light gray bars in Figure 7). The filtration experiment was then repeated (dark gray right bars in Figure 7), showing similar results to the first round, demonstrating that it is possible to regenerate the resin by thoroughly washing the Pb with a cost-effective EDTA solution.

[0206] To detect the metal selectivity of our device, similar filtration experiments were performed with equimolar mixtures of ZnCl2 + Pb(NO3)2 and CaCl2 + Pb(NO3)2 and human serum (HBS) spiked with 25 mM Pb(NO3)2 (Figure 8). The Pb concentration, as well as the Ca or Zn concentrations in the first two experiments, revealed that 1f and 8f do not capture these essential metals, as minimal amounts of Zn and Ca were detected in the filtrate. Notably, these devices removed Pb to the same extent as in the first experiment in the absence of additional metal salts, demonstrating the metal selectivity of our device. Similar results were achieved with Pb-spiked HBS (Figure 8).

[0207] [References] Sears, Margaret E. Chelation: Harnessing and Enhancing Heavy Metal Detoxification - A Review. 2013. The Scientific World Journal, Volume 2013, Article ID 219840, 13 pages White, C. J.; Yudin, A. K. Contemporary Strategies for Peptide Macrocyclization. Nat. Chem. 2011, 3 (7), 509-524. https: / / doi.org / 10.1038 / nchem.1062. Feoktistova, M.; Geserick, P.; Leverkus, M. Crystal Violet Assay for Determining Viability of Cultured Cells. Cold Spring Harb. Protoc. 2016, 2016 (4), 343-346. https: / / doi.org / 10.1101 / pdb.prot087379.

Claims

1. Formula 1: 【Chemistry 1】 (In the formula, Each R, independently of any other R, is —CH 3 and -H, R A1 and R A2 are, independently of each other, C 1-4 - alkyl or phenyl, wherein said C 1-4 -Alkyl or the phenyl is -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -SO 3 H, -COOH, -NH 2 , -CONH 2 , -NH-C(=NH)(NH 2 ), a 5-10 membered heterocycle, a cyclic hydrocarbon moiety containing 3 to 10 carbon atoms, wherein The 5- to 10-membered heterocyclic ring or the cyclic hydrocarbon moiety may be C 1-4 -alkyl, —SH, (═S), —S—C 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -SO 3 H, (=O), -COOH, -NH 2 , -CONH 2 and optionally substituted with one or more substituents selected from At least one of R A1 and R A2 contains a heteroatom S; R B1 and R B2 are independent of each other. -H, or ・-OH, -SH, -S-C 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH 2 , —NH—C 1-4 -Alkyl, -NH-C(=NH)(NH 2 ), -CONH 2 , -SO 3 H, a 5- to 10-membered heterocyclic ring or a hydrocarbon moiety containing 1 to 12 C atoms, wherein the 5- to 10-membered heterocyclic ring or the hydrocarbon moiety is selected from -OH, (=O), -SH, (=S), -S-C 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH 2 , —NH—C 1-4 -Alkyl, -NH-C(=NH)(NH 2 ), -CONH 2 , -SO 3 optionally substituted with one or more substituents independently selected from H and a 5- to 10-membered heterocycle; a linker suitable for binding to a detectable marker or solid support; a detectable marker, optionally linked by a linker, or - a linker attached to a solid support) Compound.

2. The compound is represented by formula 2, 3, 4, 5, 6 or 7 【Chemistry 2】 2. The compound of claim 1, which is a compound of formula:

3. R A1 and R A2 are each independently -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -SO 3 H, -COOH, -NH 2 , -CONH 2 , a 5- to 10-membered heterocycle, a cyclic hydrocarbon moiety containing 3 to 6 carbon atoms, C 1-4 -alkyl, where The 5- to 10-membered heterocyclic ring or the cyclic hydrocarbon moiety is C 1-4 -Alkyl, -SH, (=S), -S-C 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -SO 3 H, (=O), -COOH, -NH 2 , -CONH 2 may be optionally substituted with one or more substituents selected from 3. The compound according to claim 1 or 2.

4. R A1 and R A2 The compound according to any one of claims 1 to 3, wherein the cyclic hydrocarbon moiety is selected from cyclopentyl, cyclohexyl and phenyl.

5. R A1 and R A2 are each independently —SH, —S—CH 3 , -SeH, -Se-CH 3 , -SO 3 H, -COOH, -NH 2 , -CONH 2 substituted by one or two substituents independently selected from imidazolyl, mercaptoimidazolyl, thiofuranyl, indolyl and phenyl; 1-3 alkyl, where The compound of any one of claims 1 to 4, wherein the phenyl may be optionally substituted with one or more substituents selected from -SH and -SeH.

6. R A1 and R A2 is -CH 2 -SH, -(CH 2 ) 2 -SH, -CH 2 -S-CH 3 , -(CH 2 ) 2 -S-CH 3 , -CH(SH)(-CH 2 -SH), -CH 2 -CH(SH)(-CH 2 -SH), -CH(SH)(-COOH), -CH(SH)-CH 2 -COOH, -CH 2 -CH(SH)(-COOH), -phenyl-SH, -CH 2 -SO 3 H, -(CH 2 ) 2 -SO 3 H-CH 2 -COOH, -(CH 2 ) 2 -COOH, -CH 2 -NH 2 , -(CH 2 ) 2 -NH 2 , -CH 2 -CONH 2 , -(CH 2 ) 2 -CONH 2 , -CH 2 -imidazolyl, -CH 2 -mercaptoimidazolyl and -CH 2 6. The compound of claim 1, wherein the aryl, aryl, phenyl, aryl ...

7. R B1 and R B2 teeth, -H, or ・-OH, -SH, -S-C 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH 2 , —NH—C 1-4 -Alkyl, -NH-C(=NH)(NH 2 ), -CONH 2 , -SO 3 H, a 5- to 10-membered heterocyclic ring or a hydrocarbon moiety containing 1 to 12 C atoms (wherein the 5- to 10-membered heterocyclic ring or the hydrocarbon moiety is -OH, (=O), -SH, (=S), -S-C 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH 2 , —NH—C 1-4 -Alkyl, -NH-C(=NH)(NH 2 ), -CONH 2 , -SO 3 and optionally substituted with one or more substituents independently selected from H and a 5- to 10-membered heterocycle. The compound of any one of claims 1 to 6, independently selected from:

8. R B1 and R B2 teeth, -H, or ・-OH, -SH, -S-C 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH 2 , —NH—C 1-4 -Alkyl, -NH-C(=NH)(NH 2 ), -CONH 2 , -SO 3 H, 5- to 10-membered heterocycle, cyclopentyl, cyclohexyl, phenyl or C 1-8 Alkyl (wherein cyclopentyl, cyclohexyl, phenyl or C 1-8 Alkyl is —OH, —SH, —S—C 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH 2 , —NH—C 1-4 -Alkyl, -NH-C(=NH)(NH 2 ), -CONH 2 , -SO 3 and optionally substituted with one or more substituents independently selected from H and a 5- to 10-membered heterocycle. The compound of any one of claims 1 to 7, independently selected from:

9. R B1 and R B2 is -H, -C 3-6 -Alkyl, -CH 2 -phenyl, -SH, -(CH 2 ) m -SH, -(CH 2 ) m -COOH and -(CH 2 ) r 9. The compound of claim 1, wherein m and r are independently selected from -CONH2, and m and r are 0, 1, 2 or 3.

10. R A1 and R A2 and / or R B1 and R B2 10. The compound according to any one of claims 1 to 9, wherein the heterocycle is selected from piperidinyl, piperazinyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, pyrazolyl, imidazolyl, mercaptoimidazolyl, thiofuranyl, oxazolonyl, indolyl, mercaptopurinyl, benzothiophenyl.

11. R A1 and R A2 are the same, and / or R B1 and R B2 The compound according to any one of claims 1 to 10, wherein

12. the detectable marker is selected from a moiety comprising a dye, an affinity tag, a magnetic bead, and a radioisotope; and / or the linker is a hydrocarbon moiety containing up to 50 C atoms, in which one or more C atoms may optionally be replaced by O, S or N, and / or The solid support is a resin, a bead, an electrode surface or the bottom / wall of a reaction vessel; The compound according to any one of claims 1 to 11.

13. A pharmaceutical composition comprising the compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

14. A metal complex comprising a ligand and a metal, wherein the ligand is the compound according to any one of claims 1 to 12.

15. Formula 10: 【Transformation 3】 (In the formula, each R, independent of any other R, is independently selected from —CH 3 and —H; R A1 and R A2 are, independently of one another, C 1-4 -alkyl or phenyl, wherein said C 1-4 -alkyl or said phenyl is substituted by one or more substituents independently selected from -SH, -S-C 1-4 -alkyl, -SeH, -Se-C 1-4 -alkyl, -SO 3 H, -COOH, -NH 2 , -CONH 2 , -NH-C(═NH)(NH 2 ), a 5- to 10-membered heterocycle, a cyclic hydrocarbon moiety containing 3 to 10 carbon atoms, and wherein said 5- to 10-membered heterocycle or said cyclic hydrocarbon moiety may optionally be substituted with one or more substituents selected from C 1-4 -alkyl, —SH, (═S), —S—C 1-4 -alkyl, —SeH, —Se—C 1-4 -alkyl, —SO 3 H, (═O), —COOH, —NH 2 , —CONH 2 ; At least one of R A1 and R A2 contains a heteroatom S; R B1 and R B2 are independently -H, or - selected from -OH, -SH, -S-Ci_4-alkyl, -SeH, -Se-Ci_4-alkyl, -COOH, -NH2, -NH-Ci_4-alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, a 5- to 10-membered heterocycle or a hydrocarbon moiety containing 1 to 12 C atoms, wherein the 5- to 10-membered heterocycle or the hydrocarbon moiety is -OH, (=O), -SH, (=S), -S-Ci_4-alkyl, -SeH, -Se-Ci_4-alkyl, -COOH, -NH2, -NH-Ci_4-alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H optionally substituted with one or more substituents independently selected from H and a 5- to 10-membered heterocycle; a linker suitable for binding to a detectable marker or solid support; a detectable marker, optionally linked by a linker, or - a linker attached to a solid support) A pharmaceutical composition for the treatment of a disease comprising a compound of formula (I).

16. R A1 and R A2 are each independently C 1-4 -alkyl substituted by one or more substituents independently selected from -SH, -S-C 1-4 -alkyl, -SeH, -Se-C 1-4 -alkyl, -SO 3 H, -COOH, -NH 2 , -CONH 2 , a 5- to 10-membered heterocycle, a cyclic hydrocarbon moiety containing 3 to 6 carbon atoms, wherein 16. The pharmaceutical composition of claim 15, wherein the 5- to 10-membered heterocycle or the cyclic hydrocarbon moiety may be optionally substituted with one or more substituents selected from C 1-4 -alkyl, -SH, (=S), -S-C 1-4 -alkyl, -SeH, -Se-C 1-4 -alkyl, -SO 3 H, (=O), -COOH, -NH 2 , -CONH 2 .

17. R A1 and R A2 are each independently a C 1-3 alkyl substituted by one or two substituents independently selected from —SH, —S—CH 3 , —SeH, —Se—CH 3 , —SO 3 H, —COOH, —NH 2 , —CONH 2 , imidazolyl, mercaptoimidazolyl, thiofuranyl, indolyl, and phenyl, wherein 17. The pharmaceutical composition of claim 15 or 16, wherein the phenyl can be optionally substituted with one or more substituents selected from -SH and -SeH.

18. R B1 and R B2 are -H, or -OH, -SH, -S-Ci_4-alkyl, -SeH, -Se-Ci_4-alkyl, -COOH, -NH2, -NH-Ci_4-alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, a 5- to 10-membered heterocyclic ring or a hydrocarbon moiety containing 1 to 12 C atoms (wherein the 5- to 10-membered heterocyclic ring or the hydrocarbon moiety is -OH, (=O), -SH, (=S), -S-Ci_4-alkyl, -SeH, -Se-Ci_4-alkyl, -COOH, -NH2, -NH-Ci_4-alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H and optionally substituted with one or more substituents independently selected from H and a 5- to 10-membered heterocycle. The pharmaceutical composition according to any one of claims 15 to 17, wherein the compound is independently selected from the group consisting of:

19. A pharmaceutical composition described in any one of claims 15 to 18 for the treatment of metal poisoning.

20. A method for removing and / or detecting metals from / in a substrate, said method comprising using a compound according to any one of claims 1 to 12.

21. The method of claim 20, wherein the metal is selected from Pb, As, Cd and Hg, and / or the substrate is soil or an aqueous solution or aqueous suspension.

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