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 the cyclic tetrapeptides in the treatment of diseases, particularly metal poisoning, and in the diagnosis of said diseases. Also provided are methods for removing or detecting said metals by applying the cyclic tetrapeptides to materials such as contaminated soils and waters. [Background technology]

[0002] Toxic metals such as lead (Pb), arsenic (As), mercury (Hg) and cadmium (Cd) can be found in polluted soil or water, where they pose 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 therefore can 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 per year worldwide. Alarmingly, one in three children suffer from Pb poisoning, and even in the United States, more than 3% of children are found to have dangerous 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+ It is distributed in soft tissues, with the highest accumulation levels in the liver and kidney. 2+ Due to the similar ionic radius, Pb 2+ Pb 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 crossing the placenta.

[0006] Chelation therapy is the latest treatment for Pb poisoning. It is based on the administration of drugs called chelating agents (CAs), which should ideally 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) ability to penetrate cells and tissues. The 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 above mentioned requirements. However, despite being a temporary treatment for Pb poisoning, they have significant drawbacks, mainly low metal selectivity that results in essential metals being depleted from the body during treatment, which increases drug toxicity. Moreover, EDTA cannot cross cell membranes, limiting its use to extracellular targets. They are the first-line treatment for Pb poisoning. 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 in the treatment of metal poisoning and in its diagnosis, 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, as well as in further advantageous aspects as explained in the dependent claims, the examples, the figures and the general description of this specification. Summary of the Invention

[0009] A first aspect of the present invention relates to compounds of formula I, and in particular formula Ia: [ka] (In the formula, each R, independent of any other R, is independently selected from: -CH3 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 -alkyl, -SO3H, -COOH, -NH2, -CONH2, -NHC(=NH)(NH2), a 5-10 membered heterocycle, a cyclic hydrocarbon moiety containing 3 to 10, particularly 3 to 6, carbon atoms, wherein the 5-10 membered heterocycle 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-C1-4 -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 the cyclic hydrocarbon moiety is selected from -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 5-10 membered heterocycle; a linker suitable for binding to a detectable marker or a solid support, a detectable marker, optionally linked by a linker, or A linker attached to a solid support is) The present invention relates to a compound of the formula:

[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 an α-amino acid and βXaa represents a β-amino acid.

[0011] Cyclic tetrapeptides are suitable for binding metals. Pb 2+ The distinction between toxic metals such as arginine 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 above formulas 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 a variety of 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 moiety R, which contains functional groups such as alcohol, amide, carboxylic acid or primary amine. B This 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 accomplished by a linker, a linker attached to a solid support, or a detectable marker in. Such cyclic tetrapeptides can be used in the diagnosis of metal poisoning, in determining the degree of contamination of a substrate 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, the thiol and / or carboxylic acid moieties can be linked to the metal in their deprotonated form via suitable moieties in Pb 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 a disease.

[0021] A fourth aspect of the invention relates to the use of a compound according to the first aspect of the 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 pharma- ceutically acceptable salt thereof, and at least one pharma- ceutically acceptable carrier, diluent or excipient.

[0023] A fifth aspect of the invention provides 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 the steps of:

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

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

[0027] A seventh aspect of the invention relates to a method for detecting a metal in a substrate, comprising using a compound according to the first aspect of the 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 matrices such as contaminated water or soil.

[0029] Terms and Definitions For 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 shall control.

[0030] The terms "comprising," "having," "containing," and "including," and other similar forms and their grammatical equivalents, as used herein, are intended to be equivalent in meaning and to be non-limiting in that the item or items preceding these words are not meant to be a closed enumeration 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 the disclosure 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 directed to 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 (e.g., 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 techniques.

[0035] The term "tetrapeptide" in the context of this specification denotes a molecule consisting of four amino acids forming 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 represented in Formula 1.

[0037] Amino acid residue sequences are given from the amino terminus 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 the amino acid name or code refers to the corresponding D- or (2R)-amino acid. Sequences are written from left to right in the amino- to carboxyl-terminus direction. In accordance with standard nomenclature, α-amino acid residue sequences are named by either the three-letter or one-letter code, 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 β-carbon identical to that of 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 heterocyclyl" relates to a compound consisting of 5-10 carbon atoms, one or more carbon atoms being replaced by a heteroatom N, S or O, in particular N. Similarly, "5-6 membered heterocyclyl" consists of 5-6 carbon atoms, one or more carbon atoms being replaced by a heteroatom N, S or O, in particular N. The carbon atoms and the heteroatom or atoms 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, in particular 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 that contains a carbon-carbon single bond, a double bond and / or a triple bond, in particular 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 5-6 -Cycloalkyl relates to a saturated hydrocarbon ring having 5 or 6 carbon atoms.

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

[0044] [Figure 1]FIG. 1 shows DMSA and EDTA as benchmark drugs for Pb poisoning. [Figure 2a] Figure 1 shows the detoxification potential of the test peptides compared to the benchmark drug and glutathione (GSH) at the highest dose concentration in vivo in DH5α cells at 120 mM (10 equivalents). Values ​​are the mean + SD of more than three repeats, each performed in triplicate. [Figure 2b] Figure 1 shows the detoxification potential of the test peptides compared to the benchmark drug 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] FIG. 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] FIG. 1 shows the concentration-dependent detoxification capacity in HT-29 cells of EDTA and 8 as Ca vs. Na salts. 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 the 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 dose-dependent recovery of HT-29 cells treated with Pb(NO3)2 (2 mM) followed by Na28a, CaNa2EDTA, and Na2DMSA (1 h after addition of Pb2+ ions; values ​​were calculated relative to cells intoxicated with Pb2+ ions as a negative control). [Figure 5a] FIG. 1 shows the mean blood lead levels (BLL) of eight mice per group taken at the end of the study (day 18) and analyzed by ICP-MS. [Figure 5b] FIG. 13 shows urinary Pb from 8 mice per group (only 34 out of 40) collected at the end 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] FIG. 14 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 Pb concentrations detected by ICP-MS and calculated relative to equimolar ZnCl2+Pb(NO3)2 and CaCl2+Pb(NO3)2 solutions and original solutions of human serum spiked with Pb(NO3)2 for 0f (black bars), 1f (light grey bars) and 8f (medium grey bars) (all salt solutions are 25 mM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] A first aspect of the present invention relates to compounds of formula I, and in particular formula Ia: [ka] (In the formula, each R, independent of any other R, is independently selected from: -CH3 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-alkyl, -SO3H, -COOH, -NH2, -CONH2, -NHC(=NH)(NH2), a 5-10 membered heterocycle, a cyclic hydrocarbon moiety containing 3 to 10, particularly 3 to 6, carbon atoms, wherein the 5-10 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, -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, -SOH, -COOH, -NH, -CONH; 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, a 5-10 membered heterocycle or a hydrocarbon moiety containing 1 to 12 C atoms, wherein the 5-10 membered heterocycle or the cyclic hydrocarbon moiety is selected from -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 5-10 membered heterocycle; a linker suitable for binding to a detectable marker or a solid support, a detectable marker, optionally linked by a linker, or A linker attached to a solid support is) The present invention relates to a compound of the formula:

[0046] In some embodiments, at least one R is H and the other 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 heteroatoms of form 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 certain 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] TIFF2024508579000004.tif187143 TIFF2024508579000005.tif58139

[0051] Cyclic tetrapeptides can be formed from L- or D-amino acids or mixtures thereof. For economic reasons, in particular L-amino acids are used since 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 oriented in the same direction to capture

[0053] In certain 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 connected to the α-carbon atom by an up-wedge bond, or R A1 and R A2 are both bonded to the α-carbon atom by down wedge bonds.

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

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

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

[0057] In some embodiments, R A1 and R A2 are, independently of each other, -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 C substituted by one or more, in particular one or two, substituents independently selected from: -alkyl, -SOH, -COOH, -NH, -CONH, 5-10 membered heterocycle, cyclic hydrocarbon moiety containing 3-6 carbon atoms; 1-4 -Alkyl, especially C 1-3 Alkyl, more particularly C 1-2alkyl, wherein the 5- to 10-membered heterocycle or the cyclic hydrocarbon moiety is 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, -SOH, -COOH, -NH, -CONH, 5-10 membered heterocycle, cyclic hydrocarbon moiety containing 3-6 carbon atoms; 1-4 -Alkyl, especially C 1-3 Alkyl, more particularly C 1-2 alkyl, where the cyclic hydrocarbon moiety is 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 called benzothiofuran.

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

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

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

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

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

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

[0068] In some embodiments, the 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, where said phenyl may be optionally substituted with one or more, especially one, substituents selected from -SH and -SeH, especially -SH.

[0073] In some embodiments, R A1 and R A2is 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 an 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, where said phenyl may be optionally substituted with one or more, especially one, substituents 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: -CH2-SH, -(CH2)2-SH, -CH2-S-CH3, -(CH2)2-S-CH3, -CH(SH)(-CH2-SH), -CH2-CH(SH)(-CH2-SH), -CH(SH)(-COOH), -CH(SH)-CH2-COOH, -CH2-CH(SH)(-COOH), -phenyl-SH, -CH2-SO3H, -(CH2)2-SO3H-CH2-COOH, -(CH2)2-COOH, -CH2-NH2, -(CH2)2-NH2, -CH2-CONH2, -(CH2)2-CONH2.

[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 is independently selected from -CH2-SH, -(CH2)2-S-CH3, and -CH2-COOH.

[0079] In one embodiment, -R A1 and R A2 are the same 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, -SOH, -COOH, -NH, -CONH, 5-10 membered heterocycle, 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 -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 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 and / or optionally substituted with one or more, in particular one, substituent selected from: -alkyl, -SO3H, -COOH, -NH2, -CONH2; -R A1 -SH, -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, and -COOH, especially -SH, -SC 1-4 -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, -SOH, -COOH, -NH, -CONH, 5-10 membered heterocycle, 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, -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, substituents independently selected from: alkyl, -COOH, -NH2, -CONH2, a 5- to 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 -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 -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, in which 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; 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 the same and are 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 certain 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 certain embodiments of any of the aspects of the invention, R A1 No or R A2 The alkyl moiety in 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 -alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, a 5-10 membered heterocycle or a hydrocarbon moiety containing 1 to 12 C atoms, Here, the 5- to 10-membered heterocycle 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)(NH2), -CONH2, -SO3H and a 5-10 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 -alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, a 5-10 membered heterocycle or a hydrocarbon moiety containing 1 to 12 C atoms, Here, the cyclic hydrocarbon 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)(NH2), -CONH2, -SO3H and a 5-10 membered heterocycle; are independently selected from

[0087] In order to enhance the water solubility of the cyclic tetrapeptides according to the invention, one or both of the moieties R B1 and R B2 may contain a hydrophilic moiety. In some embodiments, at least one R B1 and R B2 is 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 certain 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 1-12 C atoms, where the 5-10 membered heterocycle or the hydrocarbon moiety is (=O), -SH, (=S), -SC 1-4 -Alkyl, -SeH, -Se-C 1-4 -Alkyl, -COOH, -NH2, -NH-C 1-4 In one embodiment, R is a moiety optionally substituted with one or more substituents independently selected from -alkyl, -NH-C(=NH)(NH), -CONH, -SOH, and a 5-10 membered heterocycle. B1 and R B2 At least one of the following is independently selected from the group consisting of -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 1-12 C atoms, where 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 5-10 membered heterocycle; 5-10 membered heterocycles are -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)(NH2), -CONH2, -SO3H 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 B2Heterocyclyl is 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 Heterocyclyl 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 The heterocyclyl of 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 is 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 -CONH2; 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 of the cyclic tetrapeptides according to the invention and / or metal complexes comprising the cyclic tetrapeptides according to the invention, the cyclic tetrapeptides may comprise a detectable marker.

[0103] In certain 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 affinity tags are Strep-tag, Glutathione-S-Transferase (GST) tag, 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 certain embodiments, the solid support is a resin, a bead, an electrode surface or the bottom / wall of a reaction vessel, in particular an electrode surface, a resin or a bead, more in particular 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-X22, in particular of formula X1-11 or X14-22. [ka] TIFF2024508579000007.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 certain 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, the thiol and / or carboxylic acid moiety binds to a metal in its deprotonated form, as shown below: 2+ (See also FIG. 3). [ka]

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

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

[0119] In certain 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 embodiments 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 a 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 invention relates to the use of a compound according to the first aspect of the 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 a medical context, the compounds according to the first aspect of the invention may be applied according to 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 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 the steps of:

[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 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 the embodiments of the first aspect of the invention.

[0135] The 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 the detection of 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 invention relates to a method for removing metals, particularly metals selected from Pb, As, Cd and Hg, more particularly Pb, from a substrate, particularly soil or an aqueous solution or suspension, comprising using a compound according to the first aspect of the 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 invention relates to a method for detecting a metal, in particular a metal selected from Pb, As, Cd and Hg, more particularly Pb, in a substrate, in particular a soil or an aqueous solution or suspension, comprising using a compound according to the first aspect of the 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, the preparation comprising 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 one embodiment, the coupling agent is 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 number 330645-87-9), HOBt / DIC (benzotriazol-1-ol, CAS number 2592-95-2) and N,N'-di(propan-2-yl)methane diimine, CAS number 693-13-0), DCC (N,N'-dicyclohexylmethane diimine, CAS number 538-75-0), DPPA (diphenylphosphoryl azide, CAS number 26386-88-9).

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

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

[0150] In one embodiment, 3 molar equivalents of base are used relative to the molar amount of the 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 a period of from 12 hours to 72 hours, particularly from 16 hours 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. It is not necessary to heat or cool the reaction mixture.

[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 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 comprising administering to the patient a compound according to the first aspect of the 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 pharmaceutically acceptable salt of said drug.Pharmaceutically acceptable salt comprises a counter ion with the opposite charge to ionized drug.Non-limiting examples of pharmaceutically acceptable anionic salt form 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 pharma- ceutically 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 Therapeutic Drugs for the Treatment of Acetylcholine Chemists, 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 pharma- ceutically acceptable salt thereof and a pharma- ceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharma- ceutically acceptable carriers, such as those described herein.

[0163] In certain embodiments of the invention, the compounds of the 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 compositions are formulated in a manner suitable for topical administration, such as aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol, etc., and contain the active agent together with one or more solubilizers, stabilizers, tonicity enhancers, buffers and preservatives known to those of skill in the art.

[0165] The pharmaceutical composition can be formulated for enteral administration, particularly oral or rectal administration.In addition, the pharmaceutical composition 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 injection, intradermal, subcutaneous or intramuscular administration.

[0167] The dosing regimen of the compounds of the present invention will vary according to 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 extent of the condition; type of concomitant therapy; frequency of treatment; route of administration; renal and hepatic function of the patient; 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 a unit dosage of about 1-1000 mg of active ingredient for a subject weighing about 50-70 kg. The therapeutically effective dosage of the compound, pharmaceutical composition or combination thereof depends on the subject's species, weight, age and personal condition, disorder or disease or its severity being treated. A physician, clinician or veterinarian of ordinary skill 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, for example, by conventional mixing, granulation, dissolving, 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 Ed, 2013 (ISBN 8123922892).

[0170] Manufacturing 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 pharma- ceutically acceptable salt thereof, as de?ned above, in a method for the manufacture of a medicament for the treatment or prevention of metal poisoning, in particular Pb poisoning, As poisoning, Cd poisoning and Hg poisoning, more in particular 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 entails administering to the patient a compound according to the first aspect of the invention, or a pharma- ceutically 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 derive. These examples are intended to illustrate the invention and are not intended to limit its scope. EXAMPLES

[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 because in addition to enhancing stability, the βAla was expected to facilitate the difficult intramolecular cyclization of the tetrapeptide. [ka]

[0174] Here, the inventors have 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) greatly outperformed a benchmark chelator (CA). Mechanistic studies of the successful peptides shed light on their biological consequences and medical potential.

[0175] We began their work by synthesizing nine side-chain protected linear peptides (Table 1, 1-9). Typically, head-to-tail cyclization occurs in dimethylformamide (DMF) as the solvent and is extremely rare for peptides shorter than pentamers (White et al., 2011). We aimed to cyclize the tetrapeptide in the absence of a high boiling point solvent such as DMF. Upon condition screening, we found the following ideal conditions: PyBOP and Hunig's base (1.5 and 3.0 equivalents, respectively) as coupling reagents and base, respectively, and ultra-high dilution of peptide (0.1 mM) in CHCl for 16-48 h until complete conversion was obtained. The cyclic peptides were then side-chain deprotected and purified without the need for HPLC, achieving purity of >95% with yields of 62-87% 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 (Fig. 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 below the minimum inhibitory concentration and then treated with various concentrations ranging from 0.1 to 10 equiv. of the CA to be investigated. Cell viability was determined by colony counting or by crystal violet (Feoktistova et al., 2016) for bacteria and human cells, respectively, and compared to intoxicated cells that were not treated with any CA as a negative control. Mainly, performing both assays proved to be very valuable, since the in vivo assay examines CAs on solidified medium, eliminating limitations originating from the low solubility of the compounds. On the other hand, testing the compounds in human cells, which cannot be performed with insoluble compounds, is more relevant for medical purposes.

[0178] Of the nine peptides, four performed outstandingly in detoxifying intoxicated E. coli compared to the 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 in its preferred unique one-sided configuration; Cys, DCys, Met and Asp, respectively. Treatment with 1 increased recovery by more than eight-fold, whereas substitution of one of the Lcys with DCys reduced the detoxification ability of peptide 2. This is likely due to the fact that Pb 2+This points out the requirement that the binding moieties capturing the Pb should point in the same direction. Surprisingly, homo-functionalized peptides 3-5 showed 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 showed little detoxification capacity. We conclude that in addition to the expected enhanced proteolytic stability, cyclization also fosters pre-organization of the ligand that confers its metal affinity by improving its coordination properties.

[0179] Despite their high activity in vivo, peptides 1, 2 and 6 exhibited low 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 by 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 not satisfactory (Figure 2a). Their low 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 for their ability to rescue intoxicated human cells in vitro (Figure 2b). Among all compounds, peptide 8 was found to have a by far superior ability to rescue Pb with a recovery rate of 334±42%, compared to 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 (Figures 2b, 2c). 2+We also observed similar patterns for most compounds between the two assays, suggesting that the effects and chelation mechanisms are similar in both systems.

[0181] Administration of EDTA as a Ca 2+ To reduce the unwanted depletion of ions, the Na-salt of 8 was changed to Na2CaEDTA. Therefore, in the case of 8, it was tested whether the counter cation also influenced 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-salt of 8. 2+ The ability to bind to Ca, which does not bind to EDTA, 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 denotes the side chain of an α- or β-amino acid. The side chain can be protected by an appropriate protecting group (R'). The 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 with TFA in CHCl for 5 rounds of 1 min each. Cyclization is obtained by reacting the side-chain protected peptide with PyBOP (as coupling reagent) and Hunig's base (DIPEA; as base) in a ratio of 1.5 equivalents of PyBOP and 3 equivalents of base relative to the peptide. The peptides are highly diluted (0.1 mM) to avoid dimerization, and the solvent is CHCl alone. The reaction mixture is incubated overnight (16-48 h). The side chains are deprotected with a TFA cocktail that is 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 precipitation with Cl because TFA is a poison. - The ions are reacted with HCl to replace the TFA anions. The 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 for: 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 for: 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 for: 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 study 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 cultured at OD with additional TMM up to a total volume of 5 mL. 600 Dilute to 0.03 and measure the OD 600 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 h.

[0187] Aqueous stock solutions of each CA were plated onto freshly prepared agar LB plates such that the final concentration of each compound was equal to 0.5, 1, 2, 5, and 10 equivalents compared to the amount of Pb(NO3)2 in the 50 μL pre-poisoning medium. Stock solutions were prepared such that plating 30 μL of each solution and uniform spreading provided the desired amount of CA. To two additional plates, 30 μL of H2O was added.

[0188] 50 μL of pre-poisoned culture was spread evenly on each CA-containing plate 5 h before adding metal to the culture. Pb-containing culture was also placed on one of the two H2O-containing plates and labeled as a negative control. Finally, 50 μL of non-poisoned culture was placed on the second H2O-containing plate and labeled as a positive control. All plates (positive and negative controls and five plates for each test compound) were then incubated overnight at 37°C. Plates were then photographed and colonies were counted (with 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 repetitions performed in triplicate.

[0190] In vitro recovery test 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). 96-well plates were prepared so that each well contained 10,000 cells in 100 μL of medium and 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 H2O was added to the positive control wells. Sixty minutes after the addition of the metals, 10 μL of each solution of the 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 H2O was added to the positive control wells without metals and to the negative control wells with metals but no CA. Each condition was performed in triplicate. The plates were incubated for a further 23 hours at 37°C and 5% CO2, 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), and the plates were 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, the plates were gently shaken (60 rpm) for 20 min, and then their absorbance at 560 nm was read on 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 repetitions 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). 96-well plates were prepared so that each well contained 10,000 cells in 100 μL of medium and cells were allowed to adhere overnight. All wells except the positive control were added with 10 μL of each solution of the test CA (2.4, 6, 12, 24, 48 and 120 mM) to reach final concentrations of 0.2, 0.5, 1, 2, 4 and 10 mM. 10 μL of H2O was added to the positive control wells. 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, 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), and the plates were 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, the plates were gently shaken (60 rpm) for 20 minutes, and then their absorbance at 560 nm was read on 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 repetitions 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 revealed the best results 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) aged 6–8 weeks were fed a 20 mM Pb(OAc)2 solution as the only 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 Pb(OAc)2 once daily for 7 days, except group 1, which served as a negative control. -1 were treated with either CaNa2EDTA, DMSA or 8a at concentrations of 0.01–0.001 (Table 3).

[0198] Blood samples (100 μL) were collected before dosing the mice on days 10–15 and 2 days after the last dose on day 18, 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 on the final day clearly show that 8a is more effective than the two SOCs when administered both orally and IV (Table 3, FIG. 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] Pb content in urine from 34 (of 40; Figure 5B) mice collected on the last 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 urine from groups 4 and 5 are consistent with the decreased BLLs in these groups compared to groups 1–3. Comparing IV and oral administration of the peptide, Pb was excreted 2.9 and 2.8-fold, respectively, compared to the untreated group. The peptide also allowed elevated removal of Pb compared to 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] In addition, a negative control (0f) was synthesized in which the second PEG2 was acetylated. All three devices were then subjected to Pb(NO3)2 acetylation from a 25 mM Pb(NO3)2 solution. 2+ They were tested for their ability to trap ions. One hour after addition of the metal solutions to the device, the solutions were filtered and the Pb concentration in each of them was quantified by ICP-MS. The effectiveness was calculated by dividing the concentration of each solution by the concentration found in the original solution as 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 grey 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 quantified to demonstrate effective resin regeneration (light grey bars in Figure 7). The filtration experiment was then repeated (dark grey right bars in Figure 7), showing similar results to the first round, demonstrating that it is possible to regenerate the resin by extensively 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, since the amount of Zn and Ca detected in the filtrate was minimal. Notably, these devices removed Pb to the same extent as in the first experiment in which no additional metal salts were present, demonstrating the metal selectivity of our device. Similar results were also achieved in 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: 【Chemical 1】 wherein Each R is independently of any other R, -CH 3 and is independently selected from -H, R A1 and R A2 are, independently of each other, C 1-4 -alkyl or phenyl, where the C 1-4 -alkyl or the 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 ), 5- to 10-membered heterocyclic rings, and cyclic hydrocarbon moieties containing 3 to 10 carbon atoms, where The 5- to 10-membered heterocyclic ring 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 and may be optionally substituted with one or more substituents selected therefrom at least one of R A1 and R A2 contains the heteroatom S; R B1 and R B2 are independent of each other · is -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 carbon 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 H and is optionally substituted with one or more substituents independently selected from 5- to 10-membered heterocyclic rings, or · a linker suitable for binding to a detection marker or a solid support, · optionally a detection marker linked by a linker, or · a linker bound to a solid support) compound.

2. The compound according to claim 1, wherein the compound is a compound of formula 2, 3, 4, 5, 6 or 7 【Chemical 2】 .

3. R A1 and R A2 are, independently of one another, -SH, -S-C 1-4 -alkyl, -SeH, -Se-C 1-4 -alkyl, -SO 3 H, -COOH, -NH 2 , -CONH 2 , one or more substituents independently selected from a 5- to 10-membered heterocyclic ring, a cyclic hydrocarbon moiety containing 3 to 6 carbon atoms, and are 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 optionally substituted with one or more substituents selected from: 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 at R and R is selected from cyclopentyl, cyclohexyl and phenyl.

5. R A1 and R A2 are, independently of each other, C 3 alkyl substituted by one or two substituents independently selected from -SH, -S-CH 3 , -SeH, -Se-CH 3 , -SO 2 H, -COOH, -NH 2 , -CONH 1-3 , imidazolyl, mercaptoimidazolyl, thiophenyl, indolyl and phenyl, where The compound according to 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 selected independently from -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 -phenyl, and is a compound according to any one of claims 1 to 5

7. R B1 and R B2 are, · is -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 carbon atoms (wherein the 5- to 10-membered heterocyclic ring or the hydrocarbon moiety is independently 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 H and is optionally substituted with one or more substituents independently selected from 5- to 10-membered heterocyclic rings) The compound according to any one of claims 1 to 6, independently selected from

8. R B1 and R B2 are, · is -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 optionally substituted with one or more substituents independently selected from -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 and one or more substituents independently selected from 5- to 10-membered heterocycles)) The compound according to any one of claims 1 to 7, independently selected from

9. R B1 and R B2 are independently selected from -H, -C 3-6 -alkyl, -CH 2 -phenyl, -SH, -(CH 2 ) m -SH, -(CH 2 ) m -COOH and -(CH 2 ) r -CONH2, and m and r are 0, 1, 2 or 3. The compound according to any one of claims 1 to 8.

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

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

12. The detectable marker is selected from the group consisting of dyes, affinity tags, magnetic beads and radioisotopes, and / or the linker is a hydrocarbon moiety containing up to 50 carbon atoms, wherein one or more carbon atoms may be optionally replaced by O, S or N, and / or the solid support is a resin, beads, 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 consisting of a ligand and a metal, wherein the ligand is the compound according to any one of claims 1 to 12.

15. Formula 10: 【Chemical Formula 3】 wherein each R is independently selected from -CH 3 and -H, independently of any other R, R A1 and R A2 are, independently of one another, C 1-4 -alkyl or phenyl, where the C 1-4 -alkyl or the 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 heterocyclic ring, a cyclic hydrocarbon moiety containing 3 to 10 carbon atoms, where the 5- to 10-membered heterocyclic ring or the cyclic hydrocarbon moiety may be optionally substituted by 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 the heteroatom S; R B1 and R B2 are, independently of one another · -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, where the 5- to 10-membered heterocyclic ring or the hydrocarbon moiety is optionally substituted by one or more substituents independently 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 H and a 5- to 10-membered heterocyclic ring, or · a linker suitable for binding to a detection marker or a solid support, · a detection marker, optionally linked by a linker, or · a linker bound to a solid support), A pharmaceutical composition for the treatment of a disease, comprising a compound of). **Claim 16**: RA1 and RA2 are, independently of each other, C1-4-alkyl substituted by one or more substituents independently selected from -SH, -S-C1-4-alkyl, -SeH, -Se-C1-4-alkyl, -SO3H, -COOH, -NH2, -CONH2, a 5- to 10-membered heterocyclic ring, and a cyclic hydrocarbon moiety containing 3 to 6 carbon atoms, where the 5- to 10-membered heterocyclic ring or the cyclic hydrocarbon moiety may be optionally substituted by one or more substituents selected from C1-4-alkyl, -SH, (=S), -S-C1-4-alkyl, -SeH, -Se-C1-4-alkyl, -SO3H, (=O), -COOH, -NH2, -CONH2. The pharmaceutical composition according to claim 15. **Claim 17**: RA1 and RA2 are, independently of each other, C1-3alkyl substituted by one or two substituents independently selected from -SH, -S-CH3, -SeH, -Se-CH3, -SO3H, -COOH, -NH2, -CONH2, imidazolyl, mercaptoimidazolyl, thiophenyl, indolyl, and phenyl, where the phenyl may be optionally substituted by one or more substituents selected from -SH and -SeH. The pharmaceutical composition according to claim 15 or 16. **Claim 18**: RB1 and RB2 are · -H, or · -OH, -SH, -S-C1-4-alkyl, -SeH, -Se-C1-4-alkyl, -COOH, -NH2, -NH-C1-4-alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, a 5- to 10-membered heterocyclic ring, or a hydrocarbon moiety containing 1 to 12 C atoms (where the 5- to 10-membered heterocyclic ring or the hydrocarbon moiety may be optionally substituted by one or more substituents independently selected from -OH, (=O), -SH, (=S), -S-C1-4-alkyl, -SeH, -Se-C1-4-alkyl, -COOH, -NH2, -NH-C1-4-alkyl, -NH-C(=NH)(NH2), -CONH2, -SO3H, and a 5- to 10-membered heterocyclic ring) independently selected from. The pharmaceutical composition according to any one of claims 15 to 17.

19. The pharmaceutical composition according to any one of claims 15 to 18 for the treatment of metal poisoning.

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

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

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