Platinum complexes, related compositions, and their use in cyanide countermeasures

Abstract

Platinum-thioester complexes, compositions containing such platinum-thioester complexes, methods for treating cyanide poisoning and / or exposure therewith, and related combination therapies and kits therefor.

Description

[Technical Field]

[0001] Priority This application is related to and claims priority to U.S. Provisional Patent Application No. 63 / 63 / 939,647, filed July 29, 2022. The contents of the aforementioned application are incorporated by reference in their entirety into this disclosure.

[0002] Technical Field The present disclosure relates generally to platinum-containing complexes and methods for treating cyanide poisoning (e.g., providing an antidote) by administering platinum complexes to a subject exposed to cyanide (e.g., cyanide gas).

[0003] Government funding This invention was made with government support under NS112107 (CM and RP) awarded by the National Institutes of Health (NIH) and under P200A150136 awarded by the US Department of Education Graduate Assistance in Areas of National Need. The government has certain rights in this invention. [Background technology]

[0004] Cyanide anion (CN - ) are highly toxic due to their ability to disrupt electron transport in cells, which can prevent cells from aerobically producing adenosine triphosphate (ATP) for energy. Cyanide is a persistent threat from accidental and malicious misuse due to its ease of production as a toxic gas and widespread access across multiple industries. Exposure to cyanide can be fatal even in small amounts if inhaled, ingested, or through direct contact, killing organisms as diverse as insects, fish, and humans within seconds to hours.

[0005] The most common cause of smoke inhalation-related deaths is suspected to be the result of cyanide poisoning. Cyanide can be released into smoke when carbon- and nitrogen-rich materials in everyday household products are burned. Anseeuw et al., Cyanide poisoning by fire smoke inhalation: a European expert consensus, European J Emergency Med 20: 2-9 (2013). House fires resulting in the combustion of plant-based materials, including plastic and wood, can also be a source of cyanide. For example, cyanide released by the burning of soundproofing plastic has been implicated in a high morbidity and mortality event at a nightclub in Brazil, which claimed the lives of over 4,200 individuals in a single event. Cyanide and cyanide derivatives are often used in many industries, including metal polishing, mining, photo processing, and chemical production for products such as pesticides. Bhattacharya & Flora, Cyanide toxicity and its treatment, Handbook of Toxicology of Chemical Warfare Agents, 301-314 (2015); Use in Mining, International Cyanide Management Code (ICMI) for the Manufacture, Transport, and Use of Cyanide in the Production of Gold. Approximately 1.1 million tons of cyanide are produced annually for industrial use. Use in Mining, supra. Cyanide production is easily accomplished using readily available raw materials such as ammonia and methane. Grabow et al., Descriptor-based analysis applied to HCN synthesis from NH3 and CH4, Angewandte Chemi 50(20): 4601-4605 (2011). In the presence of acid, hydrogen cyanide is volatile and can quickly fill the air with lethal gas.

[0006] Due to its widespread availability and easy gas conversion, cyanide poses a threat for malicious misuse (e.g., as a chemical weapon) that induces high mortality and morbidity rates. Examples of such attacks include the Tokyo subway attack, in which terrorists placed cyanide salts and acid in trash cans in an attempt to fill the station with lethal gas, and the 1978 incident in Jonestown, Guyana. Kristof, "How Tokyo barely escaped even deadlier subway attack," New York Times, May 18, 1995; Conroy, "An apocalyptic cult, 900 dead: remembering the Jonestown massacre, 40 years on," The Guardian, November 17, 2018.

[0007] Cyanide gas is difficult to detect and has a rapid onset of toxicity, often becoming fatal within minutes at doses as low as 2 mg / kg. By the time emergency responders arrive and administer treatment, the toxic effects of cyanide may already be underway. In cases of fatal acute cyanide exposure, mortality and morbidity can occur within the first 30 to 60 minutes, thus representing an unmet medical need for fast-acting countermeasures.

[0008] Cyanide is a systemic poison that inhibits cellular respiration through reversible inhibition of cytochrome c oxidase in mitochondria. Levels NRC (US) S on AEG, Hydrogen cyanide: acute exposure guideline levels, National Academies Press (2002); Leavesley et al., Interaction of cyanide and nitric oxide with cytochrome c oxidase: implications for acute cyanide toxicity, Toxicological Sciences 101(1): 101-111 (2008). This inhibition can manifest clinically as arrhythmias, seizures, behavioral disturbances, or even an imbalance in normal oxygenated / deoxygenated hemoglobin homeostasis.

[0009] The rapid effects of cyanide on physiological function necessitate rapid treatment options to reduce morbidity and mortality. Approximately 50% of mice exposed to constant airborne cyanide concentrations as low as 177 parts per million (ppm) died within 30 minutes of exposure. Alarie, Toxicity of Fire Smoke, Critical Reviews in Toxicology 32: 259-289 (2022).

[0010] FDA-approved treatments for cyanide exposure include hydroxocobalamin, sodium nitrite, and sodium thiosulfate. Dicobalt edetate is also used in Europe, but only in cases of severe cyanide poisoning due to the compound's adverse effects. However, current cyanide countermeasures are administered by intravenous (IV) infusion or inhalation. For example, 5 grams of hydroxocobalamin typically requires administration over 15 minutes via IV infusion, sometimes requiring repeated dosing. CYANOKIT® Hydroxocobalamin for Injection, Food and Drug Administration, Reference ID: 4369589, for intravenous infusion. In some cases, higher doses may be required over a longer period, which can significantly increase the risk of comorbidities, including drug-induced renal dysfunction. Furthermore, strong evidence supports that rapid delivery of cyanide scavengers after exposure can significantly improve survival. Thompson et al., Modest and variable efficacy of pre-exposure hydroxocobalamin and dicobalt edetate in a porcine model of acute cyanide salt poisoning, Clinical Toxicology (Philia) 58: 190-200 (2019). However, current IV infusion of scavengers requires skilled medical staff and at least several minutes to deliver the full dose. Meillier & Heller, Acute cyanide poisoning: hydroxocobalamin and sodium thiosulfate treatment with two outcomes following one exposure event, Case Reports in Medicine (2015). Therefore, existing treatments can require significant time to achieve full administration and pharmacological action, making them suboptimal due to the rapid onset of toxicity observed in cyanide exposures. In cyanide exposure situations with the potential for mass casualties, establishing multiple IV infusion lines by first responders is not feasible.

[0011] A further complicating factor is that conventional compounds used to treat cyanide exposure (e.g., hydroxocobalamin) have relatively low aqueous solubility (27.3 ng / mL), which can be a confounding factor with the multiple dosing often required. Furthermore, the numerous toxic side effects of conventional treatments leave uncertainty regarding the best response options in emergencies. A further complication is that conventional active scavenger agents are stoichiometric reactants with cyanide, which can increase the required dosage of the active pharmaceutical ingredient and impose additional restrictions on the route of administration. Hydroxocobalamin's mechanism of action involves direct binding of cyanide at the metal center in a 1:1 stoichiometry. Hamel, Review of acute cyanide poisoning with a treatment update, Critical Care Nurse 31: 72-82 (2011).

[0012] The discovery and development of fast-acting cyanide countermeasures with increased solubility and reduced side effects that can be delivered via a single bolus intramuscular (IM) administration remains an important area of investigation. Summary of the Invention

[0013] Platinum (Pt)-based compounds comprising bidentate ligands are provided. In certain embodiments, the compounds have the structure of formula (I):

[0014] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer thereof, wherein Pt is platinum(II), each L1 and L2 is a ligand, each L1 forms a bidentate ligand, each L2 forms a bidentate ligand, each bidentate ligand contains N, S, or both N and S coordinated to platinum(II), at least one of the ligands is a leaving group, and at least two of the ligands directly bonded to platinum(II) each independently contain an alkyl, carboxamide, amine, aminosulfide, carboxylate, carboxyester, carbonyl, or a thioether containing any combination of the foregoing; each n is independently from about 1 to 5].

[0015] In certain embodiments, the compound has the structure of formula (II):

[0016] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer thereof [In the formula, Pt is platinum(II), each L1 and L2 is a ligand, each L1 forms a bidentate ligand, each L2 forms a bidentate ligand, each bidentate ligand contains N, S, or both N and S coordinated to platinum(II), at least one of the ligands is a leaving group, and at least two of the ligands each independently contain an alkyl, carboxamide, carboxyester, amine, aminosulfide, carboxylate, carbonyl, or thioether containing any combination of the foregoing; R1 is C 1~3 Alkyl, C 1~3 Haloalkyl, C 2~4 Alkenyl, C 2~4 Alkynyl, and C 6~10 aryl or absent; each n is independently from about 1 to 5].

[0017] In certain embodiments, the compound has the structure of formula (III):

[0018] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer thereof, wherein Pt is platinum(II), each L1 and L2 is a ligand, each L1 forms a bidentate ligand, each L2 forms a bidentate ligand, each bidentate ligand contains N, S, or both N and S coordinated to platinum(II), at least one of the ligands is a leaving group, and at least two of the ligands directly bonded to platinum(II) contain alkyl, carboxamide, amine, aminosulfide, carboxylate, carbonyl, or a thioether containing any combination of the foregoing; Each R1 is C 1~3 Alkyl, C 1~3 Haloalkyl, C 2~4 Alkenyl, C 2~4 Alkynyl, and C 6~10 aryl or absent; each n is independently from about 1 to 5].

[0019] Each L can be a leaving group. Each L can be a leaving group. In certain embodiments, each L and L is a leaving group.

[0020] In certain embodiments, each R of formula (II) or (III) is absent. 1~3 It can be alkyl.

[0021] The compound can include a cis configuration. The compound can include a trans configuration. The first ligand (e.g., bidentate) can include a sulfide and the second ligand (e.g., bidentate) includes an amide.

[0022] At least one thioester ligand of the compound can comprise an amino sulfide. The thioester ligands can each comprise an amino sulfide.

[0023] In certain embodiments, the bidentate ligands can independently comprise 5- or 6-membered bidentate ligands.

[0024] The compound has the following structure:

[0025] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of any of the foregoing structures.

[0026] The compound may comprise the following structure, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof.

[0027] [ka]

[0028] The compound may comprise the following structure, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof.

[0029] [ka]

[0030] In certain embodiments, at least one of the ligands comprises a methyl thioether group. In certain embodiments, at least one of the thioether ligands comprises methionine or S-methylcysteine, optionally comprising one or more amidated carboxylates. The bidentate ligands can independently comprise 5- or 6-membered bidentate ligands, and at least one of such bidentate ligands can comprise a carboxylate or carboxamide substituent.

[0031] In certain embodiments, at least one of the ligands comprises HCP-AKN, cisplatin-AKN, (salylCys)2Pt, (SMePenicillamine)2P, (Cilastatin)2Pt, or (bridgedMet2)Pt, MetPt(taurine)2.

[0032] Pharmaceutical compositions are also provided. The pharmaceutical compositions herein can comprise the compounds herein, or any of their pharmaceutically acceptable salts, N-oxides, solvates, tautomers or stereoisomers, and pharmaceutically acceptable carriers and / or excipients. The pharmaceutical compositions can further comprise pharmaceutically acceptable additives. The compositions can be suitable for intramuscular injection.

[0033] Also provided is the use of a pharmaceutical salt, N-oxide, solvate, tautomer, or stereoisomer of a compound herein, a pharmaceutical salt, N-oxide, solvate, tautomer, or stereoisomer thereof, or a pharmaceutical composition herein in the manufacture of a medicament for treating a disease or condition in a subject. In certain embodiments, the disease or condition is cyanide poisoning or cyanide exposure. The medicament may be formulated for intramuscular administration. The medicament may be formulated in a single bolus dose. The medicament may be formulated at a pH of about 5 or greater (such as about 5, 5, or greater than 5). The medicament may be stored at a pH of about 5 or less (such as about 5, 5, or greater than 5 (>5)).

[0034] Also provided is a method for treating cyanide poisoning or cyanide exposure in a subject. In certain embodiments, the method for treating cyanide poisoning or cyanide exposure in a subject comprises administering to the subject a first therapy comprising a therapeutically effective amount of: a compound herein, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of a compound herein, or a pharmaceutical composition herein.

[0035] In certain embodiments, the administering step comprises intramuscular injection. In certain embodiments, the administering step comprises administering a therapeutically effective amount of a first therapy comprises administering a single dose.

[0036] The method can further include administering a second therapy to the subject, the second therapy comprising administering to the subject a therapeutically effective amount of one or more of glyoxylate, hydroxocobalamin, methemoglobin, riboflavin, methotrexate, 4-dimethylaminophenol (4-DMAP), dicobalt edetate, glucose, activated charcoal, and ethylenediaminetetraacetic acid (EDTA) disodium cobalt; intravenous isotonic fluid; and / or oxygen therapy. The first and second therapies can be administered sequentially. The first and second therapies can be administered concurrently. The second therapy can be glyoxylate, and the therapeutically effective amount of the first therapy can be about 3.0 to 5.5 mg / kg (based on the subject's body weight). The therapeutically effective amount of the first therapy can be about 3.5 mg / kg (based on the subject's body weight).

[0037] At least one ligand of the compound or pharmaceutical composition can include one or more carboxamide substituted amino acid ligands.

[0038] In certain embodiments, the administering step comprises intramuscularly injecting the compound, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of the compound, or pharmaceutical composition into the subject, wherein the compound, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of the compound, or pharmaceutical composition reaches a peak concentration in the subject at or about 7 to 9 minutes after administration (e.g., at or about 7 minutes to 9 minutes, at or about 7 minutes to 9 minutes, or at or about 7 minutes to 9 minutes). In certain embodiments, after being administered intramuscularly to the subject, the compound, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of the compound, or pharmaceutical composition undergoes pH-induced isomerization, resulting in a cyanide scavenging rate that is reduced compared to the cyanide scavenging rate of the compound, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of the compound, or pharmaceutical composition within 1 hour of administration to the subject.

[0039] Also provided are combination therapies for treating cyanide poisoning or exposure in a subject. In certain embodiments, the combination therapies for treating cyanide poisoning or exposure in a subject include administering to the subject a therapeutically effective amount of a cyanide chelator and a therapeutically effective amount of an agent for ameliorating cyanide-induced oxidative stress in the subject. In certain embodiments, the cyanide chelator includes a platinum(II) thioether comprising a bidentate ligand.

[0040] Cyanide chelators include the compounds of the present invention; pharmaceutically acceptable salts, N-oxides, solvates, tautomers, or stereoisomers of the compounds of the present invention; and compounds having the following structure, or pharmaceutically acceptable salts, N-oxides, solvates, tautomers, or stereoisomers:

[0041] [ka] or the pharmaceutical composition herein.

[0042] The agent for ameliorating cyanide-induced oxidative stress in a subject can be glyoxylate or an analog or functional fragment thereof. The agent for ameliorating cyanide-induced oxidative stress in a subject can include a ketone or aldehyde compound selected from the group consisting of glyoxylate, glyceraldehyde, acetaldehyde, dihydroxyacetone, pyruvate, methylglyoxal, and alpha-ketoglutarate.

[0043] Also provided is a kit for treating cyanide poisoning or cyanide exposure. In certain embodiments, the kit for treating cyanide poisoning or cyanide exposure comprises a compound of the present invention, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer of a compound of the present invention, a compound having the following structure, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer:

[0044] [ka] and a pharmaceutically acceptable carrier and / or excipient. The drug injection device includes one or more fluid chambers pre-filled with a first formulation comprising:

[0045] The first formulation can contain a targeted effective dose of the compound, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer, for intramuscular injection. The pre-filled fluid chamber can be a syringe or cartridge. The formulation can have a pH value of 5 or less. The drug injection device can be an autoinjector or a hand-held injector.

[0046] The kit can further include one or more fluid chambers pre-filled with a second formulation. The second formulation can include a therapeutically effective amount of an agent for ameliorating cyanide-induced oxidative stress in a subject and a pharmaceutically acceptable carrier and / or additive. The agent for ameliorating cyanide-induced oxidative stress in a subject can be glyoxylate or an analog or functional fragment thereof.

[0047] The disclosed embodiments and other features, advantages, and aspects contained herein, as well as problems related to achieving the same, will become apparent in light of the following detailed description of various exemplary embodiments of the present disclosure, which will be better understood when read in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0048] [Figure 1] FIG. 1 shows the structures of the starting materials (compounds 1-5) and dimethyl sulfoxide (DMSO) complexes described herein, as well as the structure of the Pt(CN) 4 2− ion. [Figure 2]FIG. 1 depicts Scheme 1, a structural representation of the Pt amine sulfide-containing complexes isolated and pharmacologically evaluated in the studies described herein, where the structures represent the major isomers assigned based on heteronuclear NMR spectra. [Figure 3] Figure 1 shows x-ray fluorescence (XRF) spectral data of Pt(IV) and Pt(II) excited core absorbance. The platinum-related peaks were integrated relative to a 6 keV manganese chloride internal standard. [Figure 4] 1 is a table showing the weight percent (% w / w) composition of each platinum compound as measured by XFR. Manganese chloride served as an internal standard for quantification. [Figure 5] Table showing measures evaluated by monitoring the response to cyanide in vitro. Observed rate constants were obtained under pseudo-first-order conditions using 10 molar equivalents of cyanide, product formation was monitored at 255 nm for Pt(CN)4 2-, cyanide was quantified using an ion-selective electrode measuring the reduction of free cyanide after 10 minutes, and XRF values were used to adjust for platinum content and correct for concentration. High-performance liquid chromatography (HPLC) data were collected 24 hours after preparation, and equilibrium was presumed to have been established at the time of collection (ID = compound number, and [ka] = Rate data for Pt(IV) compounds 2 and 4 were established from the disappearance of the signal at 275 nm (astronomical units / min). [Figure 6-1]Figure 6 shows data on the reaction between platinum and cyanide monitored by high-performance liquid chromatography (HPLC). Figure 6A shows compound 6, and cyanide was quantified using a Resetek Ultra IBD (Restech Corporation, Houston, TX). Figures 6B and 6C show photodiode arrays (200–300 nm) of compound 6 and Pt(CN) 2- shown in Figure 6A, respectively. Figure 6C shows a strong charge-transfer band at 260 nm consistent with the formation of Pt(CN) 2-. Figure 6D shows data on compound 6 being titrated with 1–10 molar equivalents of cyanide. [Figure 6-2] Figure 6 shows data on the reaction between platinum and cyanide monitored by high-performance liquid chromatography (HPLC). Figure 6A shows compound 6, and cyanide was quantified using a Resetek Ultra IBD (Restech Corporation, Houston, TX). Figures 6B and 6C show photodiode arrays (200–300 nm) of compound 6 and Pt(CN) 2- shown in Figure 6A, respectively. Figure 6C shows a strong charge-transfer band at 260 nm consistent with the formation of Pt(CN) 2-. Figure 6D shows data on compound 6 being titrated with 1–10 molar equivalents of cyanide. [Figure 6-3] 6E shows the calibration curve for Pt(CN)4-2, where each point contains n = 3 injections of standard material (error bars are small and not visible), and FIG. 6F shows the calibration curve for Compound 6, where each point has n = 3 injections of standard (error bars are small and not visible). [Figure 7-1]Figure 7 shows Pt nuclear magnetic resonance (NMR) spectra of compound 6 (51 mM in 200 mM NaPi 10% DO, pH 7.5) titrated with cyanide at 291.5 K. Figure 7A depicts the spectrum before the addition of cyanide, Figure 7B depicts the spectrum after the addition of approximately 50 mM cyanide, and Figure 7C depicts the spectrum after the addition of 200 mM (final concentration) cyanide. The observed signals correspond to the reactant (PtMet2) with a Pt chemical shift of approximately -3650 ppm and the final product [Pt(CN)4 2-] with a Pt chemical shift of -4699 ppm. [Figure 7-2] Figure 7 shows Pt nuclear magnetic resonance (NMR) spectra of compound 6 (51 mM in 200 mM NaPi 10% DO, pH 7.5) titrated with cyanide at 291.5 K. Figure 7A depicts the spectrum before the addition of cyanide, Figure 7B depicts the spectrum after the addition of approximately 50 mM cyanide, and Figure 7C depicts the spectrum after the addition of 200 mM (final concentration) cyanide. The observed signals correspond to the reactant (PtMet2) with a Pt chemical shift of approximately -3650 ppm and the final product [Pt(CN)4 2-] with a Pt chemical shift of -4699 ppm. [Figure 7-3] Figure 7 shows Pt nuclear magnetic resonance (NMR) spectra of compound 6 (51 mM in 200 mM NaPi 10% DO, pH 7.5) titrated with cyanide at 291.5 K. Figure 7A depicts the spectrum before the addition of cyanide, Figure 7B depicts the spectrum after the addition of approximately 50 mM cyanide, and Figure 7C depicts the spectrum after the addition of 200 mM (final concentration) cyanide. The observed signals correspond to the reactant (PtMet2) with a Pt chemical shift of approximately -3650 ppm and the final product [Pt(CN)4 2-] with a Pt chemical shift of -4699 ppm. [Figure 7-4] Figure 7D shows H NMR spectral data related to the competitive reaction of compound 3 and compound 6 with KCN. The methyl signals at 2.08 ppm (*) and 2.67 ppm (**) indicate the release of methionine and DMSO, respectively. [Figure 8] FIG. 1 shows data related to the Pt amine-sulfide compound 11 reaction with potassium cyanide (KCN) monitored by H NMR at 298 K. A: 1 mM compound 11 alone in 50 mM NaPi, pH 7.5, 10% DO; B: with 1 mM KCN added; C: with 5 mM (final concentration) KCN added. [Figure 9] FIG. 1 shows the H NMR spectra of 1 mM compound 3 (cisplatin-DMSO) without added cyanide (bottom) and with added 5 mM cyanide (top). [Figure 10] FIG. 1 shows the H NMR spectra of 1 mM compound 5 without added cyanide (bottom) and with added 5 mM cyanide (top). [Figure 11] FIG. 1 shows the H NMR spectra of 1 mM compound 7 with no added cyanide (bottom), 1 mM added cyanide (middle), and 5 mM added cyanide (top). [Figure 12] FIG. 1 shows the H NMR spectra of 1 mM compound 8 with no added cyanide (bottom), 1 mM added cyanide (middle), and 5 mM added cyanide (top). [Figure 13] FIG. 1 shows the H NMR spectra of 1 mM compound 9 with no cyanide added (bottom), 1 mM cyanide added (middle), and 5 mM cyanide added (top). [Figure 14] FIG. 1 shows the H NMR spectra of 1 mM compound 10 with no added cyanide (bottom), 1 mM added cyanide (middle), and 5 mM added cyanide (top). [Figure 15] FIG. 1 shows the H NMR spectra of 1 mM compound 11 with no added cyanide (bottom), 1 mM added cyanide (middle), and 5 mM added cyanide (top). [Figure 16]Figure 1 shows the H NMR spectra for the reaction of compound 9 (50 μM Pt(MetNH)Cl, pH ∼12) (bottom) 50 minutes after preparation and compound 9 with cyanide added (50 μM Pt(MetNH)Cl, +500 μM KCN, pH ∼12) (top) 60 minutes after preparation. The H NMR spectra show no observable change between the cyanide-added and cyanide-free samples (50 μM compound 9), confirming the lack of reaction within 1 hour of preparation. [Figure 17] Figure 1 shows the C NMR spectra of the reaction between compound 6 and cyanide in 95% rabbit serum / % D2O. A shows data from rabbit serum spiked with 400 μM C-KCN, and B shows data from 100 μM compound 6. The signal labeled with an asterisk at 117 ppm was generated from KCN, which exists primarily as HCN at neutral pH. The signal at 125 ppm was assigned to Pt(CN)4 2-, while the satellite from the Pt coupling was not clearly observed, likely due to low signal-to-noise ratio and line broadening. Semi-quantification of Pt(CN)4 2- was based on the signal at 117 ppm (for 400 μM), consistent with the expected complete scavenging of cyanide by compound 6. [Figure 18] FIG. 1 shows graphical data related to cardiotoxicity testing of Pt compounds in zebrafish (P<0.00005 vs. dofetilide control). [Figure 19] 1 is a table describing the basic scheme for testing lethal exposure by inhalation in a mouse model. [Figure 20-1]Figure 20A depicts the protocol for a non-lethal rabbit cyanide exposure study conducted using compounds 3 and 6 herein. Figures 20B-20D show data generated by the study described in Figure 20A, Figures 20B and 20C show data related to the restoration of normal homeostatic levels of blood hemoglobin in rabbits treated intramuscularly (IM) with compound 3 and compound 6, respectively, after cyanide injection, and Figure 20D shows the results of platinum blood plasma analysis determined by inductively coupled plasma mass spectrometry (ICP-MS) using the area under the curve (AUC) as the mean of n=3 replicates calculated for the first 60 minutes after treatment (maximum recorded concentration (e.g., C) from a single injection combined with the time observed (T)). [Figure 20-2] Figures 20B-20D show data generated from the study described in Figure 20A, Figures 20B and 20C show data related to the restoration of normal homeostatic levels of blood hemoglobin in rabbits treated intramuscularly (IM) with Compound 3 and Compound 6, respectively, after cyanide injection, and Figure 20D shows the results of platinum blood plasma analysis determined by inductively coupled plasma mass spectrometry (ICP-MS) using the area under the curve (AUC) as the mean value of n=3 replicates calculated for the first 60 minutes after treatment (maximum recorded concentration (e.g., Cmax) from a single injection combined with the time observed (Tmax)). [Figure 20-3] Figures 20E-20J are graphs depicting blood plasma concentrations of total platinum content collected by ICP-MS in rabbits dosed with either Compound 3 or Compound 6 in the non-lethal cyanide model described herein. [Figure 20-4] Figures 20E-20J are graphs depicting blood plasma concentrations of total platinum content collected by ICP-MS in rabbits dosed with either Compound 3 or Compound 6 in the non-lethal cyanide model described herein. [Figure 20-5]Figures 20E-20J are graphs depicting blood plasma concentrations of total platinum content collected by ICP-MS in rabbits dosed with either Compound 3 or Compound 6 in the non-lethal cyanide model described herein. [Figure 21] FIG. 1 shows graphs related to monitoring the stability of compound 4 by 195Pt NMR and 1H NMR. [Figure 22] FIG. 1 shows mass spectral data for compound 3 using an LTQ orbitrap. [Figure 23] FIG. 1 shows mass spectral data for compound 4 using an LTQ orbitrap. [Figure 24] FIG. 1 shows mass spectral data for compound 5 using an LTQ orbitrap. [Figure 25] FIG. 1 shows mass spectral data for compound 6 using an LTQ orbitrap. [Figure 26] FIG. 1 shows mass spectral data for compound 7 using an LTQ orbitrap. [Figure 27-1] FIG. 27A shows the mass spectrum data of compound 8. [Figure 27-2] FIG. 27B shows mass spectral data for compound 8 using an Advion Expression spectrometer in electrospray ionization positive ion (top) and negative ion (bottom) modes. [Figure 28] FIG. 1 shows mass spectral data for compound 9 using an LTQ orbitrap. [Figure 29] FIG. 1 shows mass spectral data for compound 10 using an LTQ orbitrap. [Figure 30] FIG. 1 shows mass spectral data for compound 11 using an LTQ orbitrap. [Figure 31] FIG. 1 shows 1H NMR spectral data (500 MHz) for a fresh solution of 15.6 mg of compound 6 in 500 μL of 90 mM NaPi, pH 8.0 (final pH ∼5.7), 10% DO at 292 K. [Figure 32] FIG. 1 shows the structures of Pt(II) compounds 1′-4′ described herein. [Figure 33-1] Figures 33A and 33B show osmolality curves for compound 6' (+2NaCl) (Figure 33A) and compound 9 (+2NaCl) (Figure 33B). Data were taken from a single stock solution, which was then diluted with purified water. [Figure 33-2] Figures 33A and 33B show osmolality curves for compound 6' (+2NaCl) (Figure 33A) and compound 9 (+2NaCl) (Figure 33B). Data were taken from a single stock solution, which was then diluted with purified water. [Figure 34-1] Figures 34A and 34B show, as representative examples, the reaction of compound 13 (+2NaCl) (Figure 34A) or compound 6 (+2NaCl) (Figure 34B) with 1:40 Pt:CN in purified water. Samples were incubated for a minimum of 24 hours, and once the signal was stable and consistent with the day 0 reaction rate, Pt(CN)4-2 was quantified by UV-Vis. [Figure 34-2] Figures 34A and 34B show the response of compound 13(+2NaCl) (Figure 34A) or compound 6(+2NaCl) (Figure 34B) at 1:40 Pt:CN in purified water, as a representative example. Samples were also incubated for a minimum of 24 hours, and once the signal stabilized and matched the reaction kinetics on day 0, Pt(CN) was quantified by UV-Vis. Figures 34C and 34D show the response of compound 13(+NaCl) (Figure 34C (bars represent 48 hours)) and compound 6(+NaCl) (Figure 34D), respectively, at 1:5, 1:10, and 1:80 Pt:CN in purified water with KCN. [Figure 34-3] Figures 34C and 34D show the reaction of compound 13 (+NaCl) (Figure 34C (bar represents 48 hours)) and compound 6 (+NaCl) (Figure 34D) with KCN at 1:5, 1:10, and 1:80 Pt:CN in purified water, respectively, as representative examples. [Figure 35]FIG. 1 shows data relating to the UV-Vis spectrum of compound 13 after addition of 100× molar equivalents of NaOH to monitor spectral changes over a 710 minute period. [Figure 36-1] Figure 36A shows the graphical data from combining compound 9 with 50 molar equivalents of NaOH and waiting 240 minutes, with the peak occurring at 245 nm. Figure 36B shows the graphical data from combining compound 9 with varying amounts of NaOH at increasing NaOH concentrations, with the signal at 245 nm. [Figure 36-2] Figure 36C shows the spectral analysis of Compound 9 when incubated in pH 6.8 phosphate buffer at room temperature. Compound 9 showed a new form labeled as Peak II by HPLC after approximately 175 minutes (blue trace (bottom line)) was Compound 9 in purified water, with absorbance detected at 220 nm. [Figure 36-3] Figure 36D is a graph of the absorbance change for compound 9 in the presence of KCN, monitored at 245 nm. Compound 9 was prepared in pH 7.26 phosphate buffer at 18.8 °C. The data shown are the results of the absorbance increase at 245 nm. Scans were taken every 0.1, 1, and 30 minutes throughout the course of the analysis. The data were linearly fitted with a semi-logarithmic plot, suggesting that the process follows first-order kinetics. The apparent half-life for these conditions is 2.6 hours with an R = 0.9962. [Figure 37-1] Figures 37A and 37B show graphs of the time-dependent changes in compound 13 in phosphate buffer, pH 7.3, upon reaction with KCN at 19°C (Figure 37A) and 37°C (Figure 37B). Data were acquired by monitoring the signal at 241 nm. [Figure 37-2]Figures 37A and 37B show graphs of the time-dependent changes in compound 13 in phosphate buffer, pH 7.3, upon reaction with KCN at 19°C (Figure 37A) and 37°C (Figure 37B). Data were acquired by monitoring the signal at 241 nm. [Figure 38] Figures 38A and 38B show H NMR spectral data demonstrating the transformation of compound 9 at room temperature and pH 7.15 (day 0, labeled B, and day 3, labeled A). Figure 38A shows the downfield region for the amino and amide NH, suggesting their subtle changes over time. Figure 38B shows the upfield region showing changes for the ligand side chains. [Figure 39] FIG. 1 shows 1H NMR spectral data monitoring the change in free -SMe for compound 9 upon incubation in pH 7 phosphate buffer over a period of 3 weeks with HCl added to adjust the solution pH to approximately 2.5. [Figure 40] Figure 1 shows data from compound 9 incubated in pH 6.8 phosphate buffer at room temperature for 3 days. The data show primarily "Peak II" at T=0, which reacts slowly with 2 mM compound 9 and 8 mM KCN to form Pt(CN)-2. [Figure 41-1] Figures 41A-41D show HPLC stability results for compounds 6, 9, 12, and 13, respectively, after each compound was reacted with 4 molar equivalents of KCN for 10 minutes prior to injection into the HPLC and the Pt(CN)4 2- produced was quantified. [Figure 41-2] Figures 41A-41D show HPLC stability results for compounds 6, 9, 12, and 13, respectively, after each compound was reacted with 4 molar equivalents of KCN for 10 minutes prior to injection into the HPLC and the Pt(CN)4 2- produced was quantified. [Figure 42]Figure 1 shows HPLC stability results for compound 6 after reaction with 4 molar equivalents of KCN for 10 minutes prior to injection into the HPLC and quantification of the Pt(CN)4 2- produced to monitor reactivity over 42 days. Data shown per time point is the average of triplicates. [Figure 43] FIG. 1 shows the titration curve of approximately 5 μmol of compound 6 with 5 mM NaOH after fresh preparation in water versus aging at room temperature over 7 days. [Figure 44] FIG. 1 shows the titration curve of approximately 5 μmol of compound 9 with 5 mM NaOH, which shows the lack of a clear equivalence point, indicating a chemical process more complex than the titration of an ionic group. [Figure 45] Figure 1 shows zebrafish data representing the detoxification activity (EC100) in aqueous conditions of each compound formulation (compounds 6, 9, 12, and 13). Each result is the concentration of platinum required for 100% (n=5) survival in the presence of 100 µM KCN, the concentration that resulted in death after 1 hour in the control group. Survival times reported were measured 4 hours after cyanide exposure; compounds were prepared 3-7 days prior to use. [Figure 46] Figures 46A and 46B show graphical data from a study in which rats were dosed with Compound 6α via intraperitoneal (IP) injection using various dosages. Figure 46A shows the mean blood urea nitrogen (BUN) concentrations for male and female cohorts, both showing significant (p<0.0005) signs of acute kidney injury (AKI) after 5 days at 218 μmol / kg (42.5 mg Pt / kg). Figure 46B shows the mean creatinine (CREA) concentrations for male and female cohorts, both showing significant levels (p<0.05 and p<0.0005, respectively) compared to vehicle after 1 and 5 days. Analysis was performed using ordinary two-way ANOVA with Sidak's multiple comparisons comparing cell means across rows and columns. [Figure 47]Figures 47A and 47B are graphs showing BUN (Figure 47A) and CREA (Figure 47B) levels in cohorts of rats after treatment with Compound 6 to assess the role, if any, of osmolality. Two formulations of Compound 6, and vehicle for comparison purposes, were also tested. [Figure 48] 1 is a graph of the weight change of rats dosed with increasing amounts of Compound 6α. [Figure 49-1] Figures 49A-49C illustrate the changes observed in the levels of AKI markers BUN, CREA, and PHOS in response to five-fold doses of each of compounds 6, 9, 12, and 13, showing the indicated toxicity observed at the highest dose of 218 μmol / kg (42.5 mg Pt / kg). Compound 9 was assessed at two pH values. Analysis was performed using ordinary two-way ANOVA with Sidak's multiple comparisons comparing cell means across rows and columns. [Figure 49-2] Figures 49A-49C illustrate the changes observed in the levels of AKI markers BUN, CREA, and PHOS in response to five-fold doses of each of compounds 6, 9, 12, and 13, showing the indicated toxicity observed at the highest dose of 218 μmol / kg (42.5 mg Pt / kg). Compound 9 was assessed at two pH values. Analysis was performed using ordinary two-way ANOVA with Sidak's multiple comparisons comparing cell means across rows and columns. [Figure 50] Figures 50A and 50B are graphs showing BUN and glucose (GLU) levels (Figures 50A or 50B, respectively) as examples of markers of renal dysfunction. Analysis was performed using ordinary two-way ANOVA with Sidak's multiple comparisons comparing cell means across rows and columns. [Figure 51] Figure 1 shows the total platinum in plasma concentration versus time profiles measured by ICP-MS in rats for compounds 6 and 9. The expanded initial concentration versus time profiles (inset) illustrate the apparent difference in early distribution between the two compounds. [Figure 52] FIG. 1 shows structures of various ligands for incorporation into the compounds herein. [Figure 53] FIG. 1 illustrates the workflow and performance metrics that can be applied to select and propose new ligands and new models. [Figure 54] FIG. 1 illustrates the workflow and performance metrics that can be applied to formulate Pt(II)-glyoxylate combinations. [Figure 55] Graph of data from a combination treatment containing a Pt(II) cyanide scavenger and glyoxylate, which rescued a lethal zebrafish model after sensitization (glyoxylate EC100 = 32 μM; PCP EC100 = 62 μM; 15 μM PCP plus 8 μM glyoxylate (100% survival); 32 μM PCP plus 4 μM glyoxylate (100% survival)). [Figure 56] Figure 1 shows an example of C NMR data that allows for the measurement of cyanohydrin formation (pyruvate in this case). The Kd (μM) for each metabolite is: glyoxylate 1.7, glyceraldehyde 6, alpha-ketoglutarate 80, pyruvate 90, dihyroxyacetone 200, and glucose >3000. [Figure 57] Graph of survival in cyanide-poisoned pigs after IM administration of glyoxylate (Glyoxylate improves survival). Kaplan-Meier plot of survival in control (n=8; labeled A) vs. glyoxylate (n=6; labeled B) treated animals (p<0.001). [Figure 58-1]Figures 58A and 58B are graphs of plasma biomarker data reflecting improved intracellular and extracellular redox balance in glyoxylate-treated pigs. In pigs treated with cyanide (gray box) and then at t=0, 10 mg / kg glyoxylate was administered intramuscularly (labeled A; n=6) or 20 mg / kg hexachloroplatinate was administered intramuscularly (labeled B; n=3), and serial plasma samples were collected to measure 1) the lactate:pyruvate ratio, 2) a circulating marker in approximate equilibrium with cellular NADH:NAD+, and 2) the cysteine:cystine ratio, a plasma biomarker of extracellular oxidative stress. [Figure 58-2] Figures 58A and 58B are graphs of plasma biomarker data reflecting improved intracellular and extracellular redox balance in glyoxylate-treated pigs. In pigs treated with cyanide (gray box) and then at t=0, 10 mg / kg glyoxylate was administered intramuscularly (labeled A; n=6) or 20 mg / kg hexachloroplatinate was administered intramuscularly (labeled B; n=3), and serial plasma samples were collected to measure 1) the lactate:pyruvate ratio, 2) a circulating marker in approximate equilibrium with cellular NADH:NAD+, and 2) the cysteine:cystine ratio, a plasma biomarker of extracellular oxidative stress. [Figure 59] FIG. 1 illustrates the workflow and performance metrics that may be applicable for evaluation in the pig model described herein. [Figure 60] Figure 1 shows an example of preliminary data for Pt blood levels after IM injection of the listed doses in a pig model. The agent (Met2Pt) was delivered in a 6 mL formulation. These data indicate the feasibility of meeting the listed performance criteria. DETAILED DESCRIPTION OF THE INVENTION

[0049] While the concepts of the present disclosure have been illustrated and described in detail in the description herein, it is understood that the results in the description are to be considered exemplary and not restrictive in character, that only illustrative embodiments have been shown and described, and that all changes and modifications that fall within the spirit of the disclosure are desired to be protected.

[0050] Cyanide poses a risk to human health, at least as an agent in chemical warfare, suicide, occupational exposure, and smoke inhalation. Cyanide potently inhibits cytochrome c oxidase and potentially other metabolic enzymes, thereby unleashing a cascade of metabolic perturbations. More specifically, cyanide inhibits complex IV of the respiratory chain by binding to the ferric ion in cytochrome a3. Cyanide fixation in cytochrome c oxidase prevents its reoxidation by oxygen, thereby causing electron congestion in the electron transport chain. Complex I is then trapped in a reduced state and therefore cannot reduce nicotinamide adenine dinucleotide (NADH) to regenerate NAD+, which is required for the tricarboxylic acid (TCA) cycle. As the NADH / NAD+ ratio increases, negative feedback inhibition of the TCA cycle ensues. Electron transport and oxidative phosphorylation can cease, resulting in a shift in cellular metabolism from aerobic to anaerobic.

[0051] The cessation of aerobic metabolism, not the other way around, can lead to a fatal deficiency in oxygen consumption. Therefore, cyanide is most toxic to organs with high metabolic requirements, such as the brain and heart. Milligram amounts of cyanide can cause convulsions, seizures, cardiovascular collapse, and death within minutes of exposure, while lower doses can cause a range of debilitating, long-term pathologies, including Parkinson-like syndromes due to irreversible neuronal death in selected brain regions.

[0052] The body has natural defense mechanisms to eliminate cyanide. The cyanide detoxification pathway is via the sulfotransferase rhodanese, which leads to the sulfurization of cyanide to form the less toxic compound thiocyanate, which is excreted in the urine.

[0053] Although there are several conventional antidotes available for cyanide poisoning, their formulations and mechanisms of action require intravenous (IV) infusion in a hospital setting, requiring at least 15 to 20 minutes to administer a single dose, and are not modifiable for mass casualty incidents or field operations.

[0054] In addition, like other metallodrugs, platinum-based drug products are known to have risks of nephrotoxicity, ototoxicity, cardiotoxicity, and peripheral neuropathy. Oun et al., The side effects of platinum-based chemotherapy drugs: a review for chemists, Dalton Transactions 47: 6645-6653 (2018). Acute kidney injury (AKI) is commonly encountered in patients receiving cisplatin, and it is highly dependent on dose, dosing frequency, and cumulative dose. Miller et al., Mechanisms of cisplatin nephrotoxicity, Toxins 2: 2490-2518 (2010). Coadministration of methionine and other sulfur-containing agents has also been demonstrated to modulate cisplatin AKI. Jones et al., Thiol and thioether suppression of cis-platinum-induced nephrotoxicity in rats bearing the Walker 256 carcinosarcoma, Anticancer Research 9: 1937-1941 (1989); Jones et al., Thioether suppression of cisplatin nephrotoxicity in the rat, Anticancer Research 11: 449-453 (1991). It is hypothesized that cisplatin's modulation of platinum-induced AKI is due to reduced reaction with thiol-containing amino acids, peptides, and proteins in the cytosol. Stankovic et al., Antioxidant supplementation in the treatment of neurotoxicity induced by platinum-based chemotherapeutics - a review, IJMS 21: 7753 (2020).

[0055] In a previous study, an in vivo screen for chemical agents that rescue zebrafish from toxic levels of cyanide exposure led to the discovery of platinum-based complexes. The proposed mechanism of action was based on the known interaction of platinum with cyanide, which forms a stable complex. A list of 36 organoplatinum complexes was tested, including drugs such as cisplatin and oxaliplatin. Each sample required heating in dimethyl sulfoxide (DMSO) before dilution in phosphate-buffered saline to reveal meaningful rescue activity. Interestingly, the cyanide-protecting activity of these materials all shares a common requirement: the samples must be pretreated in DMSO before dilution and use in cyanide rescue.

[0056] DMSO reactivity with platinum II (Pt(II))-based therapeutics is well known. Cisplatin formulated with DMSO, when administered via the intraperitoneal (IP) route, has been shown to rescue mice treated with lethal doses of cyanide. Using this formulation approach, hexachloroplatinate (HCP) was further tested in rabbit and pig models of cyanide toxicity via IP and IM administration to demonstrate efficacy in survival from lethal cyanide exposure. Evidence of platinum cyanate formation in the circulation and reversal of metabolic blockade of the Krebs cycle was observed. Both DMSO-treated cisplatin and HCP reversed the cyanide-induced oxyhemoglobin symptoms monitored in the rabbit circulation, demonstrating a reduction in toxicant levels. These studies established the necessary proof of concept that cyanide-reactive Pt complexes may display antidote properties in these animal models.

[0057] The prior use of platinum in pharmaceuticals in clinical settings provides some guidance for the design of practical cyanide countermeasures. Cisplatin, oxaliplatin, and carboplatin are widely used anticancer drugs administered exclusively by IV infusion. Cisplatin's dose-limiting toxicity is 20 times greater than that of HCP, while its solubility is 33 times less than that of HCP. Interestingly, the use of DMSO with cisplatin and related drugs is known to reduce anticancer efficacy and toxic side effects. However, several conventional compounds are associated with dose-limiting toxicities.

[0058] Furthermore, FDA-approved treatments that can act by directly binding cyanide, namely hydroxocobalamin, do not reverse the biological and metabolic damage caused by cyanide, while glyoxylate or other metabolic agents that counteract the effects of cyanide do not eliminate cyanide from the body.

[0059] In view of the above, compounds and pharmaceutically acceptable salts thereof are provided that are soluble, exhibit reduced toxicity (e.g., are non-toxic) compared to conventional cyanide antidotes, are effective, and are amenable to mass distribution and administration to affected individuals to prevent morbidity and mortality resulting from cyanide exposure. In certain embodiments, the compounds herein are therapeutically effective when administered intramuscularly (IM). In certain embodiments, the compounds exhibit time- and / or pH-dependence on cyanide reactivity for both in vitro and in vivo efficacy.

[0060] Additionally, in certain embodiments, the platinum-based compound contains an aminosulfide-containing bidentate ligand to direct the reaction of the Pt(II) complex with cyanide. As used herein, "bidentate ligand" refers to a molecule or ion that donates an electron pair to the core metal ion of the compound to form a coordinate covalent bond. Such compounds have been shown to increase the addition rate to generate tetracyanoplatinate(II). Importantly, this reactivity directly translates into an enhanced ability of Pt(II) to rescue cyanide toxicity, at least in zebrafish, mouse, and rabbit models. Variation in the ligand composition opens new avenues for identifying additional top candidates for next-generation cyanide countermeasures.

[0061] Yet further combinations are provided in which the chelators herein (e.g., the Pt(II) complexes described herein) are combined with the administration of metabolic regulators to address not only cyanide scavenging but also amelioration of cyanide-induced oxidative stress in the body.

[0062] compound The compounds herein are cyanide scavengers capable of activating and directly binding to cyanide anions. The compounds include coordination complexes having at least the following components: (1) a core metal atom or ion that is a coordination center, and (2) one or more bidentate ligands (including ligands L1 or L2 as described in connection with Formulas (I)-(III)) bound to the core metal atom or ion.

[0063] The core metal atom or ion can be a platinum atom (Pt) or a Pt ion. The core can be charged (e.g., the core metal atom can be a positively charged metal atom). The core can be charged Pt. The core can be a platinum(II) (Pt(II)) atom. The term "platinum(II)" or "Pt(II)" refers to a platinum ion having a +2 charge. The Pt(II) ion is hexadentate and bound only to neutral (uncharged) ligands. The term "ligand" refers to a molecule that participates in a coordinate bond with the Pt(II) ion. The term "uncharged" indicates that the ligand is not charged (i.e., anionic or cationic), either inherently at the atoms comprising the core structure or as a group attached to the core structure. In certain embodiments, two of the six coordination sites of Pt(II) are occupied by at least one bidentate bicyclic ligand. The ability of a bidentate ligand to bridge and / or form two bonds with the core metal of a compound can increase the stability and coordination of the complex.

[0064] Unlike cobalt, which directly binds cyanide at a 1:1 stoichiometry (the framework of conventional hydroxocobalamin, cobinamide (the naturally occurring precursor of cobalamin (vitamin B12)), and Co(III) porphyrin and Schiff base complexes), Pt(II) can bind an additional four equivalents of cyanide, thus providing increased potency compared to conventional compounds. Nath et al., Cisplatin analogs confer protection against cyanide poisoning, Cell Chemical Biology, 24: 565-575 (2017); Cronican et al., A comparison of the cyanide-scavenging capabilities of some cobalt-containing complexes in mice, Chemical Research Toxicology 31: 259-268 (2018); Chan et al., Cobinamide is superior to other treatments in a mouse model of cyanide poisoning, Clinical Toxicology (Philia) 48: 709-717 (2010). More specifically, cobalt scavengers bind only 1 or 2 molar equivalents of cyanide. Lopez-Manzano et al., Cyanide scavenging by a cobalt Schiff-Base macrocycle: a cost-effective alternative to Corrinoids, Chemical Research Toxicology (Philia) 29: 1011-1019 (2016).

[0065] When administered via IM injection, the Pt(II) complexes herein have proven effective at platinum concentrations as low as 0.090 mmol / kg in a mouse cyanide inhalation model. Behymer et al., Identification of platinum (II) sulfide complexes suitable as intramuscular cyanide countermeasures, Chemical Research in Toxicology 35(11): 1983-1996 (2022). Thus, in certain embodiments, Pt(II)-based complexes provide IM-available agents with high potency per molar equivalent of metal, while simultaneously reducing overall molecular weight compared to conventional cobalt-based scavengers. Id.

[0066] At least one or more of the ligands of the compound are leaving groups (e.g., groups that can be displaced / substituted by cyanide). In certain embodiments, at least three of the ligands are leaving groups to the extent that the non-leaving ligands do not interfere with the cyanide reaction with the Pt core or create toxic complexes with Pt(II). In certain embodiments, four ligands are leaving groups. The concept of ligand release upon cyanide addition can be exploited to provide additional beneficial effects without the added toxicity burden.

[0067] One or more of the ligands can comprise a metabolic modulator (i.e., modulate the reactivity of the platinum center of the compound to facilitate reaction with hydrogen cyanide). Selected ligand structures can be used to modulate or adjust the reactivity of the platinum center of the compound for reaction with hydrogen cyanide. In certain embodiments, this can promote selectivity toward cyanide while eliminating the risk of potential toxicity (e.g., nephrotoxicity). For example, without limitation, the ligand can comprise one or more sulfurs (e.g., sulfides) to promote the sulfur-directed activity of Pt(II) toward cyanide addition and / or ligand substitution in vivo. In certain embodiments, at least two of the ligands comprise sulfides such that the compound comprises a disulfide.

[0068] Table 1 lists some non-limiting examples of platinum compounds as described herein that are tunable (i.e., can be used to modulate and / or enhance selectivity towards cyanide).

[0069] [Table 1]

[0070] In certain embodiments, a ligand is selected for inclusion in a bidentate ligand and / or compound if it possesses one or more of the following characteristics: it is generally well tolerated in vivo as a free ligand, it enhances affinity for Pt(II) and stability in biological matrices, and it confers some renal protection in a rat model of cisplatin toxicity or similar models (e.g., similar to cilastatin and taurine).

[0071] The bidentate ligands of the compound or pharmaceutically acceptable salt can be the same or different, or a mixture thereof. The ligands can include molecules that complex with the platinum core of the compound (e.g., to form a bidentate ligand). Functional groups on the ligands are in a bridging relationship that allows for bidentate-oriented bonding with Pt(II).

[0072] The compound can include a bicyclic bidentate ligand bound to the platinum core. The bidentate ligands of the compound can independently include 5- or 6-membered bidentate ligands bound to the platinum. At least one bidentate ligand can include a 5-membered bidentate ligand bound to the platinum core. At least one bidentate ligand can include a 6-membered bidentate ligand bound to the platinum core. In certain embodiments, the bidentate ligands of the compound independently include 5- or 6-membered bidentate ligands, and at least one of the bidentate ligands includes a carboxylate or carboxamide substituent. In such embodiments, the compound can be a trans-directing sulfur ligand on Pt(II) that is effective in vivo when delivered intramuscularly (IM).

[0073] At least one or two of the ligands of the bidentate ligand can be a thioether (e.g., at least one ligand can comprise an aminosulfide). At least one thioester ligand can comprise an aminosulfide. In certain embodiments, the thioether-containing ligand can further comprise an alkyl, carboxyamide, amine, aminosulfide, carboxylate, carboxyester, carbonyl, or any combination of the foregoing, to the extent that the thioether comprises a carbon-bonded sulfide and the ligand does not interfere with the cyanide-Pt core cyanide reaction or form a toxic complex with Pt(II).

[0074] The ligand can include an amino acid. The ligand can include methionine. The ligand can include methionine sulfoxide. The ligand can include N-acetyl-L-methionine (Ac-methionine). The ligand can include S-methyl-L-cysteine (SMeCys). The ligand can include reduced glutathione. The ligand can include SMeCys sulfoxide. The ligand can include S-methylated glutathione. The ligand can include any carbon-bonded sulfide.

[0075] In certain embodiments, the first ligand of the bidentate ligand comprises a sulfide and the second ligand of the bidentate ligand comprises an amide.

[0076] At least one of the ligands can include a carboxyamide. At least one of the ligands can include an amine. The amine can be an amino sulfide. In certain embodiments, at least one of the ligands includes a carboxylate or a carbonyl. The ligand can include a carboxy ester.

[0077] In certain embodiments, one or more of the ligands can exert protective effects through co-administration of ketone and aldehyde compounds, such as glyoxylate, glyceraldehyde, acetaldehyde, dihydroxyacetone, pyruvate, methylglyoxal, and alpha-ketoglutarate. Nielson et al., Glyoxylate protects against cyanide toxicity through metabolic modulation, Scientific Reports 12: 4982 (2022). Cytochrome c oxidase levels recovered more rapidly in rabbits treated with glyoxylate compared with alpha-ketoglutarate. Additional mechanistic studies revealed that the glyoxylate rescue effect depends on lactate dehydrogenase activity, providing evidence that glyoxylate rapidly oxidizes to form oxalate. Overall, cyanide chelation is a component of glyoxylate cyanide protection, but additional metabolic contributions appear to play a role in its enhanced efficacy.

[0078] The observations regarding alpha-ketoglutarate were not surprising in light of previous work in the field. The mechanism of action of this agent has been attributed primarily to the chelation of cyanide in the form of a reversible covalent bond to form cyanohydrin. Although glyoxylate dosing is high on a per mole cyanide basis, efficacy is improved by more than 10-fold. More importantly, this enhanced efficacy translated into in vivo efficacy with significant results in both mouse inhalation and rabbit models when glyoxylate was administered via IM. Id. Cytochrome c oxidase levels rapidly recovered in rabbits.

[0079] One or more of the ligands can include a ligand such as methionine, which can form an S,N-chelate with platinum and exist in solution as multiple isomers. The ligands of each bidentate ligand can be selected to affect the assay method for the compound's cyanide scavenging, thereby affecting the compound's detoxification efficacy. Behymer et al. (2022), supra.

[0080] Additionally, ligand selection can take into account that certain structures exhibit a pH dependence with respect to cyanide reactivity: the rate of association of amines with platinum, for example, increases at higher pH, leading to loss of a proton and favoring Pt-N bond formation. Summa et al., Thermodynamic and kinetic studies on reactions of Pt(II) complexes with biologically relevant nucleophiles, Inorganic Chem 45: 2948-2959 (2006); Appleton et al., S,O- versus S,N-chelation in the reactions of the cis-diamminediaqua platinum (II) cation with methionine and S-methylcysteine, Inorganic Chem 27: 130-137 (1988); Appleton et al., Nitrogen-15 and platinum-195 NMR spectra of platinum ammine complexes: trans- and cis-influence series based on platinum-195-nitrogen-15 coupling constants and nitrogen-15 chemical shifts, Inorganic Chemistry 24: 4685-4693 (1985). This can be utilized to facilitate compound safety and prevent toxicity (e.g., dose-limiting toxicity).

[0081] The ligand may be substituted. The ligand may be unsubstituted. In certain embodiments, the ligand is substituted with a carboxyamide. In certain embodiments, the ligand is substituted with a carboxylate and / or a carboxyester. The ligand may be substituted with one or more amidated carboxylates.

[0082] The ligand may be optionally substituted. "Optionally substituted" means unsubstituted or substituted. As used herein, the term "substituted" means that a hydrogen atom has been removed and replaced with a substituent. It is understood that substitution at a given atom is limited by the valence of the atom.

[0083] At least one of the ligands can include a methyl thioether group. At least one of the thioether ligands can include methionine or S-methyl cysteine, optionally including one or more amidated carboxylates.

[0084] At least one of the ligands may be substituted with a halogen (i.e., the halogen is not directly coordinated to the Pt core). "Halogen," by itself or as part of another substituent, means a fluorine (Fl), chlorine (Cl), bromine (Br), or iodine (I) atom, unless otherwise specified. For example, the halogen may be bonded to a carbon in the ligand bridge of a bidentate ligand.

[0085] The ligands (i.e., the leaving group and / or those that remain conjugated after attachment) can be selected to enhance the reactivity of the metal center. For example, but not limited to, at least one of the ligands can be a methylthioether group.

[0086] In certain embodiments, the ligands of the compounds are substituted with carboxamides or esteramides. The structure and / or size of certain non-linking substituents on the ligands of the compounds can affect the reactivity of the platinum core with respect to cyanide anions.

[0087] In certain embodiments, the compound comprises a complex that includes: (1) a core metal atom; (2) at least one ligand (L1 and / or L2) coordinated to the core metal atom that is a leaving group; and (3) one or two ligands (L1 and / or L2) coordinated to the core metal atom that remain conjugated to the positively charged metal atom after binding of the cyanide atom (e.g., groups that remain conjugated to the metal in the presence of cyanide).

[0088] In some embodiments, the compound has the structure of Formula (I):

[0089] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer thereof, wherein Pt is platinum (e.g., in the Pt(II) oxidation state); each L1 and L2 is a ligand, each L1 forms a bidentate ligand, each L2 forms a bidentate ligand, each bidentate ligand contains N, S, or both N and S coordinated to Pt, at least one of the ligands is a leaving group, and at least two of the ligands directly bonded to Pt(II) each independently contain an alkyl, a carboxamide, an amine, an aminosulfide, a carboxylate, a carboxyester, a carbonyl, or a thioether containing any combination of the foregoing; each n is independently from about 1 to 5].

[0090] In certain embodiments of Formula (I), Pt is platinum(II).

[0091] The compound has the structure of formula (II):

[0092] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer thereof, wherein Pt is platinum (e.g., in the Pt(II) oxidation state); each L1 and L2 is a ligand, each L1 forms a bidentate ligand, each L2 forms a bidentate ligand, each bidentate ligand contains N, S, or both N and S coordinated to platinum, at least one of the ligands is a leaving group, and at least two of the ligands directly bonded to platinum each independently contain an alkyl, carboxamide, amine, aminosulfide, carboxylate, carboxyester, carbonyl, or a thioether containing any combination of the foregoing; R1 is C 1~3 Alkyl, C 1~3 Haloalkyl, C 2~4 Alkenyl, C 2~4 Alkynyl, and C 6~10 aryl or absent; each n is independently from about 1 to 5].

[0093] In certain embodiments of Formula (II), Pt is platinum(II).

[0094] The compound has the structure of formula (III):

[0095] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer thereof, wherein Pt is platinum (e.g., in the Pt(II) oxidation state); each L1 and L2 is a ligand, each L1 forms a bidentate ligand, each L2 forms a bidentate ligand, each bidentate ligand contains N, S, or both N and S coordinated to platinum, at least one of the ligands is a leaving group, and at least two of the ligands directly bonded to platinum(II) contain alkyl, carboxamide, amine, aminosulfide, carboxylate, carboxyester, carbonyl, or a thioether containing any combination of the foregoing; Each R1 is independently absent or C 1~3 Alkyl, C 1~3 Haloalkyl, C 2~4 Alkenyl, C 2~4 Alkynyl, and C 6~10 aryl, or absent; and n is independently about 1 to 5.

[0096] In certain embodiments of Formula (III), Pt is platinum(II).

[0097] It will be appreciated that when no R is present in formula (III), formula (III) is equivalent to formula (I). When one R is absent in formula (III), formula (III) can be equivalent to formula (II).

[0098] The ligands (L1 and L2) of Formulas (I)-(III) can be any of the ligands described herein. One or more of the ligands of Formulas (I)-(III) can be or include a sulfide. In certain embodiments, at least two of the ligands of Formulas (I)-(III) are sulfides (e.g., the compound includes a disulfide).

[0099] The ligands L1 and L2 (and thus the bidentate ligands) of the compound or pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer can be the same or different or a mixture thereof.

[0100] At least one of the ligands (L1 and / or L2) of formulas (I)-(III) can include a carboxyamide or a carboxyester. At least one of the ligands can include an amine. The amine can be an aminosulfide. In certain embodiments, at least one of the ligands includes a carboxylate or a carbonyl.

[0101] In certain embodiments, the first L1 comprises a sulfide ligand and the second L1 comprises an amide ligand. Additionally or alternatively, the first L2 can comprise a sulfide and the second L2 can comprise an amide.

[0102] The compounds of formula (II) and (III) can include one or more spacers (R1). The spacer (R1) can be C 1~3 The spacer can be alkyl. The spacer can be branched. The spacer can be unbranched or linear (e.g., a linear alkyl group). Some examples of linear alkyl groups include methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl groups. The spacer can comprise an alkyl group, an alkenyl group, a haloalkyl group, an alkynyl group, or an aryl group. The spacer can comprise a peptide, a peptidoglycan, a polyethylene glycol (PEG) linker, a PEG derivative linker, or a combination of two or more of the foregoing. In some embodiments, the PEG linker is (-CHCH-O-). n where n is an integer between 1 and 16, inclusive. In certain embodiments, the PEG linker's n is an integer between 1 and 4, inclusive. In certain embodiments, the spacer can comprise an optionally substituted C 1~3 In certain embodiments, the spacer is an optionally substituted C alkyl. 1~3 In certain embodiments, the spacer is an optionally substituted C 2~4 In certain embodiments, the spacer is an optionally substituted C alkenyl. 2~4In certain embodiments, the spacer is an optionally substituted C 6~10 It is aryl.

[0103] Spacers are C2~C 18 The term "fragment" as used herein refers to a molecule that has been modified to allow for linkage in a compound as either a monovalent or bivalent linkage, such as in the case of R in formula (II) or (III). The use of the term "fragment" does not require that, from a synthetic standpoint, the molecule it refers to be created during the preparation of the compound. It is a description of a moiety within a compound, regardless of how it is created.

[0104] In certain embodiments, the spacer (R1) is absent.

[0105] The spacer can be designed to adjust the reactivity of the compound towards cyanide anions.

[0106] In certain embodiments, the compound has the following structure:

[0107] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof (e.g., a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer of any one of the foregoing structures).

[0108] The compound has the following structure:

[0109] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of any of the foregoing structures.

[0110] In certain embodiments, the compound comprises HCP-AKN, cisplatin-AKN, (salylCys)2Pt, (SMePenicillamine)2P, (cilastatin)2Pt, or (bridgedMet2)Pt, MetPt(taurine)2.

[0111] In certain embodiments, the compound comprises the following structure, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof:

[0112] [ka]

[0113] In certain embodiments, the compound comprises the following structure, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof:

[0114] [ka]

[0115] In certain embodiments, processes for making the compounds herein are provided. For example, compounds 6-11 can be prepared as specified in Example 1, and / or compounds 6, 9, 12, and 13 can be prepared as specified in Example 10. Similarly, other compounds herein can be prepared according to the processes of Examples 1 and / or 10, as well as other such processes known in the art.

[0116] In certain embodiments, the compound is formulated as a prodrug. The term "prodrug" refers to a derivative of a compound that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide an active compound, particularly the Pt(II)-thioether complexes disclosed herein. Examples of prodrugs include, but are not limited to, derivatives and metabolites of the compounds herein that contain biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs. In certain embodiments, at least one ligand of the compound is substituted with a carboxamide or asteramide.

[0117] Specific prodrugs of compounds with a carboxyl functional group are lower alkyl esters of the carboxylic acid. Carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs can typically be prepared using well-known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers GmbH).

[0118] Formulation of compounds as prodrugs can incorporate hydrolyzable groups that allow the compounds to be inactivated in vivo. This can be beneficial in mitigating the challenge of dose-limiting toxicity, allowing compounds to be developed that are effective (i.e., can be dosed at sufficient concentrations to achieve a therapeutic effect) but do not present a significant risk of toxicity (e.g., have a low risk of acute kidney injury (AKI)).

[0119] A compound (or a pharmaceutically acceptable salt thereof) may contain one or more chiral centers or may otherwise exist as multiple stereoisomers, such as enantiomers, diastereomers, toposomers, isomers, and enantiomerically or diastereomerically enriched mixtures. Unless otherwise specified, all stereoisomeric forms of a compound are contemplated, and it is intended that a configuration depicted in a particular configuration (e.g., cis or trans) is not limited thereto unless explicitly so stated, but should instead be read to encompass all stereoisomers thereof. In certain embodiments, the compounds herein comprise a cis configuration. In certain embodiments, the compounds herein comprise a trans configuration.

[0120] "Isomers" refer to structural, geometric, and stereoisomers. The term "geometric isomer" refers to E or Z geometric isomers (e.g., cis or trans) of an alkene double bond. If a compound contains an alkene double bond, unless otherwise specified, it is intended to include both E and Z geometric isomers (e.g., cis or trans) and / or optical isomers. Likewise, all possible isomers and their racemic and optically pure forms, as well as all tautomeric forms, are intended to be included.

[0121] "Tautomer" means a compound that can exist in equilibrium between two isomeric forms. Such compounds may differ in the bond connecting two atoms or groups and in the positions of those atoms or groups in the compound.

[0122] Those skilled in the art will further recognize that a compound may be "deuterated," meaning that one or more hydrogen atoms may be replaced with deuterium. Because deuterium and hydrogen have nearly identical physical properties, deuterium substitution is the smallest structural change that can be made. Deuteration is well known to those skilled in the art.

[0123] The compound (or its pharmaceutically acceptable salt) can exist in a non-solvated form and a solvated form, including a hydrated form. Generally, a solvated form is equivalent to a non-solvated form. The compound can exist in multiple crystalline or amorphous forms. Generally, all physical forms are equivalent for the intended use. The formula includes pharmaceutically acceptable salts (e.g., acid addition and base salts), hydrates, and / or solvates.

[0124] Furthermore, in each of the foregoing and following embodiments, the formulas should be understood to include and represent not only all pharmaceutically acceptable salts of the conjugate, but also any and all hydrates and / or solvates of the conjugate formula or its salts. Indeed, hydrates, solvates, and N-oxides of the conjugate are also contemplated. The term "solvate" refers to a conjugate or a salt thereof that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. When the solvent is water, the solvate is a hydrate.

[0125] It will be appreciated that certain functional groups, such as hydroxy, amino, and similar groups, form complexes and / or coordination compounds with water and / or various solvents in various physical forms of the conjugate, and therefore, the above formulas should be understood to include and represent various hydrates and / or solvates thereof.

[0126] It is also to be understood that in each of the foregoing and following embodiments, the formulas include and represent any and all crystalline forms, partially crystalline forms, and non-crystalline and / or amorphous forms of the conjugate.

[0127] pharmaceutically acceptable salts "Pharmaceutically acceptable salts" of compounds are contemplated. The term "pharmaceutically acceptable salts" refers to salts whose counterions can be used in pharmaceutical preparations. In various embodiments, such salts include, but are not limited to, 1) acid addition salts that can be obtained by reacting the free base of the parent compound with inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, oxalic acid, (D) or (L) malic acid, maleic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or 2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, such as an alkali metal ion, alkaline earth ion, or aluminum ion, or coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, trimethamine, N-methylglucamine, and the like. Pharmaceutically acceptable salts are well known to those of skill in the art, and any such pharmaceutically acceptable salts are contemplated in connection with the embodiments described herein.

[0128] In various embodiments, suitable acid addition salts are formed from acids that form non-toxic salts. Illustrative examples include acetate, aspartate, benzoate, besylate, bicarbonate / carbonate, bisulfate / sulfate, borate, camsylate, citrate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, saccharate, stearate, succinate, tartrate, tosylate, and trifluoroacetate.

[0129] In various embodiments, suitable base salts are formed from bases that form non-toxic salts. Illustrative examples include arginine salts, benzathine salts, calcium salts, choline salts, diethylamine salts, diolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, olamine salts, potassium salts, sodium salts, tromethamine salts, and zinc salts. Hemisalts of acids and bases, such as hemisulfate salts and hemicalcium salts, can also be formed.

[0130] Pharmaceutical Compositions, Routes of Administration, and Dosage Further provided are pharmaceutical compositions comprising the compounds described herein, pharmaceutically acceptable salts, N-oxides, solvates, tautomers, or stereoisomers of the compounds described herein, and pharmaceutically acceptable carriers or excipients. The term "pharmaceutically acceptable carrier" refers to one or more suitable solid or liquid fillers, excipients, or encapsulating substances suitable for administration to humans or other vertebrates. The term "carrier" refers to a natural or synthetic organic or inorganic material that combines with an active ingredient to facilitate application. The carrier can be an additive. The selection of a carrier can depend on factors such as the specific mode of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form. Pharmaceutical compositions suitable for delivering the compounds described herein and methods for their preparation can be found, for example, in Remington: The Science & Practice of Pharmacy, 21st edition (Lippincott Williams & Wilkins, 2005).

[0131] The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient.

[0132] The components of the compositions are capable of being commingled with the compounds, and with each other, such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

[0133] The composition can include, for example, cremophor, polysorbate, nanoparticles, polymers, or hydrogels. In certain embodiments, the pharmaceutical composition includes multiple compounds and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition further includes at least one additional pharmaceutically active agent. The at least one additional pharmaceutically active agent can be an agent useful in the treatment of cyanide poisoning.

[0134] Pharmaceutical compositions can be prepared by combining one or more compounds with a pharmaceutically acceptable carrier and, optionally, one or more additional ingredients (e.g., pharmaceutically active ingredients). Formulations can be administered in a pharmaceutically acceptable solution, which can routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

[0135] Pharmaceutically acceptable carriers can include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., and combinations thereof. Carriers can be suitable for parenteral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Examples of such carriers (or additives) include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycol. One or more other active agents can also be incorporated into the pharmaceutical composition.

[0136] Pharmaceutical compositions can be formulated as liquids, for example, suspensions or solutions. Liquid formulations can include water, ethanol, PEG, propylene glycol, methylcellulose, or suitable oils, and one or more emulsifiers and / or suspending agents. Liquid formulations can be prepared by reconstituting solids. In certain embodiments, the composition is suitable for intramuscular injection (i.e., contains a suitable formulation).

[0137] Pharmaceutical formulations (e.g., for IM administration) include aqueous solutions of the active compound in water-soluble form. In addition, suspensions of the active compound can be prepared as appropriate oily injection suspensions. Aqueous suspensions can contain the compound alone or in combination with one or more other active agents, mixed with suitable additives. Additives include suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents such as naturally occurring phospholipids, for example, lecithin; condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate; condensation products of ethylene oxide with long-chain aliphatic alcohols, such as heptadecaethyleneoxcycetanol; condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, for example, polyoxyethylene sorbitol monooleate; or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyoxyethylene sorbitan monooleate. Aqueous suspensions can also contain one or more preservatives, such as ascorbic acid or ethyl, n-propyl, or p-hydroxybenzoates, and one or more colorants. In certain embodiments, aqueous suspensions may further comprise a suitable lipophilic solvent or vehicle, including fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0138] Alternatively, pharmaceutical compositions can be in powder form for constitution with suitable vehicle, such as sterile pyrogen-free water, before use.Dispersible powders and granules suitable for preparing aqueous suspension by adding water can provide active ingredient mixed with suspending agent, dispersing or wetting agent, and one or more preservatives.Additional additives, such as coloring agents, can also be present.

[0139] Suitable emulsifiers include naturally occurring gums, such as gum acacia or gum tragacanth; naturally occurring phospholipids, such as soybean lecithin; and esters, including partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.Isotonizing agents, such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, can be included in the composition.Prolonged absorption of injectable compositions can be achieved by including one or more agents for delaying absorption, such as monostearate salts and gelatin, in the composition.

[0140] For use in therapy or treatment, an effective amount of the compound or composition can be administered to a subject by any mode that delivers the compound as desired. Administering the composition can be accomplished by any means known to those skilled in the art. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, direct injection, inhalation, and topical.

[0141] In certain embodiments, the compound can be administered directly into the bloodstream, intramuscularly, or into an internal organ. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intranasal, and subcutaneous. Suitable means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques. When it is desired to deliver the compound and / or composition to the entire body, the compound and / or composition can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. In certain embodiments, the compound and / or composition herein can be formulated for intramuscular injection by a single bolus injection.

[0142] Injectable preparations may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with added preservatives. "Dose" and "dosage" are used interchangeably herein. The compositions may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulating agents such as suspending, stabilizing, and / or dispersing agents.

[0143] Parenteral formulations are typically aqueous solutions (preferably at a pH of 3 to 9) which may contain carriers or additives such as salts, carbohydrates, and buffers, although for some applications they may be more suitably formulated as sterile non-aqueous solutions or as a dry form for use in conjunction with a suitable vehicle such as sterile pyrogen-free water.

[0144] Liquid formulations can be adapted for parenteral administration of compounds.Under sterile conditions, for example, by lyophilization under sterile conditions, parenteral formulations can be easily prepared by using standard pharmaceutical techniques well known to those skilled in the art.The solubility of compounds can be increased by using appropriate formulation techniques, such as incorporating solubility enhancers.

[0145] Preparations for parenteral administration can be formulated for immediate and / or modified release. The compound can be administered in a sustained release formulation, for example, in a composition containing a slow-release polymer. The compound can be prepared with a carrier that will protect it from rapid release, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-polyglycolic acid copolymer (PGLA). Methods for preparing such formulations are generally known to those skilled in the art.

[0146] Sterile injectable solution can be prepared by incorporating the compound alone or with one or more other active ingredients in the required amount in suitable solvent, and when necessary, with one or combination of above-mentioned raw materials.Usually, dispersion is prepared by incorporating compound into sterile vehicle, and this contains dispersion medium and any additional raw materials mentioned above.In the case of the sterile powder that is used to prepare sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, thereby producing the powder of active ingredient plus any additional desired raw material from its previously sterile-filtered solution, or raw material can be sterilized and filtered together.

[0147] The pharmaceutical composition can be formulated as a liquid, microemulsion, liposome, or other ordered structure suitable for high drug concentration.The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof.Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.

[0148] The compound, or pharmaceutical composition containing the compound, may be administered continuously, where appropriate.

[0149] Methods of Treatment, Combination Therapies, and Uses Provided is the use of any of the compounds provided herein, pharmaceutical salts, N-oxides, solvates, tautomers or stereoisomers thereof, and / or pharmaceutical compositions in the manufacture of a medicament for treating a disease or condition in a subject. The disease or condition can be cyanide poisoning or cyanide exposure.

[0150] The medicament may be formulated for intramuscular administration (e.g., intramuscular injection). In certain embodiments, the medicament may be formulated in a single bolus dosage.

[0151] The medicament may be formulated at a pH of about 5 or greater (such as at a pH of about 5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, etc.). The medicament may be formulated at a pH of 7. The medicament may be formulated at a pH of 6.8. The medicament may be formulated at a pH of 5.8.

[0152] After formulation, the medicament may be stored, for example, at a pH of about 5 or less (such as at a pH of about 5, 5.0, 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, or at a pH below 4.0).

[0153] In some embodiments, the compounds described herein, as well as pharmaceutically acceptable salts, N-oxides, solvates, tautomers, and stereoisomers (whether part of a pharmaceutical, composition, etc.), contain a positively charged platinum core and two to six ligands, at least one of which is a leaving group. That is, the platinum core is amendable to nucleophilic attack by a cyanide ion, whereby the cyanide ion binds to the platinum core and displaces the leaving group ligand. In some embodiments, each platinum complex can bind one to six, one to five, one to four, or one to three cyanide anions. That is, each compound described herein can bind one, two, three, four, five, or six cyanide anions. In certain embodiments, the compounds described herein can bind four cyanide anions.

[0154] Due to the ability of the platinum core to bind cyanide anions, the compound, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof, can be used as an antidote for cyanide poisoning. In some embodiments, a method for treating (or preventing) cyanide poisoning or cyanide exposure in a subject (e.g., a subject experiencing or at risk of experiencing cyanide poisoning) is provided. In some embodiments, the method includes administering to the subject a first therapy comprising a therapeutically effective amount of any compound described herein (e.g., a Pt-based compound), a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof, or any of the pharmaceutical compositions described above. In certain embodiments, at least one ligand of the compound, pharmaceutical salt, N-oxide, solvate, tautomer, or stereoisomer, or pharmaceutical composition comprises an amino acid ligand substituted with one or more carboxamides. The therapeutically effective amount of the first therapy can include administering a single dose.

[0155] The therapeutically effective amount of the first therapy can be about 3.0-5.5 mg / kg (based on the subject's body weight). The therapeutically effective amount of the first therapy can be about 3.5 mg / kg (based on the subject's body weight).

[0156] The step of administering the first therapy can be carried out through an intraperitoneal (IP) injection of the compound or pharmaceutical composition to the subject. The step of administering the first therapy can include an intramuscular injection of the compound, a pharmaceutical salt, N-oxide, solvate, tautomer or stereoisomer thereof, or a pharmaceutical composition to the subject.

[0157] In certain embodiments, the administering step comprises intramuscular administration, and the compound, pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer thereof, or pharmaceutical composition reaches a peak concentration (C) in the subject at or between about 7-9 minutes (e.g., within 7 minutes to about 9 minutes, within about 7 minutes to 9 minutes, or within 7-9 minutes). max As used herein, "maximum concentration" or "Cmax " is a pharmacokinetic parameter known to those skilled in the art and refers to the peak concentration of a compound / active agent (such as the platinum complexes herein) in the serum of a subject following administration of a compound or pharmaceutical composition herein to the subject by intramuscular, intranasal, subcutaneous, intravenous, or other parenteral route.

[0158] In certain embodiments, after a compound, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer thereof, or pharmaceutical composition is administered intramuscularly to a subject, the compound, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer thereof, or pharmaceutical composition undergoes pH-induced isomerization, resulting in a reduced cyanide scavenging rate relative to the cyanide scavenging rate of the compound or pharmaceutical composition within 1 hour of administration to the subject.

[0159] The method can further include administering a second therapy to the subject. In certain embodiments, the second therapy can include administering to the subject a therapeutically effective amount of one or more of glyoxylate, hydroxocobalamin, methemoglobin, riboflavin, methotrexate, sulfanegen, 4-dimethylaminophenol (4-DMAP), dicobalt edetate, glucose, activated charcoal, and ethylenediaminetetraacetic acid (EDTA) disodium cobalt; an intravenous isotonic fluid; and / or oxygen therapy (e.g., providing supplemental oxygen to the subject).

[0160] The second therapy can comprise an agent for improving cyanide-induced oxidative stress in the subject.The agent for improving cyanide-induced oxidative stress can be, for example, glyoxylate.In certain embodiments, the second therapy is glyoxylate, and the therapeutically effective amount of the first therapy is 3.5mg / kg (based on the subject's body weight).

[0161] In certain embodiments, the first and second therapies are administered sequentially and / or alternatingly with one another. In certain embodiments, the first and second therapies are administered concurrently.

[0162] Because cyanide inhibits the mitochondrial enzyme cytochrome c oxidase, cells of an organism poisoned by cyanide are unable to use oxygen to produce ATP, a condition also known as cellular hypoxia. In some embodiments, the method includes identifying a subject exposed to cyanide. Subjects in need of treatment using the methods or compounds described herein can be identified by those skilled in the art using known methods. Early symptoms of cyanide poisoning can include headache, dizziness, rapid heartbeat, shortness of breath, and vomiting. These early symptoms can be followed by seizures, bradycardia, hypotension, loss of consciousness, cardiac arrest, and death. In survivors, the consequences of cyanide poisoning can be long-term and can include chronic respiratory disease (e.g., chronic obstructive pulmonary disease (COPD), asthma, or pulmonary hypertension), blindness, damage to or loss of vital organ function (e.g., heart, lungs, kidneys, and brain), cognitive deficits, and cardiac, neurological, and metabolic dysfunction. In some embodiments, administration of the Pt-based compounds described herein protects against or induces rapid reversal of cyanide-induced pathophysiological changes.

[0163] In one example, metabolic dysfunction caused by cyanide includes metabolic disruption (e.g., decreased production) of bile acids and purine nucleobases, nucleosides, and nucleotides. Some examples of bile acids include glycochenodeoxycholic acid, taurocholic acid, and taurochenodeoxycholic acid. Examples of purine nucleosides and nucleotides include inosine, deoxyadenosine, deoxyguanosine, adenosine, and guanosine. Examples of purine nucleobases include purine, adenine, guanine, hypoxanthine, xanthine, theobromine, and uric acid. In another example, metabolic dysfunction caused by cyanide includes increased concentrations of tricarboxylic acid (TCA) cycle metabolites, since cyanide slows their consumption. Examples of TCA cycle metabolites include α-ketoglutarate, succinate, fumarate, and malate.

[0164] In some embodiments, cyanide poisoning in a subject can be caused by inhaling smoke from a fire, exposure to pesticides, administration of the drug nitroprusside, an industrial accident, or exposure to chemical warfare. Cyanide is easily absorbed through the dermis, bronchi, and digestive routes and rapidly distributes to tissues throughout the body, causing multi-organ toxicity, particularly in organs with high demand for ATP, such as the brain and heart. Exposure to milligram amounts of the poison can cause symptoms that appear within minutes of exposure. In some embodiments, none of the methods of use or treatment, or combination therapies, referred to herein, include the use of cisplatin or carboplatin.

[0165] In some embodiments, the detoxification activity (EC 100 ) is from about 5 μM to about 150 μM, from about 7 to about 100 μM, or from about 10 to about 150 μM.

[0166] The methods herein can reduce, and even substantially reduce, systemic and off-target toxicity. "Off-target toxicity" refers to organ or tissue damage that is undesirable to the physician or other individuals treating the subject, or any other effect on the subject that is a potential adverse indicator for the treating physician (e.g., AKI).

[0167] In some embodiments, the compounds herein can be used in cyanide sensing (e.g., in sensors for cyanide anions present in a subject). When the compounds herein bind to cyanide, they are converted into different chemical compounds. Thus, differences in physical properties (e.g., detectable in UV-vis or IR absorbance) between the parent compound and the CN adduct can be measured and used to determine the presence of cyanide in a subject.

[0168] Also provided is a combination therapy for treating cyanide poisoning or exposure in a subject, the combination therapy comprising administering to the subject (a) a therapeutically effective amount of a cyanide chelator and (b) a therapeutically effective amount of an agent for ameliorating cyanide-induced oxidative stress in the subject.

[0169] The cyanide chelator can comprise a platinum(II) thioether comprising a bidentate ligand. The cyanide chelator can be any compound described herein, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof, any of the pharmaceutical compositions described herein, or a compound having the following structure:

[0170] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer.

[0171] An agent for ameliorating cyanide-induced oxidative stress can be, for example, glyoxylate or an analog or functional fragment thereof (to the extent that such functional fragment or analog can ameliorate cyanide-induced oxidative stress in a subject when administered). "Analog" in this context means a compound that is structurally similar to another compound but has slight differences in its structure or properties (e.g., modifications or substitutions at certain parts of the molecule that are not present in the other compound). "Functional fragment" in this context means a chemical structural unit or component of a larger molecule that retains the unique function or functional activity and / or reactivity of the larger molecule. A functional fragment can be linear, branched, or cyclic, an oligomer, or a low molecular weight organic molecule. A functional fragment is completely contained within the composition of the original larger molecule.

[0172] In certain embodiments, the agent for ameliorating cyanide-induced oxidative stress in a subject comprises a ketone or aldehyde compound selected from the group consisting of glyoxylate, glyceraldehyde, acetaldehyde, dihydroxyacetone, pyruvate, methylglyoxal, and alpha-ketoglutarate.

[0173] In certain embodiments, the second therapy is glyoxylate. In certain embodiments, the second therapy is glyoxylate and the therapeutically effective amount of the first therapy is 3.5 mg / kg (based on the subject's body weight).

[0174] The terms "treat," "treating," "treated," and "treatment" (in reference to a disease or condition such as cyanide poisoning) are used to describe a method for obtaining beneficial or desired results, such as a clinical outcome, which may include, but is not limited to, one or more of: ameliorating the condition associated with the disease or condition, curing the disease or condition, reducing the severity of the disease or condition, increasing the quality of life of a person suffering from the disease or condition, prolonging survival, and / or preventative treatment. In particular, in reference to cyanide poisoning, the terms "treat," "treating," "treated," or "treatment" may additionally mean reducing the oxygen concentration in the subject's blood, reducing the cyanide concentration in the subject's blood, stabilizing the subject, preventing the progression of cyanide poisoning, or any other effect on the subject that would be considered by a physician to be a therapeutic or preventative treatment of cyanide poisoning. More specifically, curative treatment refers to either the alleviation, improvement and / or elimination, reduction and / or stabilization of signs / symptoms (e.g., inability to progress to a more advanced stage), and delay in the progression of signs / symptoms of a particular disorder or condition. Preventative treatment refers to any of the following: halting onset, reducing the risk of onset, reducing the incidence, delaying onset, reducing onset, and increasing the time to onset of symptoms of a particular disorder or condition. Desirable effects of treatment may include, but are not limited to, preventing the onset of a disease or condition, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease or condition, reducing the rate of disease or condition progression, improving or alleviating the symptoms or condition, and improving prognosis. In some embodiments, the compounds and compositions herein can be used to delay the onset of cyanide poisoning or slow (or even stop) the progression of cyanide poisoning.

[0175] The term "patient" or "subject" includes humans and non-human animals, such as companion animals (such as dogs and cats) and livestock animals. Livestock animals are animals that are raised for food production. The subject to be treated is preferably a mammal, particularly a human.

[0176] As used herein, the term "administering" includes all means of introducing compounds and pharmaceutical compositions comprising same into a patient. Examples include, but are not limited to, parenteral, systemic / intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, intrasternal, intraarterial, intraperitoneal, epidural, intraurethral, intranasal, buccal, ocular, sublingual, intravaginal, rectal, etc. In certain embodiments, the route of administration is intramuscular.

[0177] Illustrative means of parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques, as well as any other means of parenteral administration recognized in the art. Parenteral formulations are typically aqueous solutions, which may contain additives such as salts, carbohydrates, and buffers (preferably at a pH within the range of about 3 to about 9). The preparation of parenteral formulations under sterile conditions may be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0178] The compounds can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient, in a variety of forms adapted to the selected route of administration. For example, pharmaceutical compositions can be formulated for and administered via parenteral, intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, topical, inhalation, and / or subcutaneous routes. Indeed, the compounds, or compositions containing them, can be administered directly into the bloodstream, intramuscularly, or into internal organs.

[0179] The compounds / compositions can be administered via infusion or injection (e.g., using needle (including microneedle) injectors and / or needle-free injectors). Single-dose injections are often beneficial in treating cyanide poisoning, and the compounds and compositions of the present invention provide significant efficacy when administered intramuscularly in a single-dose injection. Solutions of the compositions can be aqueous solutions, optionally mixed with non-toxic surfactants, and / or can contain carriers or additives such as salts, carbohydrates, and buffers (preferably at a pH of 3 to 9).

[0180] The percentage of the compound and preparation in the pharmaceutical composition can vary and can be between about 1 and about 99% by weight of the active ingredient, as well as binders, additives, disintegrants, lubricants, and / or sweeteners (as known in the art). The amount of the compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

[0181] In some embodiments, the compound is administered as a composition comprising one or more pharmaceutically acceptable carriers, adjuvants, excipients, additives, vehicles, or a combination of any of the foregoing.

[0182] The term "therapeutically effective amount," as used herein, refers to an amount of a compound that elicits the biological or medicinal response in a tissue system, animal, or human desired by a researcher, veterinarian, physician, or other clinician (e.g., the desired therapeutic effect), including alleviation of the symptoms of the disease or condition being treated. In one aspect, a therapeutically effective amount is one that will treat or alleviate the disease or symptoms of the disease at a reasonable benefit / risk ratio applicable to any medical treatment. However, it will be understood that the total daily usage of the compound may be decided by the attending physician within the scope of sound medical judgment. In the treatment of cyanide poisoning, the desired therapeutic effect can range from preventing the condition from being experienced to inhibiting the progression of the condition and ameliorating the symptoms experienced during acute cyanide poisoning. Desirably, administration of a therapeutically effective amount will scavenge a substantial amount of cyanide from the subject's blood in a short period of time, preferably to the point of eradication.

[0183] The specific therapeutically effective dose level of the compounds herein for any particular patient will depend on various factors, including the stage / severity of the condition; the specific composition used; the patient's age, weight, general health, sex, and dietary habits; the time and route of administration; the duration of treatment; drugs used in combination with or concurrently with the compound and / or pharmaceutical composition; and similar factors well known to researchers, veterinarians, physicians, or other clinicians of ordinary skill. For example, the dose of the compounds herein may range from about 3.0 to 5.5 mg per kg of patient weight (about 3.0 mg to about 5.5 mg, about 3.0 mg to about 5.5 mg, or about 3.0 mg to about 5.5 mg, etc.). Thus, the absolute amount of the compound contained in a given unit dosage form can vary widely and will depend on factors such as the age, weight, and physical condition of the subject, as well as the method of administration.

[0184] A wide range of acceptable dosages is contemplated herein, depending on the route of administration.Dosages can be single or divided, and can be administered according to a variety of protocols, including qd (once a day), bid (twice a day), tid (three times a day), or even every other day, once a week, once a month, once a quarter, etc.In each of these cases, it is understood that the therapeutically effective amount described herein corresponds to the total daily, weekly, monthly, or quarterly dose, as determined by the administration example, or alternatively, the administration protocol.

[0185] In clinical use, any compound may be administered in an amount equal to or equivalent to 0.2 to 2,000 milligrams (mg) of compound per kilogram (kg) of subject body weight per day. The compound may be administered at a dose equal to or equivalent to 2 to 2,000 mg of compound per kg of subject body weight per day. The compound may be administered at a dose equal to or equivalent to 5 to 2,000 mg of compound per kg of subject body weight per day. The compound may be administered at a dose equal to or equivalent to 20 to 2,000 mg of compound per kg of subject body weight per day. The compound may be administered at a dose equal to or equivalent to 100 to 2,000 mg of compound per kg of subject body weight per day. The compound may be administered at a dose equal to or equivalent to 200 to 2,000 mg of compound per kg of subject body weight per day. If a precursor or prodrug of a compound is to be administered, it is administered in an amount equivalent to, i.e., sufficient to deliver, the amounts of compound described above.

[0186] The compound or its pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer formulation can be administered to a human subject in an effective amount. Typical dosage ranges are from about 0.01 micrograms / kg to about 5.5 mg / kg of body weight per day. The dosage of the drug to be administered will likely depend on variables such as the type and severity of the disorder, the overall health of the specific subject, the specific compound being administered, the additives used to formulate the compound, and its administration route. Routine experimentation can be used to optimize the dosage and frequency of administration for any specific compound or its pharmaceutically acceptable salt.

[0187] The compound or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof may be administered at a concentration ranging from about 0.001 microgram / kg to about greater than 500 mg / kg. For example, concentrations may be 0.001 microgram / kg, 0.01 microgram / kg, 0.05 microgram / kg, 0.1 microgram / kg, 0.5 microgram / kg, 1.0 microgram / kg, 10.0 microgram / kg, 50.0 microgram / kg, 100.0 microgram / kg, 500 microgram / kg, 1.0 mg / kg, 5.0 mg / kg, 10.0 mg / kg, 15.0 mg / kg, 20.0 mg / kg, 25.0 mg / kg, The dose can be 30.0 mg / kg, 35.0 mg / kg, 40.0 mg / kg, 45.0 mg / kg, 50.0 mg / kg, 60.0 mg / kg, 70.0 mg / kg, 80.0 mg / kg, 90.0 mg / kg, 100.0 mg / kg, 150.0 mg / kg, 200.0 mg / kg, 250.0 mg / kg, 300.0 mg / kg, 350.0 mg / kg, 400.0 mg / kg, 450.0 mg / kg, up to about greater than 500.0 mg / kg, or any increment therein, with all values and ranges between these values and ranges being understood to be encompassed.

[0188] The compound or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof can be administered at a dosage ranging from about 0.2 milligrams / kg / day to about greater than 100 mg / kg / day. For example, dosages can range from 0.2 mg / kg / day to 100 mg / kg / day, 0.2 mg / kg / day to 50 mg / kg / day, 0.2 mg / kg / day to 25 mg / kg / day, 0.2 mg / kg / day to 10 mg / kg / day, 0.2 mg / kg / day to 7.5 mg / kg / day, 0.2 mg / kg / day to 5 mg / kg / day, 0.25 mg / kg / day to 100 mg / kg / day, 0.25 mg / kg / day to 50 mg / kg / day, 0.25 mg / kg / day to 25 mg / kg / day, 0.25 mg / kg / day to 10mg / kg / day, 0.25mg / kg / day to 7.5mg / kg / day, 0.25mg / kg / day to 5mg / kg / day, 0.5mg / kg / day to 50mg / kg / day, 0.5mg / kg / day to 25mg / kg / day, 0.5mg / kg / day to 20mg / kg / day, 0.5mg / kg / day to 15mg / kg / day, 0.5mg / kg / day to 10mg / kg / day, 0.5mg / kg / day to 7.5mg / kg / day, 0.5mg / kg / day to 5mg / kg / day, 0.75mg / kg / day to 50mg g / kg / day, 0.75mg / kg / day to 25mg / kg / day, 0.75mg / kg / day to 20mg / kg / day, 0.75mg / kg / day to 15mg / kg / day, 0.75mg / kg / day to 10mg / kg / day, 0.75mg / kg / day to 7.5mg / kg / day, 0.75mg / kg / day to 5mg / kg / day, 1.0mg / kg / day to 50mg / kg / day, 1.0mg / kg / day to 25mg / kg / day, 1.0mg / kg / day to 20mg / kg / day, 1.0mg / kg / day to 15 mg / kg / day, 1.0 mg / kg / day to 10 mg / kg / day, 1.0 mg / kg / day to 7.5 mg / kg / day, 1.0 mg / kg / day to 5 mg / kg / day, 2 mg / kg / day to 50 mg / kg / day, 2 mg / kg / day to 25 mg / kg / day, 2 mg / kg / day to 20 mg / kg / day, 2 mg / kg / day to 15 mg / kg / day, 2 mg / kg / day to 10 mg / kg / day, 2 mg / kg / day to 7.5 mg / kg / day, or 2 mg / kg / day to 5 mg / kg / day.

[0189] The compound may be administered at a dosage ranging from about 0.25 milligrams / kg / day to about 25 mg / kg / day. For example, dosages may be 0.25 mg / kg / day, 0.5 mg / kg / day, 0.75 mg / kg / day, 1.0 mg / kg / day, 1.25 mg / kg / day, 1.5 mg / kg / day, 1.75 mg / kg / day, 2.0 mg / kg / day, 2.25 mg / kg / day, 2.5 mg / kg / day, 2.75 mg / kg / day, 3.0 mg / kg / day, 3.25 mg / kg / day, 3.5 mg / kg / day, 3.75 mg / kg / day, 4.0 mg / kg / day, 5.0 mg / kg / day, 6.0 mg / kg / day, 7.0 mg / kg / day, 8.0 mg / kg / day, 9.0 mg / kg / day, 10.0 mg / kg / day, 11.0 mg / kg / day, 12.0 mg / kg / day, 13.0 mg / kg / day, 14.0 mg / kg / day, 15.0 mg / kg / day, 16.0 mg / kg / day, 17.0 mg / kg / day, 18.0 mg / kg / day, 19.0 mg / kg / day, 20.0 mg / kg / day, 21.0 mg / kg / day, 22.0 mg / kg / day, 23.0 mg / kg / day, 24.0 mg / kg / day, 25.0 mg / kg / day, 26.0 mg / kg / day, 27.0 mg / kg / day, 28.0 mg / kg / day, 29.0 mg / kg / day, 30.0 mg / kg / day, 31.0 mg / kg / day, 32.0 mg / kg / day g / day, 4.25mg / kg / day, 4.5mg / kg / day, 4.75mg / kg / day, 5mg / kg / day, 5.5mg / kg / day, 6.0mg / kg / day, 6.5mg / kg / day, 7.0mg / kg / day, 7.5 mg / kg / day, 8.0mg / kg / day, 8.5mg / kg / day, 9.0mg / kg / day, 9.5mg / kg / day, 10mg / kg / day, 11mg / kg / day, 12mg / kg / day, 13mg / kg / day, 14m g / kg / day, 15mg / kg / day, 16mg / kg / day, 17mg / kg / day, 18mg / kg / day, 19mg / kg / day, 20mg / kg / day, 21mg / kg / day, 22mg / kg / day, 23mg / kg / day, 24mg / kg / day, 25mg / kg / day, 26mg / kg / day, 27mg / kg / day, 28mg / kg / day, 29mg / kg / day, 30mg / kg / day, 31mg / kg / day, 32mg / kg / day, 33 mg / kg / day, 34 mg / kg / day, 35 mg / kg / day, 36 mg / kg / day, 37 mg / kg / day, 38 mg / kg / day, 39 mg / kg / day, 40 mg / kg / day, 41 mg / kg / day, 42 mg / kg / day, 43 mg / kg / day, 44 mg / kg / day, 45 mg / kg / day, 46 mg / kg / day, 47 mg / kg / day, 48 mg / kg / day, 49 mg / kg / day, or 50 mg / kg / day.

[0190] The compound, its pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer, or precursor thereof, can be administered at concentrations ranging from 0.01 micromolar to 500 micromolar or more. For example, doses can be 0.01 micromolar, 0.02 micromolar, 0.05 micromolar, 0.1 micromolar, 0.15 micromolar, 0.2 micromolar, 0.5 micromolar, 0.7 micromolar, 1.0 micromolar, 3.0 micromolar, 5.0 micromolar, 7.0 micromolar, 10.0 micromolar, 15.0 micromolar, 20.0 micromolar, 25.0 micromolar, 30.0 micromolar, 35.0 micromolar, 40.0 micromolar, 50.0 micromolar, 60.0 micromolar, 70.0 micromolar, 80.0 micromolar, 90.0 micromolar, 100.0 micromolar, 110.0 micromolar, 120.0 micromolar, 130.0 micromolar, 140.0 micromolar, 150.0 micromolar, 160.0 micromolar, 170.0 micromolar, 180.0 micromolar, 190.0 micromolar, 210.0 micromolar, 220.0 micromolar, 230.0 micromolar, 240.0 micromolar, 250.0 micromolar, 260.0 micromolar, 270.0 micromolar, 280.0 micromolar, 290.0 micromolar, 300.0 micromolar, 310.0 micromolar, 320.0 micromolar, 330.0 micromolar, 340.0 micromolar, The concentration can be 45.0 micromolar, 50.0 micromolar, 60.0 micromolar, 70.0 micromolar, 80.0 micromolar, 90.0 micromolar, 100.0 micromolar, 150.0 micromolar, 200.0 micromolar, 250.0 micromolar, 300.0 micromolar, 350.0 micromolar, 400.0 micromolar, 450.0 micromolar, up to about greater than 500.0 micromolar, or any increment therein, and it is understood that all values and ranges between these values and ranges are intended to be encompassed.

[0191] The compound, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof, or precursor thereof, can be administered at a concentration ranging from 0.10 micrograms / mL to 500.0 micrograms / mL. For example, concentrations can be 0.10 micrograms / mL, 0.50 micrograms / mL, 1 microgram / mL, 2.0 micrograms / mL, 5.0 micrograms / mL, 10.0 micrograms / mL, 20 micrograms / mL, 25 micrograms / mL, 30 micrograms / mL, 35 micrograms / mL, 40 micrograms / mL, 45 micrograms / mL, 50 micrograms / mL, 60.0 micrograms / mL, 70.0 micrograms / mL, 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 110.0 micrograms / mL, 120.0 micrograms / mL, 130.0 micrograms / mL, 140.0 micrograms / mL, 150.0 micrograms / mL, 160.0 micrograms / mL, 170.0 micrograms / mL, 180.0 micrograms / mL, 190.0 micrograms / mL, 200.0 micrograms / mL, 25 micrograms / mL, 30 micrograms / mL, 35 micrograms / mL, 40 micrograms / mL, 45 micrograms / mL, 50 micrograms / mL, 60.0 micrograms / mL, 210.0 micrograms / mL, 220.0 micrograms / mL, 230.0 micrograms / mL, 240.0 micrograms / mL, 250.0 micrograms / mL, 260.0 micrograms / mL, 270. mL, 80.0 micrograms / mL, 90.0 micrograms / mL, 100.0 micrograms / mL, 150.0 micrograms / mL, 200.0 micrograms / mL, 250.0 micrograms / mL, 250.0 micrograms / mL, 300.0 micrograms / mL, 350.0 micrograms / mL, 400.0 micrograms / mL, 450.0 micrograms / mL, up to about greater than 500.0 micrograms / mL, or any increment therein, it being understood that all values and ranges therebetween are intended to be encompassed.

[0192] Multiple infusions may be required to effectively treat a subject. For example, two, three, four, five, six, or more separate infusions may be administered to a patient at intervals of about 24 hours to about 48 hours, or every 3, 4, 5, 6, or 7 days. Infusions may be administered weekly, biweekly, or monthly. Monthly administrations may be repeated for 2 to 6 months or more, such as 9 months to 1 year.

[0193] The dosage administered for the compounds for treating cyanide poisoning will follow dosage and scheduling regimens practiced by those skilled in the art. Determining an effective amount or dose is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0194] Kits for treating cyanide poisoning or exposure Also provided is a kit for treating cyanide exposure or cyanide poisoning in a subject. The kit can include a drug injection device with one or more fluid chambers, each pre-filled with a formulation. The formulation includes (a) a cyanide chelator (e.g., any of the compounds described herein, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer of any of the compounds herein), or a compound having the following structure:

[0195] [ka] or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer (each an "active agent"), and (b) a pharmaceutically acceptable carrier and / or excipient (a "carrier"). The pre-filled fluid chamber can be, for example, a syringe or cartridge. In certain embodiments, the active agent and carrier of the formulation can be separated within the kit (e.g., for ease of storage). For example, the active agent can be contained in a first fluid chamber and the carrier in a second fluid chamber, with the two being mixed as a step prior to administration to a subject. In certain embodiments, the formulation can be stored pre-mixed within the fluid chamber.

[0196] The term "cyanide exposure," as used herein, refers to a condition in which a subject is either at risk of exposure to cyanide or its derivatives or has been exposed to cyanide or its derivatives (e.g., through skin contact, eye contact, inhalation, ingestion, or any other mechanism by which the subject comes into contact) but has not yet experienced detectable symptoms. "Cyanide poisoning," as used herein, refers to and includes conditions caused by cyanide, such as cellular hypoxia and associated symptoms including headache, dizziness, tachycardia, shortness of breath, and vomiting. The term "cyanide poisoning" also includes conditions in which a subject experiences acute or chronic symptoms of exposure to cyanide or its derivatives, regardless of whether those symptoms are readily detectable, such as, but not limited to, cyanide-induced bradycardia, hypotension, loss of consciousness, cardiac arrest, and death. In cases in which the poisoned subject survives, the term also includes long-term conditions caused by cyanide, such as chronic respiratory disease, blindness, cognitive deficits, and pathophysiological changes such as cardiac, neurological, and metabolic dysfunction. The terms cyanide poisoning and cyanide exposure are used interchangeably herein unless expressly stated to the contrary.

[0197] The drug injection device can be any injection device suitable for administering the compounds herein. In certain embodiments, the injection device is configured for injection through one or more needles. The drug injection device can be a handheld syringe. The drug injection device can be an autoinjector. In certain embodiments, the drug injection device is suitable for deployment in the field (e.g., in a mass casualty setting). The drug injection device can be an autoinjector or a handheld syringe.

[0198] The formulation can contain a targeted effective dose of the compound, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof. For example, each fluid chamber can contain a single bolus dose containing a therapeutically effective amount of an active agent (i.e., the compound, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof) ready for immediate administration to a subject. The formulation can contain a targeted effective dose of the compound, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof, for intramuscular injection.

[0199] The formulation can have a pH value of 5 or less. In certain embodiments, the formulation in each fluid chamber can comprise about 1 to 5 mL (e.g., about 1 mL to about 5 mL of formulation, about 1 mL to 5 mL of formulation, or 1 mL to about 5 mL of formulation). In certain embodiments, the formulation in each fluid chamber can be about 3 mL or less.

[0200] Optionally, the kit can include instructions, either as a package insert or as a label, indicating the quantities of the components to be administered, guidelines for administration, and / or guidelines for mixing the components.

[0201] The kit can optionally include, if desired or beneficial, an additional therapeutic agent (e.g., a therapeutically effective amount thereof), such as another cyanide antidote or an agent for ameliorating cyanide-induced oxidative stress in a subject, in the second formulation. In certain embodiments, the second formulation includes a therapeutically effective amount of an agent for ameliorating cyanide-induced oxidative stress in a subject and a pharmaceutically acceptable carrier and / or additive. The agent for ameliorating cyanide-induced oxidative stress in a subject can include, for example, a ketone or aldehyde compound selected from the group consisting of glyoxylate, glyceraldehyde, acetaldehyde, dihydroxyacetone, pyruvate, methylglyoxal, and alpha-ketoglutarate. The agent for ameliorating cyanide-induced oxidative stress in a subject can include glyoxylate or an analog or functional fragment thereof.

[0202] overview All patents, patent application publications, journal articles, textbooks, and other publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains.

[0203] In the above description, numerous specific details are set forth to provide a thorough understanding of the present disclosure. It should be understood that particular examples may be implemented without some or all of these specific details, and that the present disclosure is not limited to any particular biological system, cancer, or organ or tissue, which may, of course, vary and still be applicable in light of the data provided herein.

[0204] Additionally, various techniques and mechanisms of this disclosure sometimes describe a connection or coupling between two components. Terms such as bonded, connected, coupled, connected, and similar terms with their inflectional forms are used interchangeably unless a distinction is noted or otherwise made clear from the context. These words and expressions do not necessarily convey a direct connection, but include connections via intermediate components. It should be noted that a connection between two components does not necessarily imply a direct and uninterrupted connection, as there may be various other components between the two components of interest. As a result, unless otherwise noted, a connection does not necessarily imply a direct and uninterrupted connection.

[0205] Furthermore, it will be understood that the present disclosure is presented in this manner for illustrative purposes only, and that the principles and embodiments described herein may be applied to compounds and / or compositional components having configurations other than those specifically described herein. Indeed, it is expressly contemplated that the components of the compositions and compounds of the present disclosure may be tailored in furtherance of their desired application.

[0206] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the chemical and biological arts. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this application, the preferred methods and materials are described herein.

[0207] The term "about," when referring to a number or numerical value or range (including, e.g., positive integers, fractions, and percentages), means that the number or numerical range referred to is approximate within experimental variability (or within statistical experimental error), and thus the number or range can vary by 1% to 15% of the stated number or numerical range (e.g., ±5% to 15% of the recited value), but would be considered equivalent (e.g., having the same function or result) to the recited value by one of ordinary skill in the art.

[0208] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formula, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included.

[0209] The present disclosure may suitably be practiced in the absence of any element or limitation not specifically disclosed herein. Thus, for example, each instance herein of any of the terms "comprising," "consisting essentially of," and "consisting" (and related terms such as "comprise" or "comprises" or "having" or "including") can be replaced with the other mentioned term. Similarly, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods and / or steps of the type described and / or that will become apparent to those skilled in the art upon reading this disclosure. The term "substantially" can allow for a degree of variation within a value or range, e.g., within 90%, within 95%, or within 99% of a stated value or limit of a stated range.

[0210] Unless explicitly stated otherwise, the depicted structures include all stereochemical forms of the structure, i.e., right-hand (R) and left-hand (S) configurations of each asymmetric center. Accordingly, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures are within the scope of the disclosure.

[0211] As used herein, the term "composition" generally refers to any product that includes more than one ingredient, such as a compound herein and a carrier.

[0212] "Alkyl" generally refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, such as from 1 to 15 carbon atoms (e.g., C1-C 15 The disclosure of "alkyl" provided herein is intended to include independent listings of saturated "alkyl" unless otherwise specified. Alkyl can contain 1 to 13 carbon atoms (e.g., C1-C 13 alkyl). An alkyl can contain 1 to 8 carbon atoms (e.g., C1-C8 alkyl). An alkyl can contain 1 to 5 carbon atoms (e.g., C1-C5 alkyl). An alkyl can contain 1 to 4 carbon atoms (e.g., C1-C4 alkyl). An alkyl can contain 1 to 3 carbon atoms (e.g., C1-C3 alkyl). An alkyl can contain 1 to 2 carbon atoms (e.g., C1-C2 alkyl). An alkyl can contain 1 carbon atom (e.g., C1 alkyl). An alkyl can contain 5 to 15 carbon atoms (e.g., C5-C 15alkyl). An alkyl can contain 5 to 8 carbon atoms (e.g., C5-C8 alkyl). An alkyl can contain 2 to 5 carbon atoms (e.g., C2-C5 alkyl). An alkyl can contain 3 to 5 carbon atoms (e.g., C3-C5 alkyl). In other embodiments, an alkyl group is selected from methyl, ethyl, 1-propyl (n-propyl), 1-methylethyl (iso-propyl), 1-butyl (n-butyl), 1-methylpropyl (sec-butyl), 2-methylpropyl (iso-butyl), 1,1-dimethylethyl (tert-butyl), and 1-pentyl (n-pentyl). An alkyl is attached to the rest of the molecule by a single bond.

[0213] "Alkoxy" refers to a radical attached through an oxygen atom of the formula --O-alkyl, where alkyl is an alkyl chain as defined above.

[0214] "Alkylene" or "alkylene chain" generally refers to a straight or branched divalent alkyl group having from 1 to 12 carbon atoms, e.g., methylene, ethylene, propylene, i-propylene, n-butylene, etc., connecting the remainder of the molecule to a radical group.

[0215] "Aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbon atoms from 5 to 18 carbon atoms, and at least one of the rings in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2) π-electron system according to the Hückel theory. Ring systems from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin, and naphthalene.

[0216] "Arylalkyl" is R c is an alkylene chain as defined above; c-aryl radicals such as methylene, ethylene, etc. The alkylene chain portion of the aralkyl radical is optionally substituted as described above for an alkylene chain.

[0217] The term "heteroalkyl" refers to an alkyl group, as defined above, in which one or more skeletal carbon atoms of the alkyl have been replaced with a heteroatom (with the appropriate number of substituents or valences, e.g., -CH- can be replaced with -NH- or -O-). Each substituted carbon atom is independently replaced with a heteroatom, e.g., carbon is replaced with nitrogen, oxygen, selenium, or other suitable heteroatom. In some instances, each substituted carbon atom is independently replaced with oxygen, nitrogen (e.g., -NH-, -N(alkyl)-, or -N(aryl)-, or another substituent contemplated herein), or sulfur (e.g., -S-, -S(=O)-, or -S(=O)-). The heteroalkyl is attached to the remainder of the molecule at a carbon atom of the heteroalkyl. The heteroalkyl is attached to the remainder of the molecule at a heteroatom of the heteroalkyl. Heteroalkyl is a C1-C 18 Heteroalkyl is a C1-C 12 Heteroalkyl is a C1-C6 heteroalkyl. Heteroalkyl is a C1-C4 heteroalkyl. Heteroalkyl can include alkoxy, alkoxyalkyl, alkylamino, alkylaminoalkyl, aminoalkyl, heterocycloalkyl, heterocycloalkyl, and heterocycloalkylalkyl, as defined herein.

[0218] The terms "protein," "polypeptide," and "peptide" are used interchangeably to refer to compounds containing amino acids joined through peptide bonds.

[0219] It is recognized that various modifications are possible within the scope of the present disclosure. Thus, while the present disclosure has been specifically disclosed in the context of preferred embodiments and optional features, those skilled in the art may resort to modifications and variations of the concepts disclosed herein. Such modifications and variations are deemed to be within the scope of the present disclosure as claimed herein.

[0220] Therefore, it is intended that the present specification and the appended claims encompass all modifications and variations that are apparent to those skilled in the art based on this disclosure. For example, when a treatment method or therapy involves administering more than one treatment, compound, or composition to a subject, it will be understood that the order, timing, number, concentration, and volume of administration are limited only by the medical requirements and limitations of the treatment (i.e., two treatments can be administered to a subject, for example, simultaneously, sequentially, sequentially, alternatingly, or according to any other regimen).

[0221] Additionally, in describing exemplary embodiments, the present disclosure may have presented a method and / or process as a particular sequence of steps. Insofar as the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As will be appreciated by those skilled in the art, other sequences of steps may be possible. Thus, the particular order of steps disclosed herein should not be construed as a limitation on the claims. Additionally, claims directed to a method and / or process should not be limited to performing those steps in the written order; those skilled in the art will readily appreciate that the sequence may vary and still remain within the spirit and scope of the present disclosure.

[0222] Furthermore, the use of headings and subheadings is for ease of reference given the length of the document, and the statements under a heading or subheading (such as the subheadings in the Detailed Description) are not intended to limit the invention to only the subject matter specified under that particular heading or subheading. [Example]

[0223] The following examples serve to illustrate the present disclosure and are not intended to limit the scope of the claimed invention in any way.

[0224] material and method The general methodology and materials used in the examples described herein are provided in this section. Any study-specific modifications to these general materials and methods will be specified in the specific examples.

[0225] Materials and Equipment. Na2[PtCl4].nH2O (n ∼3, Premion™, Pt4 2.4% min) was purchased from Alfa Aesar (Tewksbury, MA). All other chemicals, solvents, TLC plates (silica gel), and silica gel were purchased from MilliporeSigma (Sigma-Aldrich, Burlington, MA) or Ambeed (Arlington Heights, IL) and used as received. A Smart Evaporator, an instrument capable of removing DMSO at atmospheric pressure below 50 °C, was obtained from Biochromato Co., Ltd. (Kanagawa, Japan).

[0226] Platinum X-ray Fluorescence (XRF). An Epsilon 4 spectrometer (Malvern Panalytical, Malvern, UK) equipped with a silver anode X-ray tube was used for these measurements. X-ray fluorescence spectra were collected from 1 mL aqueous samples containing the platinum compound from Example 1 above as the analyte and manganese chloride as the internal standard, pipetted onto Mylar foil (ChemPlex, Palm City, FL), and embedded in 32 mm polyethylene sample cups (ChemPlex, Palm City, FL). Each sample was irradiated for 20 minutes. Epsilon 4 software was used to calculate the parts per million of each element from each spectrum. The parts per million (ppm) value for platinum was divided by the ppm value for manganese. This ratio was converted to milligrams of platinum per milliliter by comparison with a standard curve generated using sodium hexachloroplatinate hexahydrate.

[0227] The standard K2PtCl4 run was measured at 53%, within 6% of the target amount, consistent with the variability observed for other compounds. For each test compound, the weight platinum concentration per volume was divided by the weight concentration per volume of the compound to arrive at gram % platinum.

[0228] High-Performance Liquid Chromatography (HPLC). HPLC calibration of the analytes was performed using an Agilent 1100 in reversed-phase mode using an acetonitrile:water gradient in aqueous vehicle (Figures 6E and 6F).

[0229] Platinum reaction mixtures were separated using an Agilent 1100 equipped with a Raptor Polar X 50 mm x 2.1 mm column. Approximately 1 mM stock solutions of each platinum complex were prepared in 0.1 M pH 7.5 phosphate buffer. Cyanide was added to aliquots of platinum samples at 1, 2, 4, 7, and 10 millimolar concentrations. Three aliquots of platinum stock were used for each cyanide concentration. For analysis of starting materials, purified water was used as mobile phase A and acetonitrile as mobile phase B, with a gradient of 90% mobile phase B to 50% mobile phase B over 3.5 minutes at 0.3 mL / min. An injection volume of 5 μL and absorbance at 260 nm were used. For the analysis of cyanide reaction products, purified water was used as mobile phase A, acetonitrile as mobile phase B, and 10% 200 mM ammonium formate with 0.5% v / v formic acid as mobile phase C at 0.3 mL / min with a 0–50% mobile phase B gradient over 5 min. An injection volume of 5 μL and absorbance at 260 nm were used for all analyses.

[0230] Normalized Pt(CN)4 2- is calculated as the difference between the moles of cyanide added (X) and the measured Pt(CN)4 for the titration sample set. 2- Pt(CN)4 2- divided by the range (see equation below).

[0231]

number

[0232] Ion-selective electrode (ISE) for cyanide detection. An Orion ion-selective electrode for cyanide was purchased from Thermo Fisher Scientific (Waltham, MA). The electrode was calibrated each day with freshly prepared cyanide standards ranging from 0.26 ppm to 26.0 ppm according to the product manual. Every 2 hours, the electrode drift was verified to be less than 2.0%, as specified by the pass / fail criteria in the manual.

[0233] Titration of cyanide and platinum solutions was carried out as follows: A volume of platinum mixture containing 0.1 mM platinum was adjusted to a pH above 10 using NaOH to maintain the cyanide in solution. The titration was then carried out by adding a small volume of the original solution equal to 1% v / v.

[0234] Ultraviolet-visible spectrophotometry (UV-Vis). A Cary 60 (Agilent Technologies, Inc., Santa Clara, CA) was used for all UV-Vis experiments to monitor the reaction between platinum and cyanide. All cyanide reactions were at pH 7.6 using 12.5 mM NaPi spiked with 0.8 mM KCN and approximately 0.02 mM platinum. Data collection was at 24,000 nm / s during a 0.65-second 300-220 nm averaging time interval over the first 30 seconds and once every 5 minutes. A one-phase exponential curve was used to fit the kinetic data to derive the apparent rate constant.

[0235] UV-Vis reaction rate and platinum content: The rate of change for the platinum spectrum was modeled assuming a first-order rate law. In equation (2), A is the absorbance. The rate of change (k) is calculated as ln(A ∞ -A t ) can be obtained as the slope when plotted against time (t). Tseng et al., Characteristics and applications of the Lagergren's first-order equation for adsorption kinetics, J Taiwan Institute Chem Engineers 41: 661-669 (2010). Each assay was monitored for at least 10 minutes or until the sample appeared to reach equilibrium. By rearranging equation (2), the half-life could be estimated using equation (3). After tracking the reaction for a minimum of one half-life, the observed rate constant and each observed half-life were reported.

[0236]

number

[0237] Characterization of Pt compounds by NMR. Dry powder samples were dissolved to monitor the antishielding effect on the platinum core. 195 Analyzed by Pt NMR and observed as a shift below -2800 ppm using a hexachloroplatinate reference material.

[0238] 195 Pt spectra were acquired using a Bruker DRX 500 MHz spectrometer equipped with a BBFO probe operating at room temperature. 1 H and 13 C spectra were acquired at 25 °C using the same spectrometer equipped with a cryo-TCI probe or a Bruker Avance 800 MHz spectrometer. Water-soluble Pt(II) compounds ranging from 20 to 100 mM in aqueous buffer containing 10% DO were used for the analysis. In cases of low aqueous solubility, the complex (MetPt(II)Cl) was dissolved at 50 mM in d7-DMF. 195 The Pt spectrum has a spectral width of 933 ppm, centered at -3600 ppm (or -2800 ppm for MetPt(II)Cl in d7-DMF). The excitation angle was approximately 50-60 degrees with a 0.16 s acquisition time and a 0.7 s recycle delay, and the total number of scans was 2048. An exponential window function with 50 Hz line broadening was applied to the FID before Fourier transformation, manual phasing, and automatic baseline correction.

[0239] At low pH (pH<7.0), rapid conversion between bicyclic and semicyclic isomers in the bidentate ligand was observed. 1 H spectrum assignments were not made for these compounds due to complexity. For example, compound 0 195Pt NMR showed a mixture of isomers (Figure 3), with four signals observed between -3550 and -3700 ppm depending on the pH. Norman et al. (1992), supra, proposed that the dominant peaks under acidic conditions (e.g., -3625 ppm and -3675 ppm) represent a bidentate ring-closed structure. 195 The Pt spectrum was simplified to pH values between 7 and 8 and was consistent with Norman's observations, suggesting a ring-closed dominant form.

[0240] Zebrafish Efficacy. Six-day-old zebrafish embryos were grown in baths containing a lethal dose of cyanide and varying amounts of platinum and ligand ratios (as described in further detail below). The reported efficacy is the minimum concentration of platinum required to achieve 100% survival of zebrafish embryos.

[0241] Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Digestion. In a 15 mL polypropylene centrifuge tube, 90 μL of rabbit blood sample was mixed with 800 μL of 30% v / v hydrogen peroxide (Macron Fine Chemicals; Avantor, Radnor Township, PA). Then, 800 μL of Allister ultrapure hydrochloric acid and 400 μL of Allister ultrapure nitric acid were added. Addition of the reagent to the blood sample resulted in vigorous bubbling. The solution was loosely capped and incubated at room temperature until bubbling ceased. The solution was then heated at 60°C in a water bath for 16 hours. Each sample was diluted to 10 mL with Milli-Q water and gently centrifuged at 1000 × G for 5 minutes to sediment particulates, then transferred to a new 15 mL polypropylene tube. These samples were then used for ICP-MS analysis.

[0242] Platinum quantification by ICP-MS for pharmacokinetics. A Thermo Fisher Scientific Elements II (Waltham, MA) inductively coupled plasma mass spectrometer was used for all ICP-MS analyses. Mass offsets were calculated using a multi-element standard (VWR) containing Au, Ir, Os, Pd, Pt, Re, Rh, and Ru in 10% hydrochloric acid. Samples were introduced into the mass spectrometer using an Arridus peristaltic pump. Samples were analyzed with a 90-second draw time and a 90-second sample collection time. Platinum-195 peak intensities were averaged over the sample collection time. These averages were converted to nanograms or micrograms per milliliter by comparison to a standard curve generated with sodium hexachloroplatinate in rabbit serum.

[0243] Reaction rate. Platinum was reacted with cyanide under first order conditions at Pt(CN). 42- was the product for the compounds described herein. The signal at 255 nm increased over time, indicating Pt(CN) 42- The results were transferred as Pt(CN) formation over time. 42- The observed rate constant was obtained by plotting the formation of β-glucan and fitting the curve to a first-order process. The reaction was carried out in 12.5 mM phosphate buffer, pH 7.6, supplemented with 0.8 mM KCN and 0.02 mM platinum.

[0244] Mass spectrometry (high resolution). Platinum reagents were dissolved in a 50:50 acetonitrile and water solvent. Samples were infused into an LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) using electrospray ionization in either positive or negative mode.

[0245] Mass spectrometry (low resolution). Platinum compound 8 was dissolved in a 50:50 acetonitrile and water solvent. Samples were infused into an Advion expression spectrometer (Advion, Inc., Ithaca, NY) using electrospray ionization in either positive or negative mode.

[0246] [Example 1] Formation of platinum compounds Previously, cisplatin and sodium hexachloroplatinate (HCP, Na2[PtCl6]) were found to be active as cyanide antidotes only when formulated with dimethyl sulfoxide (DMSO). Based on their known reactivity, DMSO was contemplated to act as a ligand at platinum, facilitating rapid displacement of cyanide at the metal. DMSO complexes of cisplatin reduced its toxicity and efficacy as a deoxyribonucleic acid (DNA) damaging agent. A method was used to prepare the sulfate salt of cisplatin-DMSO (compound 3) from compound 1, utilizing silver nitrate to capture the released chloride and promoting complete conversion (Figure 1).

[0247] Compound 3: Briefly, to produce compound 3, a mixture of cisplatin (3120 mg, 10.4 mmol), silver sulfate (1621 mg, 5.20 mmol), deionized water (104 mL), and DMSO (3.74 mL, 52.7 mmol) was placed in a 250 mL round-bottom flask and stirred rapidly at ambient temperature in the dark for 5 days. The heterogeneous mixture was centrifuged in a 50 mL Falcon tube to remove silver chloride and a silver-black precipitate. The supernatant was collected and concentrated to 10 mL in vacuo at 35 °C, then transferred to a 15 mL capped plastic tube and centrifuged at 7500 rpm for 2 minutes. The supernatant was centrifuged again at 14,000 rpm for 3 minutes to completely remove the silver precipitate. To recover any compound 3 from the precipitate, the solid was dissolved in water and clarified by centrifugation. The combined solution was placed in a 50 mL glass bottle and dried overnight in a smart evaporator at 35 °C. The resulting solid residue was triturated and washed with ethanol (3 mL x 4), followed by diethyl ether (3 mL x 3), and dried in vacuo to give compound 3 (4.15 g) as a light gray powder.

[0248] TLC: Rf 0.5 on silica gel, 1,4-dioxane / water = 4 / 1, UV 254 and ninhydrin. ESIMS (positive ion, solvent: water and acetonitrile), [C2H 12 ClN2OPtS] + Calculated for m / z: 342.000673; Found: 342.000084. Mass spectral data for compound 3 is shown in Figure 22 (LTQ Orbitrap used in electrospray ionization positive ion mode; solvent used was a 1:1 mixture of acetonitrile and water; m / z of 342.00084 obtained with an error of -0.48 ppm).

[0249] Two additional DMSO compounds, 4 and 5, were also prepared from compound 2 and sodium tetrachloroplatinate, respectively, for further chemical and pharmacological evaluation.

[0250] Compound 4: The preparation of Pt(IV) compound 4 was similar to the original formulation used to prepare the cyanide antidote. Compound 4 could be isolated by simple column chromatography after exposure to excess DMSO.

[0251] Briefly, to produce compound 4, HCP(Na 2 [PtCl 6 ].6H 2 A mixture of 250 mg (0.445 mmol), DMSO (252 μL, 3.55 mmol), and deionized water (252 μL) was heated at 45-50°C for 1 h in a 20 mL glass vial. The mixture was diluted with deionized water (4.5 mL) and evaporated at 30°C for 2.0 h using a smart evaporator. The process was repeated. The resulting yellow-orange residue was purified by column chromatography (silica gel, 230-400 mesh, 5 g; 2.0 cm x 3.5 cm) using a gradient of dichloromethane / acetone = 4 / 1 to 1 / 1. The recovered yellow-orange viscous liquid was first dried by blowing a stream of argon gas through it to remove residual solvent. The semisolid was pulverized and then finally dried under reduced pressure to give compound 4 (170 mg) as a yellow powder.

[0252] TLC: Rf approx. 0.4 on silica gel, dichloromethane / acetone = 1 / 1, UV 254 and ninhydrin. High-resolution ESI-MS (negative ion, mobile phase: acetone), [PtCl5(DMSO)] - Calculated for: 446.8209; Found: 446.8196. Mass spectral data for compound 4 is shown in Figure 23 (LTQ Orbitrap used in electrospray ionization negative ion mode; solvent used was a 1:1 mixture of acetonitrile and water; m / z of 447.82334 obtained with an error of -0.79 ppm).

[0253] Compound 5: Compound 5 was prepared from the reaction of DMSO with tetrachloroplatinate using a protocol similar to that used to generate compound 4. To generate compound 5, Na2[PtCl4].nHO (n ≈ 3) (200 mg, 0.416 mmol) was dissolved in deionized water (910 μL) and treated with DMSO (33 μL, 0.46 mmol). The resulting brownish homogeneous solution was kept overnight in the dark at ambient temperature. Over this period, the mixture became a yellowish solution containing needle-like solids. The solvent was removed using a smart evaporator at 40 °C until dry. The yellow solid residue was extracted with 3 / 1 acetone / methanol (3 x 1 mL) until the insoluble solid was nearly white. The entire procedure was repeated six times, and the combined yellow extracts were dried in vacuo. The recovered solid was then treated with 2 x 2 mL of ethanol and dried in vacuo to remove residual water. Compound 5 (1.08 g) was recovered as a yellow powder.

[0254] TLC: Rf 0.6 on silica gel, dichloromethane / acetone = 1 / 3, UV 254 and ninhydrin. ESI-MS (negative ion, mobile phase: acetonitrile and water) [C2H6Cl3OPtS] - Calculated for m / z: 377.885281; Found: 377.88677. Mass spectral data for compound 5 is shown in Figure 24 (LTQ Orbitrap used in electrospray ionization negative ion mode; solvent used was a 1:1 mixture of acetonitrile and water; m / z of 377.88677 obtained with an error of 3.94 ppm).

[0255] [Example 2] Ligand selection for sulfur-containing Pt(II) compounds A preliminary study was designed to guide the criteria for ligand selection for sulfur-containing Pt(II) compounds. Naturally occurring sulfhydryl, sulfide, and sulfoxide ligands / metabolites were tested as replacements for DMSO in combination with compound 2 using the zebrafish cyanide toxicity model as specified in Table 1.

[0256] Briefly, zebrafish larvae were plated in 96-well plates in hydroxyeicosapentaenoic acid (HEPE)-buffered Tuebingen E3 medium (n = 5 per well) at day 6 post-fertilization. Larvae were dosed with potassium cyanide (50 μM) and each compound (i.e., platinum complex) to be tested (1-250 μM) in each well. The cyanide concentration used represented the lethal dose (LD100) for all embryos in each well, and survival was recorded after 4 h of exposure following treatment.

[0257] The efficacy for each of these samples was determined from triplicates and quadruplicates, and no rescue was observed (NR) using a 1:10 Pt:ligand ratio of L-cysteine, L-glutathione, L-methionine sulfoxide, and L-alliin.

[0258] [Table 2]

[0259] Mixtures of compound 2 with either L-methionine or S-methyl-L-cysteine in water at 1:1 or 1:10 (Pt to ligand) ratios provided cyanide rescue in zebrafish, with a 4-fold improvement in efficacy compared to that observed in the original DMSO formulation.

[0260] In the case of compounds with free sulfhydryls (L-cysteine and reduced L-glutathione), a 1:1 mixture provided enhanced rescue potency, but this activity was lost when the ligands were in molar excess.

[0261] Finally, compound 2 in mixture with sulfoxide amino acids (L-methionine sulfoxide or L-alliin) did not provide any cyanide-rescue activity. Although promising, the possibility that compound 2 oxidizes naturally occurring sulfur-containing amino acids while complexed is a known process. As a result, sulfide ligands for Pt(II) complexes provided an alternative focus for the design of additional candidate cyanide antidotes.

[0262] [Example 3] Formation of platinum compounds by modification of carboxylate and amino groups. In previous efforts, platinum(II) (Pt(II)) complexes (containing two dimethylsulfide ligands) provided rescue of cyanide toxicity in the absence of DMSO in the zebrafish model described herein. Phosphate-buffered saline (PBS) and DMSO solvates were equally effective, demonstrating twice the potency compared to cisplatin prepared in DMSO. (2017), supra. Based on the established chemical hypothesis that sulfur ligands can direct the reactivity of nucleophilic addition to Pt(II), a series of Pt complexes containing modifications of carboxylic acid and amino groups were prepared to utilize them as improved cyanide scavenging agents. These cyanide scavengers include N-acetyl-L-methionine (compound 8), L-methionine amide (compound 9), 3-(methylthio)propylamine (compound 10), and 2-(methylthio)ethylamine (compound 11). These were prepared to compare the effect of structural variations on their reactivity with cyanide.

[0263] Sulfide ligands to platinum were expected to produce strong interactions. Generally, each of the sulfide complexes, compounds 6–11, was prepared in water and then isolated as a dry powder. No attempt was made to remove the salt product. Several isomeric species were predicted based on the ligand structure. With the exception of compound 8, all ligands possess a nitrogen center capable of bidentate coordination with Pt in a square planar configuration. Detailed assignments to date have guided the assignment of the major isomeric forms of the Pt complexes. Using NMR characterization of aqueous solutions, the major structural assignments were made as depicted in Scheme 1, shown in Figure 2.

[0264] Compound 6: To produce Compound 6, a mixture of Na2[PtCl4].nHO (2224 mg, 5.09 mmol), L-methionine (1.45 g, 9.72 mmol), and deionized water (10.2 mL) was sonicated for 5 minutes until all solids were dissolved. The homogeneous mixture was kept in the dark at ambient temperature for 2 hours and then centrifuged to remove the in situ generated platinum black particles. The pale yellow supernatant was shell-frozen and lyophilized overnight. The incompletely lyophilized mixture was refrozen and lyophilized a second time overnight to isolate Compound 6 (3543 mg) as a pale yellow powder.

[0265] TLC: Rf 0.3 silica gel, 1% NaCl in water, UV254, and ninhydrin. ESI-MS (positive ion, mobile phase: water and acetonitrile) [C 10 H 22 N2O4PtS2] 2+ Calculated value for m / z: 246.5334435; Found value: 246.53341. Mass spectral data for compound 6 is shown in Figure 25 (LTQ Orbitrap used in electrospray ionization positive ion mode; solvent used was a 1:1 mixture of acetonitrile and water; m / z of 246.53341 obtained with an error of -0.13 ppm).

[0266] Compound 7: To produce compound 7, a mixture of Na2[PtCl4].nHO (257 mg, 0.588 mmol), L-(S-methyl)cysteine (160 mg, 1.18 mmol), and deionized water (2.9 mL) was sonicated for 5 minutes or until all solids were dissolved. The homogeneous mixture was kept in the dark at ambient temperature overnight and evaporated at 35 °C using a smart evaporator. The pale green sticky residue was extensively triturated in ethanol (5 mL) using a spatula and sonication until the mixture became a uniform suspension. The solid in this mixture was isolated by high-speed centrifugation, washed with diethyl ether (2 mL, × 3), and dried to give compound 7 (295 mg) as a white powder.

[0267] TLC: 2 spots, Rf 0.3 tailing and 0.6 tailing on silica gel, 1% NaCl in water, UV254 and ninhydrin. ESI-MS (positive ion, mobile phase: acetonitrile and water), [C8H 18 N2O4PtS2] 2+ Calculated for m / z: 232.5177935; Found: 232.51769. Mass spectral data for compound 7 is shown in Figure 26 (LTQ Orbitrap used in electrospray ionization positive ion mode; solvent used was a 1:1 mixture of acetonitrile and water; m / z of 232.51769 obtained with an error of -0.44 ppm).

[0268] Compound 8: To produce compound 8, a mixture of Na2[PtCl4].nHO (654 mg, 1.50 mmol), L-(N-acetyl)methionine (580 mg, 3.03 mmol), and deionized water (4.50 mL) was sonicated for 5 minutes until all solids were dissolved. The homogeneous mixture was kept in the dark at ambient temperature overnight and evaporated at 40 °C to give a pale yellow paste-like residue. The residue was dissolved in ethanol (2 mL) and dried in vacuo (repeated twice) to give an amorphous foam-like residue, which was crushed with a spatula and dried in vacuo to isolate compound 8 as a pale yellowish-green powder (985 mg).

[0269] TLC: 2 spots, Rf approximately 0.5 and 0.7, 1,4-dioxane / water = 9 / 1, UV 254 and ninhydrin. ESI-MS (positive ion, mobile phase: acetonitrile and water), [C 14 H 26Calculated for [Ac-MetPtClN0PtS2]: 648.47; Found: 646. Mass spectral data for compound 8 are shown in Figures 27A and 27B (LTQ Orbitrap used in electrospray ionization positive ion mode (Figure 27A; solvent used was a 1:1 mixture of acetonitrile and water; 626 m / z was identified as [Ac-MetPt(HO)(HO)Na]+); Advion Expression Spectrometer used in electrospray ionization positive (top) and negative ion (bottom) modes (Figure 27B; solvent used was a 1:1 mixture of acetonitrile and water; 646 m / z was identified as [Ac-MetPtClNa]+). - ) was identified as

[0270] Compound 9: As a preliminary test to produce compound 9, a mixture of Na2[PtCl4].nH2O (119 mg, 0.272 mmol), L-methionine amide hydrochloride (81 mg, 0.44 mmol), and deionized water (1.36 mL) was briefly sonicated to create a homogeneous mixture. After 0.5 minutes, the reaction was checked by TLC, which indicated product formation. A second reaction mixture of Na2[PtCl4].nH2O (150 mg), L-methionine amide hydrochloride (102 mg), and deionized water (1.7 mL) was prepared in the same manner. Both mixtures were combined and dried overnight at 35 °C using a smart evaporator. The residue was triturated in ethanol (3 mL) using a spatula and sonicated, washed with diethyl ether (2 mL, ×2), and dried to give compound 9 (428 mg) as a beige-to-white powder.

[0271] TLC: Rf 0.15 tailing on silica gel, 1% NaCl in water, UV 254 and ninhydrin. ESI-MS (positive ion, mobile phase: acetonitrile and water), [C 10 H 24 N4O2PtS2] 2+Calculated for m / z: 490.09103; Found: 490.09107. Mass spectral data for compound 9 is shown in Figure 28 (LTQ Orbitrap used in electrospray ionization positive ion mode; solvent used was a 1:1 mixture of acetonitrile and water; m / z of 490.09107 obtained with an error of -0.08 ppm).

[0272] Compound 10: To produce compound 10, Na2[PtCl4].nHO (485 mg, 1.11 mmol) was dissolved in deionized water (5.55 mL) and 3-(methylthio)propylamine (255 μL, 2.28 mol) was added. The heterogeneous mixture was stirred at ambient temperature for 5 h. The resulting homogeneous mixture was evaporated to dryness overnight at 35 °C using a smart evaporator. The residue was triturated using a spatula, washed with IPA (3 mL x 3) using a sonicator, followed by diethyl ether (3 mL x 2), and then dried to give compound 10 (572 mg) as a beige to white powder.

[0273] TLC: Rf 0.1 tailing on silica gel, 1% NaCl in water, UV 254 and ninhydrin. ESI-MS (positive ion, mobile phase: acetonitrile and water), [C8H 22 N2PtS2] 2+ Calculated for: 404.079402; Found: 404.07977. Mass spectral data for compound 10 is shown in Figure 29 (LTQ Orbitrap used in electrospray ionization positive ion mode; solvent used was a 1:1 mixture of acetonitrile and water; m / z of 404.07977 obtained with an error of 0.91 ppm).

[0274] Compound 11: To produce compound 11, Na2[PtCl4].nHO (n ∼ 3) (134 mg, 0.307 mmol) was dissolved in deionized water (1.5 mL), followed by the addition of 2-(methylthio)ethylamine (57 μL, 0.61 mmol). The resulting heterogeneous mixture was stirred overnight at ambient temperature, followed by the addition of an equal volume of 1,4-dioxane as a cosolvent and stirring at 35–40 °C. A small precipitate containing platinum black particles formed and was removed by centrifugation. The supernatant was evaporated to dryness at 40 °C using a smart evaporator. The solid residue was washed with ethanol (1.5 mL, twice), followed by diethyl ether (1.5 mL, twice), and dried in vacuo to give compound 11 (90 mg) as a light gray solid.

[0275] TLC: 2 spots, Rf approximately 0.1 tailing and 0.2 tailing, 1% NaCl in water, UV254 and ninhydrin. ESI-MS (positive ion, mobile phase: acetonitrile and water) [C6H 18 N2PtS2] 2+ Calculated value for m / z: 188.5279635; Found value: 188.52774. Mass spectral data for compound 11 is shown in Figure 30 (LTQ Orbitrap used in electrospray ionization positive ion mode; solvent used was a 1:1 mixture of acetonitrile and water; m / z of 188.52774 obtained with an error of -0.08 ppm).

[0276] Table 2 provides a summary of the data related to the platinum compounds herein, along with their respective m / z determined by LTQ orbitrap in either negative or positive ion mode (solvent used was 1:1 acetonitrile and water).

[0277] [Table 3]

[0278] In a freshly prepared sample of compound 6 at pH 5.7, 1H NMR indicated that the complex was almost exclusively cis, with no detectable trans (Figure 31; the absence of a clear signal between 3.33 ppm and 3.5 ppm suggests that compound 6 is dominated by cis isomers without appreciable amounts of trans). A 9:1 mixture of cis to trans forms was observed after 6 days of storage at room temperature. 1 Identified by H NMR. 195 The Pt NMR resonances are reported to be centered in the high-field region of the trans phase, but were difficult to clearly define at 11.7 T. 195 The pH dependence of Pt NMR indicated that the open form dominated at low pH. In contrast, the closed form was dominant above pH 5.7. 195 Pt NMR spectra indicated a significant degree of heterogeneity, with at least four unique species. Compound 8 possessed sulfide ligation but was expected to be in the open form due to acetylation of the amine moiety. Furthermore, mass spectral analysis of the material did not point to a single component. This distinction served as a basis for comparing the role of the bidentate ligand.

[0279] [Example 4] Compound water solubility study The aqueous solubility of the compounds was assessed as a second criterion for candidate selection using methods commonly known in the art.

[0280] Neutral complexes have substantially reduced solubility in aqueous conditions, thus limiting their usefulness. For example, a 1:1 molar mixture of L-methionine and Na2[PtCl4] in water produced an insoluble precipitate, which was identified as the monoadduct (H-Met-OH)PtCl2, a known neutral Pt(II) complex. Compounds 3–5 had solubilities of greater than 50 mg / mL in water, making them all suitable for pharmacological evaluation. Compound 6 had a solubility of approximately 700 mg / mL (0.8 M) in water at 25°C.

[0281] In contrast, the mixture with 2 molar equivalents of L-methionine in water was homogeneous, indicating that the monoadduct reacted rapidly with methionine to form the bisadduct compound 6, as shown in Scheme 1 in Figure 2.

[0282] The solubility of compound 4 was also assessed in phosphate buffer. Briefly, 100 mM compound 4 in 20 mM pH 6.0 phosphate buffer was monitored for up to 18 days. During storage, samples were shielded from light to prevent accelerated decomposition due to exposure to light. Equilibrium was established after 4 days, when approximately 70% of the DMSO bound to the platinum had been released.

[0283] The DMSO-Pt interaction in compound 4 exhibited poor stability in aqueous solution as monitored by NMR (Figure 21). In contrast, Pt compounds herein containing bidentate thioether ligands such as methionine (e.g., compounds 6, 7, and 9–11) were consistently stable in solution and maintained their reactivity toward cyanide. Those compounds with the ability to form ring-closed species (e.g., compounds 6, 7, and 9–11) were also the most active cyanide scavengers. At higher pH, these compounds lack the positive charge in the primary amine that allows electron donation to the platinum d orbital, which may contribute to their resistance to hydrolysis. At lower pH values, the positively charged amine likely induces electron repulsion toward the platinum core, favoring hydroxyl or chloride ligation.

[0284] [Example 5] Platinum content and reaction with cyanide Quantification of the platinum content (% w / w) in each of the sulfur-containing compounds was obtained by X-ray fluorescence spectroscopy (XRF). An example XRF spectrum is shown in Figure 3, where the Pt(IV) and Pt(II) core excitations differ from the internal manganese standard. XRF results revealed that the bulk material for compounds 6-11 was 20-30% w / w Pt, as summarized in Figure 4. These values were used as the basis for the available platinum mass in each animal dose, regardless of any expected speciation.

[0285] The ability of each of the sulfur-containing Pt complexes (compounds 1–11) to act as cyanide scavengers was evaluated using in vitro reactions with potassium cyanide (KCN). Direct consumption of cyanide in solution was detected using three different methods.

[0286] An ion-selective (cyanide) electrode (ISE) assay was used to evaluate the consumption of free cyanide in the presence of Pt(II) complexes. After addition of 50 μM platinum-containing material in 0.5 mM cyanide at pH 10 or greater with 0.1 M ionic strength, the time before reading was limited to 10 min to better mimic conditions for rapid scavenging. A summary of the amount of cyanide reduced per mole of platinum present is shown in Figure 5. Time-dependent ultraviolet-visible spectroscopy (UV-Vis) observations of the same Pt sample at a 1:10 Pt:cyanide ratio provided an estimate of the relative rate of disappearance of the starting material. The product of the reaction of these Pt(II) compounds with excess cyanide was Pt(CN)4. 2- It was expected that the highly stable Pt(CN)4 2- The formation of was based on the strong magnetic field ligand character of the cyanide-derived ligands, which exhibited strong d-orbital splitting of the platinum core. For each of compounds 1–11, the sulfur, amino, and chloride ligands were expected to be replaced by free cyanide.

[0287] As shown in Figure 5, compounds 6, 7, 10, and 11 all appear to approach the expected consumption of 4 molar equivalents of cyanide. Product formation was also observed at 255 nm in UV-Vis assays for compounds 3-11, which indicates that Pt(CN)4 2- This is consistent with the formation of

[0288] The ISE assay conditions were maintained at high ionic strength and highly alkaline conditions to keep cyanide in solution. At longer incubation periods (>10 min), compound 4 showed a greater rate of cyanide consumption (Table 3), indicating incomplete scavenging in the ISE assay after 10 min.

[0289] [Table 4]

[0290] Compounds 8 and 9 were less reactive under the same alkaline conditions. The effect of sulfur on the reaction with cyanide is highlighted by the reduced scavenging for compounds 1 and 2 (Figure 5). ISE assays over 10 min also revealed that the sulfide complexes (compounds 6, 7, 10, and 11) were superior to the DMSO complexes (compounds 3–5) in scavenging cyanide. Similarly, the time-dependent changes in UV-Vis confirm that the most active compounds in the ISE assay also had faster apparent reaction rates. Compounds 6, 7, 9, 10, and 11 had pseudo-first-order reaction rates 10–100 times faster than the DMSO complexes observed under similar conditions, some of which were above the detection limit.

[0291] Using the protocol described in Behymer et al. (2022), supra, compounds 3 and 5–11 were prepared by reaction of cyanide with Pt(CN)4. 2- An HPLC method was developed to monitor the direct production of PtCl6 (Table 4). Four biological replicates were performed. -2 :DMSO has an EC of 62 μM 100 The following symptoms were observed.

[0292] [Table 5]

[0293] The decarboxylated form of methionine (3-(methylthio)propylamine) was effective in enhancing the efficacy of hexachloroplatinate. In addition, less of it appeared to be required to achieve the same antidote efficacy compared to the carboxylated form L-methionine.

[0294] Example data for the reaction of compound 6 with cyanide is shown in Figure 6A, where the blue trace is the starting material and the red trace is after the addition of 4 equivalents of KCN. 2- The HPLC data showed that Pt(CN)4 had a discrete retention time, the spectrum of compound 6 (Figure 6B) had no appreciable absorbance at 260 nm, and the product of the cyanide reaction exhibited a charge-transfer band (Figure 6C). 2- As a direct correlation with production, we demonstrate the depletion of compound 6 (Figure 6D). Repeat these titrations to reach the compound and product (Pt(CN)4 2- ) was quantified. The results are summarized as the maximum Pt(CN)4 observed over the titration range. 2- The results are normalized to and summarized in Figure 5.

[0295] The Pt(IV) compounds were problematic due to poor compatibility with the column chemistry. Using HPLC conditions, compounds 6-9 exhibited the most significant ability for cyanide scavenging. Close inspection of the HPLC chromatograms for compounds 10 and 11 revealed evidence of low levels of the intermediate, Pt(CN)4. 2- Possible explanations for the reduced conversion to

[0296] In a third approach to analyze the cyanide reactions with Pt compounds 3–11, proton nuclear magnetic resonance ( 1 H NMR was incorporated. The results were consistent with published observations, with the sulfide compound exhibiting a pH-dependent spectrum resulting in ring-opening and ring-closure in a bidentate form. For compound 6 at pH 7.2, 195 The Pt NMR spectrum showed two broad peaks (Figure 7A). Addition of 1 molar equivalent of KCN gave Pt(CN)4 in a ratio consistent with an unmixed ligand intermediate.2- Finally, the addition of 4 equivalents of KCN resulted in the conversion of compound 6 to Pt(CN)4 2- This resulted in quantitative conversion to (Figure 7C).

[0297] Furthermore, the competitive reaction of 1 mM compound 3 and 1 mM compound 6 with 0.4 KCN in 180 mM NaPi, pH 7.6, 10% D2O buffer at 298°K was 1 The apparent methionine release rate was estimated to be approximately 10 times that of DMSO, as monitored by 1 H NMR (Figure 7D).

[0298] Interestingly, these data support the synthesis of Pt(CN)4 from amino-sulfide Pt(II) complexes without the measurable presence of any mixed ligand intermediates. 2- On the contrary, Pt(CN)4 2- was the only Pt(II) product under equimolar amounts of cyanide and Pt(II) compounds, suggesting that the addition of the initial cyanide ligand may be rate-limiting. This feature may be an essential attribute for enhancing the efficacy of Pt(II) cyanide scavenging agents.

[0299] Each complex was also titrated with cyanide; 1 The reaction was monitored by H NMR (with a representative example for compound 11 in Figure 8). A shows 1 mM compound 11 alone in 50 mM NaPi, pH 7.5, 10% DO; B shows the addition of 1 mM KCN; and C shows the addition of 5 mM (final concentration) KCN. The ligand (2-methylthioethylamine (MTEA)) was easily identified by the characteristic sharp peaks at 2.69 ppm and 2.62 ppm for the bound form and at 2.1 ppm for the free form. In the absence of KCN, MTEA was found to be completely bound to Pt. Addition of KCN leads to quantitative release of MTEA, as the signal at 2.1 ppm gradually increases in Figure 8B and C. Complete disappearance of the bound ligand is observed in Figure 8C.

[0300] The release of the amino sulfide or DMSO ligand can be readily observed, as they have unique sets of chemical shifts and lineshapes. All reactions in compounds 6–10 showed results consistent with direct displacement of the ligand by cyanide (Figures 9–15). Similarly, further investigation of the reduction of cyanide scavenging in compound 9 under alkaline ISE conditions showed low conversion to product by proton NMR (Figure 16).

[0301] The exchange of proteins with Pt(II) metallodrugs remains an area of active investigation. Several studies have investigated the interaction of amino acid side chains with platinum drugs. Components of the biological matrix that ligate to platinum, e.g., thiol-containing metabolites such as glutathione, can reduce the ability of platinum to scavenge cyanide.

[0302] Reactions to assess the effect of biological matrices on the cyanide reactivity of compound 6 were carried out in the presence of rabbit serum (Figure 17). As a feasibility study, 100 μM of compound 6 was incubated with 400 μM of [ 13 C]KCN exposure 12 C NMR spectra were acquired (Figure 17). 13 Semi-quantification of C was performed using Pt(CN)4 with a characteristic chemical shift at 125 ppm. 2- was the major reaction product within 1 h of mixing (if not shorter, depending on the required NMR data collection time), suggesting that the serum matrix does not significantly reduce the ability of compound 6 to scavenge cyanide compared to serum-free conditions.

[0303] [Example 6] Zebrafish cyanide rescue studies Zebrafish were used to evaluate sulfur-containing Pt compounds 3–11 and prioritize candidate agents for further in vivo testing (e.g., in rodent models of cyanide toxicity). Briefly, zebrafish larvae were plated in 96-well plates in HEPE-buffered Tuebingen E3 medium (n = 5 per well) at 6 days post-fertilization. Compounds 3–11 were reconstituted in purified water, and larvae were dosed with potassium cyanide (50 μM) and each platinum complex (1–250 μM) in each well (until 100% rescue was obtained; see Table 5). This cyanide concentration represented the lethal dose (LD100) for all embryos in each well, and survival was recorded after 4 hours of exposure following treatment.

[0304] [Table 6]

[0305] Each result is the concentration of platinum required for survival (n=5, 100%) in the presence of 100 μM KCN, which resulted in death after 1 hour in the control group. Reported survival was 4 hours after cyanide exposure.

[0306] Toxicity in zebrafish is measured by the presence of platinum compounds herein at a lethal dose in half the population (LD 50 Toxicity is expressed as a percentage of the heart rate of the control group, with a lower heart rate indicating increased cardiotoxicity (n=12 per group).

[0307] Summarizing the Pt dose per molar formulated in DMSO, cisplatin (compound 1) was more potent than hexachloroplatinate (compound 2). The same trend was observed for isolated DMSO compounds 3 and 4, which were equipotent with formulations made in DMSO. These results are consistent with active Pt agent formulations containing a single DMSO ligand. This analysis further demonstrates that the Pt(II) compounds herein exhibit greater potency than Pt(IV) complexes. For comparison, isolated Pt(II)-DMSO compound 5 was also included, which exhibited similar potency to compound 3. In addition to a sulfur-directing effect due to cyanide substation on Pt, compounds 3-5 all exhibited improved aqueous solubility, consistent with contributing to their overall cyanide-rescue properties. When tested using the same zebrafish model, amino-sulfide Pt(II) compounds 6-11 revealed a similar trend, with compounds 6, 7, 10, and 11 exhibiting potency comparable to that of cisplatin-DMSO compound 3.

[0308] To assess cardiotoxicity, each compound without added cyanide was screened in a validated physiological assay using heart rate and atrioventricular (AV) conduction (Figure 18). Burns et al., High-throughput assay for small molecules that modulate zebrafish embryonic heart rate, Nature Chemical Biology 1: 263-264 (2005); Milan et al., Drugs that induce repolarization abnormalities cause bradycardia in zebrafish, Circulation 107: 1355-1358 (2003). Analysis was performed using multiple comparison tests between ventricular and atrial measures.

[0309] Briefly, Tübingen AB zebrafish embryos (bred in-house) were incubated with the indicated doses (target concentrations / doses were selected from the zebrafish cyanide rescue efficacy study described above) of each compound or vehicle alone (used as a control) for 2 hours. Heart rate was normalized to the control group. Additionally, dofetilide was used as a positive control to demonstrate AV 2:1 block. Heart rate was measured by video at 15-second intervals. Atrial and ventricular heart rates were calculated from the average pixel density over time in the region of interest. A fast Fourier transform was performed to determine heart rate and estimate AV coincidence.

[0310] The difference between the two means was not significant for all doses administered, and the EC determined for compounds 1-11 100 There was no evidence of bradycardia or AV block observed with any dose.

[0311] [Example 7] Mouse inhalation study In vivo zebrafish studies have indicated the potential for these Pt(II) compounds to act as cyanide scavengers, but in certain embodiments, availability via intramuscular injection (IM) would be beneficial. A lethal inhalation model in mice provides a rigorous test of efficacy using either intraperitoneal (IP) or IM administration. A basic scheme for this established model is presented in Figure 19. This model has also been used to test the efficacy of traditional platinum-based cyanide countermeasures. Thompson & Marrs, Hydroxocobalamin in cyanide poisoning, Clinical Toxicology 50: 875-885 (2012); Summa et al. (2006), supra.

[0312] Briefly, mice were placed in an airtight chamber and exposed to a lethal concentration of cyanide gas (LD 40 min later). 100After 15 minutes, the mice were removed, given an IM injection of antidote, and placed back into the cyanide chamber for an additional 25 minutes. Phosphate buffer was used to control the pH of each formulation of the different sulfur-containing Pt compounds. This model represents a real-life scenario of people exposed to cyanide gas in a difficult-to-access enclosed space, such as a factory or subway station, where emergency medical personnel require 15 minutes to arrive at the disaster site and 25 minutes to simultaneously treat and evacuate the victims.

[0313] Despite the efficacy of Compound 3 in the zebrafish model of Example 6, a mouse model revealed that the material was not reproducibly effective when administered by IM injection (FIG. 19). This result is consistent with previous studies using cisplatin formulated in DMSO.

[0314] When compound 4 was delivered IP, efficacy was consistent with that observed for compound 2. (Nath et al. (2017), supra; Morningstar et al., Intramuscular administration of hexachloroplatinate reverses cyanide-induced metabolic derangements and counteracts severe cyanide poisoning, FASEB Bioadvances 1: 81-92 (2019)). However, similar to compound 3, no efficacy was observed for DMSO compounds 4 and 5 when dosed up to 20 mg / kg via IM administration in cyanide-treated mice. Previously, compound 2 was found to be active in a lethal cyanide rabbit model following IM injection of 10.4 mg / kg Pt (equivalent to 41.6 mg / kg in mice). However, potential dose-limiting muscle toxicity was observed even at a Pt dose of 69 mg / kg in mice, indicating a potentially narrow therapeutic window.

[0315] The platinum amine-sulfide-containing compounds (compounds 6-11) exhibited contrasting profiles to the DMOS compounds when tested by IM administration. For compounds 6 and 7, efficacy in the mouse model showed robust relief at 17 mg / kg and 19 mg / kg, respectively (Figure 19). Similarly, compounds 9-11 (analogs of compounds 6 and 7) demonstrated similar efficacy using IM administration. These results indicate significant enhancement in detoxification activity for the five amine-sulfide Pt compounds compared to DMSO Pt compounds 2, 3, and 4.

[0316] In contrast, compound 8 appeared to have no cyanide adverse effects in a mouse model using the same IM dose range. One explanation for this loss of activity is the observed heterogeneity of Pt species in solution, which may reduce bioavailability.

[0317] Compound 9 also appears to be a less efficient agent than compounds 9-11. The results are consistent with the hypothesis that sulfur ligands on platinum may enhance the efficacy of platinum as a cyanide countermeasure. However, the physicochemical properties of the Pt ligand likely impart important attributes to the IM dosage form for enabling bioavailability.

[0318] Additionally, the stability of effective doses of Compound 6 was verified by aging formulations of Compound 6 over a 7-day period and subsequent use in the lethal inhalation mouse model described above. Briefly, solutions of Compound 6 were allowed to stand at ambient temperature for up to 7 days before being tested in the mouse inhalation model. The same dose was used in each of four studies, each with three mice, and doses were administered on days 0, 1, 3, and 7. In all cases, survival was observed, confirming that Compound 6 may be an effective cyanide countermeasure candidate with a useful shelf life (Table 6).

[0319] [Table 7]

[0320] In summary, improved efficacy was observed in Pt(II) doses compared to cisplatin-DMSO compounds. For example, the effective dose for compound 6 administered in a mouse model was 51 mg / m 2 , which is within the range of a single human dose of cisplatin used in oncology.

[0321] [Example 8] Pharmacokinetics and pharmacodynamics of non-lethal cyanide exposure in rabbits. Real-time monitoring of oxygenated / deoxygenated hemoglobin by diffuse optical spectroscopy provided a direct readout of cyanide effects in vivo. Brenner et al., Cyanide antidotes in development and new methods to monitor cyanide toxicity, In Toxicology of Cyanides & Cyanogens 309-316, John Wiley & Sons, Ltd. (2015). The platform for conducting these studies of cyanide toxicity in a rabbit model allowed for direct comparison of leading countermeasure candidates.

[0322] Rabbits were ventilated with 100% O2 throughout the experiment. A sublethal dose of cyanide was infused (0.167 mg / min) over 55 minutes, and an antidote was injected intramuscularly at the completion of the cyanide infusion. Changes in in vivo tissue oxygenation status were monitored noninvasively using continuous wave near-infrared spectroscopy (CWNIRS) for 90 minutes after antidote injection.

[0323] A comparison of the time-dependent changes in hemoglobin status was performed for compounds 3 and 6 (Figures 20A-20D). Upon IM delivery of compound 3, the rate of change in the oxygenated / deoxygenated hemoglobin ratio in the blood recovered over the next 2 hours, representing a significant response in a non-lethal rabbit model (Figure 20B). For compound 6, IM delivery resulted in a rapid change in the oxygenated / deoxygenated hemoglobin ratio over the next 15-20 minutes (Figure 20C). These data are consistent with the apparent reactivity of compound 6 with cyanide using in vitro measurements.

[0324] Considering that IM administration fails to demonstrate meaningful efficacy in the mouse inhalation model, a significant response of Compound 3 in this non-lethal cyanide toxicity rabbit model was unexpected. Analysis of total platinum content in the blood over the time course of the experiment was used to compare IM doses of Compounds 3 and 6, as indicated by the area under the curve (AUC) (Figure 20D). Dose normalization to total injected platinum yielded C max Showing that levels were comparable at higher doses.

[0325] Blood plasma concentrations of total platinum content were also collected by ICP-MS in rabbits dosed with compounds 3 or 6 in a non-lethal cyanide model (FIG. 20D). Blood samples were collected for up to 180 minutes, and data were presented as total platinum concentration in μg / mL versus time. ICP-MS sample injections were performed using an Arridus peristaltic pump as described herein, and analysis of each sample was performed using 90-second collection times. Platinum-195 peak intensities were converted to micrograms per milliliter as described above.

[0326] Most unique was the difference in T for 8.7 mg of compound 3 versus 6 mg of compound 6 in total Pt dose. max It was. T max Direct comparison between the two suggests that the absorption rate of compound 6 contributed to achieving efficacy in both mouse and rabbit models of cyanide toxicity.

[0327] [Example 9] Sites of injection toxicity in mouse models Previous studies with PtCl6 formulated in DMSO and PBS using an IM dose of 200 mg / kg in mice revealed significant levels of chemically induced tissue damage. (Morningstar et al. (2019), supra.) In contrast to these previous results observed with hexachloroplatinate in DMSO, a comparative muscle toxicity study was conducted to compare both platinum compounds 4 and 6 and to assess whether toxicity may be a general limitation of the novel Pt(II) compounds herein. Briefly, CD-1 mice were obtained from Envigo (Indianapolis, IN) at 3-4 weeks of age and weighing between 18 and 20 grams. In two separate studies, each complex was administered via IM injection into the gastrocnemius muscle at 42 mg / kg and 50 mg / kg to males (n = 2 per dose) and females (n = 2 per dose). Injected samples contained Ca. 2+ and Mg 2+ The injections were administered in 50 μL of Compound 4 or Compound 6 containing free PBS. The study was conducted at the Purdue Translational Pharmacology and Clinical Veterinary Pathology Laboratories with full IACUC approval. The injection sites were monitored for significant inflammation or bruising. At 1 and 5 days post-dose, animals from both cohorts were humanely euthanized in accordance with the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. After euthanasia, the gastrocnemius muscles were surgically removed and fixed for dissection and histopathological analysis.

[0328] In contrast to previous studies using HCP / DMSO, reduced levels of Compound 4 did not reveal significant pathological findings beyond the needle injection site.

[0329] 2.5-fold or 3-fold excess of EC in mouse models 100In IM injection of Compound 6 in rats, induction of some acute myonecrosis was observed in approximately 60% of the analyzed sections, manifested as eosinophilia or swollen microfibers. The effect was localized, indicating loss of striations and sarcoplasmic fragmentation on day 1 after injection, with improvement observed in these sections by day 5. Signs of localized inflammatory and edematous changes remained at the injection site in approximately 40% of the sections. Collective information did not indicate any significant tissue toxicity beyond the injection site induced by Compound 6.

[0330] [Example 10] Formulation of Compounds 6, 9, 12, and 13 Platinum forms strong bonds with four cyanide equivalents, thereby enhancing its potential to ameliorate the toxic effects of cyanide poisoning. Early investigations demonstrated a significant DMSO solvent effect in modulating the efficacy of cyanide toxicity in a zebrafish model. Nath et al. (2017), supra. Follow-up efforts established the efficacy of IM-administered Pt(IV)-DMSO complexes in a lethal pig model, demonstrating 100% rescue at 35.6 μmol / kg. Morningstar et al. (2019), supra. As described in the examples above, it has also been determined that Pt(II)-thioether compounds derived from cisplatin metabolites can be approximately 10-25 times more effective against cyanide than cisplatin formulated in DMSO in a lethal zebrafish exposure model. Behymer et al. (2022), supra. Additionally, these new Pt(II)-thioether compounds (e.g., compounds 6-11) were bioavailable via IM administration and provided efficacy in a lethal cyanide inhalation mouse model. Therefore, further research into the properties of Pt(II) compounds, their characteristics, and properties is warranted.

[0331] Additional compounds, including Pt(II) compounds containing new ligands, have been prepared to serve as improved cyanide scavenging agents. In addition to identifying new ligands for Pt(II) compounds, shelf-stable formulations would be beneficial for use in developing practical, safe, and effective cyanide countermeasures for field deployment. Figure 32 shows (for illustrative purposes only) representative structures of four bidentate Pt(II) compounds (Compound 6, Compound 9, Compound 12, and Compound 13) depicted in the cis configuration. Compound 6 is a six-membered bidentate complex (or pharmaceutical salt) that, in certain embodiments, comprises bis-(L-methionine(S,N)platinum(II) dichloride). Compound 6 can be, for example, a bidentate ring-closed configuration of Compound 1 described above. Compound 9 is a six-membered bidentate complex (or pharmaceutical salt) that comprises methionine and a carboxamide, in certain embodiments, comprises bis-(L-methionine(S,N)platinum(II) dichloride). Compound 9 can be, for example, a bidentate ring-closed configuration of compound 2 described above. Compound 12 is a five-membered bidentate complex (or pharmaceutical salt) containing bis-(S-methylcysteine)-(S,N)platinum(II) dichloride. Compound 13 is a five-membered bidentate complex (or pharmaceutical salt) containing bis-(S-methylcysteineamide)-(S,N)platinum(II) dichloride. Each of compounds 6, 9, 12, and 13 can be modified with functional groups including, but not limited to, one or more alkyl groups, hydroxyl groups, amino groups, carbonyl groups, carboxyl groups, etc.

[0332] Compound 6 (+2NaCl): To produce compound 6 (+2NaCl), a mixture of Na2[PtCl4].nHO (2.08 g, 5.20 mmol), methionine (1.59 g, 2.05 molar equivalents), and Milli-Q water (10.4 mL) was sonicated until all solids dissolved and stirred overnight at ambient temperature in the dark. Thin-layer chromatography (silica gel 60F254 coated) (MilliporeSigma, Burlington, MA), developed with 1% sodium chloride in water and stained with UV254, iodine, and ninhydrin, was used to ensure reaction completion. The mixture was centrifuged to remove the platinum black precipitate. The clear supernatant was lyophilized overnight to isolate compound 6, a pale yellow solid (3.66 g).

[0333] Compound 6 (without NaCl): A mixture of KPtCl (208 mg, 0.50 mmol), methionine (75 mg, 0.50 mmol), and Milli-Q water (2.0 mL) was sonicated. The mixture quickly became a dark reddish-brown homogeneous solution, then began to produce a precipitate and turn light brown, fading over time. The mixture was sonicated to dissolve all clumps of methionine, which immediately produced a precipitate, and was allowed to stir overnight at ambient temperature in the dark.

[0334] The solid was isolated from the reaction mixture and washed with Milli-Q water (0.5 mL x 3) to remove KCl, then with isopropyl alcohol (0.5 mL x 2), followed by diethyl ether (0.5 mL x 2), and dried under vacuum to yield mono(methionine)platinum dichloride (MetPtCl, 133 mg). A mixture of MetPtCl (1121 mg), methionine (411 mg, 1.02 equivalents), and Milli-Q water (2.7 mL) was sonicated to form a homogeneous solution, kept overnight in the dark at ambient temperature, and lyophilized to isolate the NaCl-free batch of compound 6 (1526 mg). The platinum content for this product was approximately 38% w / w Pt (Table 7). Absorption data was based on the vehicle. The data shown is the platinum analysis obtained by UV-Vis and KCN after 24 hours. Results are the average of n=3 replicates with an RSD of less than 0.5%, indicating good reproducibility for the UV-Vis method.

[0335] [Table 8]

[0336] Compound 9 (without NaCl): A mixture of K2PtCl4 (415 mg, 1.0 mmol), methioninecarboxamide hydrochloride (185 mg, 1.0 mmol), and Milli-Q water (1.8 mL) was sonicated until all solids dissolved. The mixture was kept in the dark at ambient temperature for 5 days. A white solid precipitate formed and was isolated by centrifuging the mixture. The solid was resuspended in ice-cold Milli-Q water (1.0 mL), centrifuged, and the supernatant was carefully removed. This water washing procedure was repeated two more times to remove the KCl. The solid was rinsed with isopropyl alcohol (1.0 mL × 2), followed by diethyl ether (1.0 mL × 2), and dried to yield mono(methionine-carboxamide)platinum dichloride (MetNH2)PtCl2, 305 mg. A mixture of (MetNH)PtCl (305 mg), methionine carboxamide hydrochloride (139 mg, 1.02 equivalents), and Milli-Q water (1.5 mL) was sonicated to form a homogeneous solution, kept overnight at ambient temperature in the dark, and lyophilized to isolate compound 2' (452 mg) as a NaCl-free batch. The platinum content for this product was approximately 33% w / w Pt (Table 8).

[0337] The data shown in Table 8 are platinum content analyses obtained by UV-Vis and potassium cyanide and are the average of triplicates with 32% and 33% Pt for batch A and batch B, respectively. These results confirm that the synthesis of compound 2' (without NaCl) can reliably produce solid material with consistent platinum content (absorbance at 255 nm was recorded after background subtraction using excipients as standards).

[0338] [Table 9]

[0339] Compound 12 (+2NaCl): Compound 3′ was prepared from NaPtCl.HO (1.20 g, 3.00 mmol), S-methylcysteine (831 mg, 2.05 mol equivalents) and Milli-Q water (6.0 mL) in a manner similar to that described above to produce compound 6 (+2NaCl), yielding 2.04 g.

[0340] Compound 13 (+2NaCl): A solution of N-tert-butoxycarbonyl-S-methylcysteine (2.49 g) in anhydrous tetrahydrofuran (26 mL) was cooled in an ice bath, and then carbonyldiimidazole (3.78 g) was added dropwise. The mixture was stirred in the ice bath for 4 hours, and then 28% ammonia solution (6.5 mL) was added dropwise, followed by stirring for an additional 3 hours and storage at 4°C overnight. The mixture was briefly concentrated by rotary evaporation and extracted with ethyl acetate (100 mL). The extract was washed with 1 M HCl (25 mL x 2) to remove imidazole. The aqueous phase was re-extracted with ethyl acetate (20 mL × 1), and the combined extracts were concentrated by rotary evaporation to produce a crude solid residue, which was washed with a 2:1 mixture of hexane / ethyl acetate, filtered, washed with a 2:1 mixture of hexane / ethyl acetate, and dried outdoors to give N-tert-butoxycarbonyl-S-methylcysteine carboxamide (Boc-SMeCysNH, 2.49 g). Boc-SMeCysNH (2.10 g) was dissolved in 9.0 mL of 1,4-dioxane with warming and sonication, followed by the addition of 4 M hydrogen chloride in 1,4-dioxane (9.0 mL, 4.0 equiv.). The mixture was kept at ambient temperature overnight. At this point, the reaction was not complete, so additional 4 M hydrogen chloride in 1,4-dioxane (4.5 mL, 2.0 equiv.) was added. After 4 hours, the addition of hexane (23 mL) promoted precipitation of the product, which settled to the bottom of the vessel. The supernatant was removed by decantation, and the solid was rinsed with diethyl ether (10 mL × 3), carefully decanting each time, and dried in vacuo to yield S-methylcysteine carboxamide hydrochloride (SMeCysNH2.HCl, 1.53 g). Compound 4' (+2NaCl) was prepared from Na2PtCl4.HO (401 mg, 1.00 mmol), SMeCysNH2.HCl (345 mg, 2.05 mol equivalents), and Milli-Q water (5.0 mL) in a manner similar to that described above to produce compound 6 (+2NaCl), yielding 631 mg.

[0341] [Example 11] Optimization studies: stability and speciation The formulation stability of four cyanide-scavenging Pt(II) compounds was evaluated. The ligands were selected to enhance the reactivity of the Pt(II) metal center by including a methylthioether group. Each compound was prepared in an aqueous medium selected for its high solubility and potential hydrolytic stability. During initial discovery efforts for these bidentate (S,N)-ligand Pt(II) compounds, the formation of several pH-dependent mixed isomers was observed, consistent with prior structural assignments. (Summa et al. (2006), supra; Norman et al. (1992), supra). However, different isomers of the bidentate chelate can alter the cyanide-binding kinetics. (Appleton et al. (1988), supra). Regarding formulation stability, the effect of formulation pH can affect in vivo efficacy, and further evaluation is warranted.

[0342] The existing risk of Pt(II)-based therapy inducing adverse side effects can create dose-limiting toxicity, especially if additional mitigation strategies are not utilized. For example, the prevalence of acute kidney injury (AKI) caused by the chemotherapy agent cisplatin alone is high, occurring in approximately 30-40% of patients receiving the agent. Volarevic et al., Molecular mechanism of cisplatin-induced nephrotoxicity, J Biomedical Sci 26: 25 (2019). Therefore, it would be beneficial to be able to modulate the reactivity of cyanide scavengers to effectively inactivate the compound, thereby reducing or even preventing toxicity in patients.

[0343] Compounds 6 and 9 were previously shown to be reactive with cyanide but demonstrated different potencies in lethal zebrafish and mouse cyanide exposure models (see Examples above). Indeed, bis-(L-methionine amido(S,N)platinum(II) dichloride (compound 9) demonstrated reduced cyanide reactivity at high pH (Behymer et al. (2022), supra). Despite their chemical similarity, compound 6 consistently had higher cyanide scavenging kinetics compared to compound 9 across all assays at neutral pH. These observations motivated further investigation of the pH dependence of this reactivity and its potential impact on in vivo efficacy.

[0344] Platinum complexes have been reported to have greater stability, resisting pH-dependent changes, due to the five-membered ring system (Lawrance, Introduction to Coordination Chemistry, John Wiley & Sons (2013)). Enhanced conformational stability potentially slows the rate of cyanide substitution. However, compound 12 demonstrated similar enhanced cyanide reactivity and in vivo efficacy as compound 6 within the resolution of the experimental method (see the examples above and Behymer et al. (2022), supra). Therefore, the potential role of the carboxamide ligand in the five-membered ring system motivated the preparation of compound 13 to serve as a complimentary test case for the effect of the carboxamide group.

[0345] Using the data in Figures 34A-34D, Pt(CN)4 from compound 13 2- An apparent rate constant for production was obtained. Furthermore, the results in both Figures 34C and 34D demonstrate that the signal was stable 5-10 minutes after cyanide addition.

[0346] An initial comparison of the UV spectra for compound 13 suggested a pH-dependent change (Figure 35) similar to that for compound 9. The observed rate constant was 0.6 min in the presence of 40:1 KCN / Pt. -1 Indeed, the cyanide reaction rate with compound 13 was observed to be slower than that with compound 6 (Figure 34B).

[0347] To assess compound stability, cyanide scavenging of carboxylate complexes (compounds 6, 9, 12, and 13) was evaluated using HPLC at all pH conditions (e.g., 4-7) for up to 14 days at room temperature. For HPLC, an Agilent 1100 equipped with a DAD and a Restek Ultra IBD 2.1 x 50 mm column for scanning wavelengths from 200 to 300 nm was used to detect and separate the formulation mixture of platinum complexes. The column was operated in reversed-phase mode with 5 μL injections at 0.3 mL / min. Baseline conditions were 65% purified water with 2 mM ammonium formate and 35% v / v acetonitrile. An elution step with a pH 3.7 solution of 2 mM ammonium formate and 0.5% v / v formic acid gradient was performed to induce ion exchange. The identity of the Pt(CN)4 peak was quantified at 260 nm and confirmed using the absorbance spectrum from 200 to 300 nm.

[0348] As noted above, stability samples were stored at room temperature over the 14-day study period. HPLC sample preparation was performed by diluting a 10 mM platinum stock to 350 μM and adding KCN to a final concentration of 1.4 mM (1:4 Pt to KCN) to react the platinum for 10 minutes at ambient temperature. The reaction solution was 12.5 mM sodium phosphate pH 7.3 buffer. Sample preparation was staggered so that each HPLC injection occurred at 10 minutes. The 10-minute reaction step was repeated in triplicate for each time point.

[0349] For the HPLC speciation assay, an Agilent 1100 equipped with a UV detector (λ = 220 nm) and an Agilent Zobax Eclipse XD8 C18 column was used with 50 μL injections. The mobile phase consisted of 88% water: 12% acetonitrile. The aqueous component consisted of 25 mM sodium phosphate pH 5.5 with 12.5 mM heptanesulfonic acid as an ion-pairing agent. The method was performed using an isocratic run at 1 mL / min for 35 minutes.

[0350] Cyanide scavenging of the carboxylate complexes (compounds 6 and 12) was maintained at room temperature for up to 14 days at all pH conditions (e.g., 4-7). In contrast, the carboxamide compounds (compounds 9 and 13) exhibited no significant cyanide scavenging activity in formulations above pH 5 when stored for several days. 2- The instability also resulted in reduced cyanide scavenging activity for carboxamide compounds 9 and 13. For example, compound 9 revealed spectral changes in the presence of sodium hydroxide (Figure 36A). In comparison, compounds 6 and 13 are carboxylate complexes and did not demonstrate the same behavior by UV in the presence of NaOH. A pH-dependent rate was demonstrated for compound 9 in Figure 36B, suggesting a possible base-catalyzed process.

[0351] The conversion rate of compound 9 at 18.8°C and pH 7.3, assessed by UV at 245 nm, was found to have a half-life of 2.6 hours (Figure 36D), while compound 13 had apparent half-lives of 3.3 hours and 9 minutes at 19°C and 37°C, respectively (Figures 37A and 37B).

[0352] HPLC analysis of compound 9 in pH 6.8 phosphate buffer at room temperature showed a gradual change over 12 hours with repeated injections, likely due to a speciation event in Figure 36C. Initially, there was a dominant form (form 1), labeled as peak I at approximately 33 minutes, which disappeared after 4 hours to give rise to a new form 2, labeled as peak II (Figures 36A-36C).

[0353] The UV and HPLC observations were generally consistent with the NMR. In Figure 38A, the peaks at 7.25 and 7.18 ppm were observed when comparing day 0 (labeled B) and day 3 (labeled A). 1 The H NMR signals were reduced and shifted upfield. In addition, new signals appeared between 5.5 and 6 ppm on day 3. Figure 38B also demonstrates the simultaneous changes. The singlet signal at 2.1 ppm was assigned as the -SMe signal, signaling that the functional group was no longer bound to platinum, and a significant increase in intensity was observed on day 3. These data suggest that compound 9 lost the interaction between the thioether and platinum. The loss of the thiomethyl bond to platinum was most visible in the signal at 2.1 ppm (consistent with the expected chemical shift for free methionine), which grew much more dominant on day 3, while the two larger signals between 2.6 and 2.55 ppm for the bound methyl group (day 0, labeled B) shifted to 2.5 ppm and became less intense.

[0354] Compound 9 was analyzed for reversibility over a 3-week period at pH 7. 1 H NMR was performed (Figure 39). Compound 9 was incubated in pH 7 phosphate buffer for 3 weeks (labeled B in Figure 39), and HCl was added to adjust the solution pH to approximately 2.5. Addition of HCl to reduce the pH to approximately 2.5 showed a gradual loss of the free -SMe signal at 2.1 ppm, confirming the reassociation of -SMe with platinum.

[0355] The functional stability of platinum complex formulations is described as their ability to retain reactivity with cyanide during storage in solution. Platinum-thioether compound 9, which produced a slower reaction rate with cyanide, was less effective in fish and mouse models, especially when stored over several days (see the Examples above and Behymer et al. (2022), supra).

[0356] To further evaluate this, compound 9 (2 mM) was incubated in pH 6.8 phosphate buffer at room temperature for 3 days. Upon addition of 4 molar equivalents of cyanide, Form I disappeared immediately, as shown in Figure 40, while the slower conversion of Form II occurred over several hours. Pseudo-first order conditions with a 1:40 molar ratio of compound 9 to cyanide in pH 7 phosphate were observed in 0.32 min. -1 Under similar conditions, when compound 9 was freshly prepared, the observed rate for cyanide was 15 min. -1 (See the Examples above and Behymer et al. (2022), supra.) Thus, the rate of cyanide scavenging for compound 9 stored at neutral pH is significantly lower than for freshly prepared solutions.

[0357] Under a similar set of conditions, the reaction rate of compound 13 with cyanide was also reduced (Figure 36D).

[0358] Thus, for compounds 9 and 13, a loss of reactivity was observed as the solution pH approached neutrality, suggesting the role of the carboxamide group, as opposed to the carboxylate group in compounds 6 and 12. To demonstrate the apparent difference in reactivity at different pH values, platinum complexes were stored in a range of buffers for up to 14 days. At different time points, samples of the formulation were reacted with 4 molar equivalents of KCN for 10 minutes to obtain the product (Pt(CN)4 2- ) was quantified by HPLC (n=3 for each pH condition).

[0359] Under all pH conditions, compounds 6 and 12 reacted with Pt(CN)4 2-The production did not show any significant change in reactivity over 14 days (Figures 41A and 41B). Monitoring of compound 6 for up to 42 days (Figure 42) suggests potential stability much longer than 14 days. As expected, carboxamide complexes 9 and 14 reacted with Pt(CN)4 at pH values above 5 over 14 days. 2- This produced a reduction in the amount of (Figures 41C and 41D).

[0360] Figure 41C and 41D show Pt(CN)4 for compounds 9 and 13 upon storage in the pH range 5-8. 2 The observed reduction in the cyanide-dependent conversion to Pt(CN)4 is consistent with the results observed in Figures 36A-36C, 38A, 38B, and 40. The pH-dependent change in the form of compounds 9 and 13 was revealed by monitoring the cyanide reaction, which revealed the formation of Pt(CN)4 from the second species formed. 2- Biphasic kinetics were observed, leading to significantly slower production of . The data are consistent with a modulation of the cyanide scavenging rates of compounds 9 and 13, resulting from pH-induced isomerization of the complexes to a second form.

[0361] Upon reconstitution of the solid preparations in water for compounds 6 and 9, acidic solutions were formed. The initial acid-base titration of compound 6 (Figure 43) reveals an apparent pKa of 2.9. At the equivalence point, approximately 2 moles of NaOH per Pt(II) is consistent with carboxylate neutralization. However, when the acidic solution of compound 6 was stored at room temperature for an additional 7 days and then titrated with NaOH, a very different profile was observed (Figure 44). The extended range of NaOH required to produce a progressive increase in pH indicated a complex process. The titration of compound 9 was also complex but did not show a clear equivalence point (Figure 44). Furthermore, leaving compound 9 in water for several days resulted in a gradual acidification of the pH over time after each titration step, which may be due to the complex undergoing transition to a new form.

[0362] The observed pH-dependent difference in stability between carboxylate (compounds 6 and 12) and carboxamide ligands (compounds 9 and 13) supports a mechanism of intramolecular isomerization, likely via amine deprotonation as a key step to facilitate ring closure. The Pt-ligand bond angle in the five-membered ring should have greater stability than the six-membered structure based on the shorter Pt-S / N bond length. Lawrance (2013), supra. As indicated by UV and HPLC, the results indicate that the rate of formation of new species is slower for compound 13 than for compound 9. In our previous work, compound 12 exhibited a slower cyanide scavenging rate than compound 6, reflecting higher conformational stability resulting in a slower substitution reaction rate by cyanide. A reduced cyanide scavenging rate was also observed for compound 13 under the same conditions. These observations confirm that S,N-chelate size appears to influence the isomerization and cyanide scavenging rates, and that five-membered ring structures are more stable than six-membered structures in binding with Pt(II).

[0363] [Example 12] In vivo efficacy of formulations Formulation pH conditions for intramuscular administration were screened to identify optimal conditions for maintaining cyanide scavenging for each Pt(II) complex. Compounds 6, 9, 12, and 13 were first tested in a lethal cyanide-sensitized zebrafish survival assay to confirm their stability for in vivo scavenging activity over several days. (Nath et al. (2017), supra.) Each formulation was prepared in buffers ranging from pH 4.3 to 7.6, and details for each formulation are summarized in Table 9. Several buffer conditions were prepared with approximately 10x platinum concentration for each compound (6, 9, 12, and 13) to ensure stable pH during shipping. The efficacy of each sample was tested after 5 days for compounds 6 and 9, and after 4 days for compounds 12 and 13, to confirm activity.

[0364] The results in Figure 45 show that compounds 6, 12, and 13 remain effective (EC 100 This demonstrates that the potency of all four compounds was ≤15 μM. Thus, all four compounds operate within an optimal pH range to react with Pt(II):CN in a 1:4 stoichiometry. Compound 9 exhibited decreased potency above pH 6.8, suggesting that speciation has a significant effect on in vivo rescue.

[0365] The zebrafish efficacy results were consistent with the in vitro findings for compound 9, as slower cyanide scavenging kinetics occurred when compound 9 was stored at near-neutral pH values. Together, these data suggest that a lower pH should be considered for formulations stored over several days.

[0366] [Table 10]

[0367] Formulations of compounds 6, 9, 12, and 13 were also tested for efficacy in the previously described lethal cyanide inhalation mouse model via intramuscular injection (see the Examples above and Behymer et al. (2022), supra, and Chan et al., The combination of cobinamide and sulfanegen is highly effective in mouse models of cyanide poisoning, Clin Toxicology (Phila) 49(5): DOI: 10.3109 / 15563650.2011.584879 (2011)).

[0368] The primary motivation was to identify suitable formulations of compounds 6, 9, 12, and 13 that retained efficacy via IM injection. Preliminary studies were performed with compounds 1-3, prepared as the +2 NaCl form (α) (described above in Example 7). To assess the impact of osmolality on IM administration, alternative procedures for reducing NaCl (β) were investigated.

[0369] Briefly, the osmolality of each sample was recorded using vapor pressure using a VAPRO® Vapor Pressure Osmometer Model 5600, using a sample volume of 10 μL. Examples of calibration and analysis for the preparation of compounds 6 and 9 are shown in Figures 33A and 33B. A linear decrease was observed as a function of concentration, with osmolality decreasing linearly with concentration with a correlation coefficient of greater than 0.99.

[0370] Compounds prepared either as +2NaCl forms (α) or as NaCl-free forms (β) were compared by testing in a lethal cyanide inhalation mouse model using IM administration. Previously, compound 6α was found to be 100% effective at 87 μmol / kg (Example 7; Behymer et al. (2022), supra). In addition, the efficacy of compound 9α was only effective in 3 of 5 mice at 87 μmol / kg. Ibid.

[0371] In Table 10, compound 6β also shows 100% survival when dosed as low as 72 μmol / kg in a lethal mouse model. A new preparation of compound 9β at pH 6.5 was able to provide survival in all four mice treated with 103 μmol / kg, and pH 4.2 showed complete rescue at 87 μmol / kg. These results suggest that lower pH provides a stable formulation.

[0372] It was hypothesized that the five-membered ring in compound 13α, with optimized ring strain, might reduce the rate of speciation and improve formulation stability. Results for compound 13α revealed total rescue at 103 μmol / kg, as indicated in Table 10. Results from a lethal cyanide-treated mouse model demonstrate that low pH can improve the efficacy of intramuscularly delivered platinum compounds 9 and 13 bearing carboxamide ligands by reducing speciation. Furthermore, formulations with reduced NaCl content and reduced osmolality also retain efficacy.

[0373] [Table 11]

[0374] Findings reveal that, at least in part, compounds 6 and 12 maintain similar kinetic activity toward cyanide across the pH range studied. However, compounds 9 and 13, when formulated above pH 5, exhibited reduced cyanide scavenging reaction rates after 3 days. Compounds 9 and 13 were stable and maintained reactivity over a pH range of ≤5. Optimal formulation pH conditions were determined for all four compounds, demonstrating rapid stoichiometric formation of Pt(CN)4, exceeding the binding of any known scavengers approved or in development. 2- This led to...

[0375] [Example 13] Platinum nephrotoxicity Using rat models, changes in blood chemistry and complete blood counts were evaluated in a dose-dependent manner to assess the tolerability of a single intraperitoneal (IP) injection of compounds 6, 9, 12, and 13. Garrett & Korstanje, Using genetic and species diversity to tackle kidney disease, Trends in Genetics 36: 499-509 (2020); Kohl et al., Evaluation of urinary biomarkers for early detection of acute kidney injury in a rat nephropathy model, J Pharmacological & Toxicological Methods 105: 106901 (2020).

[0376] Briefly, Sprague-Dawley rats (Envigo and Inotiv) weighing 225-250 grams were used in this study, which was conducted in the Purdue University Translational Pharmacology and Veterinary Clinical Pathology Laboratory. Compounds 6 and 12 were formulated in phosphate buffer. The final pH of the formulation at the time of injection was 6.5-7.0 using sodium hydroxide for final pH adjustment. Compounds 9 and 13 were administered in sodium acetate buffer (Ca2 + and Mg2 + The solution was prepared in PBS (free), with a final pH adjustment using sodium hydroxide to a target pH of 4.2. A final adjustment was made using Milli-Q water to reduce the osmolality of the solution.

[0377] Platinum and vehicle were administered by intraperitoneal (IP) injection at 5.28 ml / kg or less. Doses were allometrically scaled using the difference in body surface area between mice and rats and designed to reach five times the effective dose in the lethal cyanide mouse model, but using IP administration (Table 11).

[0378] [Table 12]

[0379] Blood samples were drawn 1 and 5 days after injection and processed for a comprehensive metabolic panel and a complete blood count panel. At the end of the study, animals were euthanized in accordance with the Public Health Service Policy on the Humane Treatment and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals. All procedures were performed in accordance with the Animal Welfare Act and the International Association for the Assessment and Accreditation of Laboratory Animal Care regulations and guidelines. The Purdue University IACUC Committee approved all experimental protocols (1405001069).

[0380] Animals treated with up to five times the effective dose (218 μmol / kg) of compound 6 exhibited significant increases in both blood urea nitrogen (BUN), creatinine (CREA), and phosphate (PHOS), indicative of acute kidney injury (AKI) (Figures 46A and 46B). Briefly, each animal received compound 6α in pH 6.5 phosphate buffer at an injection volume of 2.12 ml / kg or less. Each group of 44 to 218 μmol / kg (8.5 to 28.3 mg Pt / kg) had n = 6 rats, while vehicle and 42.5 mg Pt / kg contained n = 12 rats.

[0381] In addition, a form of Compound 6β with reduced NaCl in the formulation was used to assess the potential impact of osmolality. Compound 6 was administered to the first cohort (n=11) at a small volume (SV) of 2.2 ml / kg and to the second cohort (n=6) at a large volume (LV) of 5.28 ml / kg. Analysis was performed using a two-way ordinary ANOVA with Sidak's multiple comparisons comparing cell means across rows and columns. No significant differences were observed in AKI markers between animals treated with the highest dose of Compound 6α or Compound 6β, thus reducing the role of osmolality (Figures 47A and 47B).

[0382] Additionally, to assess body weight changes and resulting toxic stress, animals received Compound 6 in pH 6.5 phosphate buffer at injection volumes of 2.2 ml / kg or less. Each group of 8.5 to 28.3 mg Pt / kg had n = 6 rats, while the vehicle and 42.5 mg Pt / kg groups contained n = 12 rats. Animals dosed with the highest doses of Compound 6α or Compound 6β lost approximately 15% body weight after 5 days, consistent with some level of toxic stress caused by the Pt(II) species (Figure 48). Compound 12 also demonstrated significant increases in BUN, CREA, and PHOS levels after 5 days at five times the effective dose (223 μmol / kg). Although mean marker levels appeared lower than Compound 6 at similar doses, these results were not significantly different.

[0383] As shown in Figures 49A-49C, the carboxamide-containing ligands Compounds 9 and 13 exhibited strikingly different effects on blood chemistry and complete blood counts. Compound 9 was tested in both pH 4.3 and 6.5 formulations at five times the effective dose in mice (218 μmol / kg). No significant differences in AKI markers were observed with either formulation of Compound 9 (9α and 9β) compared to the control group.

[0384] Rats treated with compound 13 at pH 4.3 and 218 μmol / kg had BUN, CREA, and PHOS levels similar to the control group and significantly lower than rats dosed with compound 12. Thus, both compounds 9 and 13 have a significantly reduced risk of inducing AKI at five times the effective dose when compared to their carboxylate counterparts, compounds 6 and 12.

[0385] The data confirm that the free carboxylate of each amino acid rapidly generates the cyanide-scavenging Pt(II) compounds 6 and 12 but may also increase AKI at higher doses, which may reduce the therapeutic index. Alternatively, the carboxamides on the bidentate amino acid ligands of compounds (e.g., compounds 9 and 13) exhibited reduced scavenging kinetics when formulated above pH 5; however, optimized pH formulations of the carboxamide compounds not only maintained cyanide reactivity but also significantly reduced the risk of platinum-induced toxicity (i.e., AKI).

[0386] Although the study was not adequately powered to assess significant sex differences, females appeared to be less prone than males to increased AKI markers with Pt(II) compounds and 12 (Figures 50A and 50B). There is substantial data from animal model studies highlighting sex differences associated with cisplatin-induced toxicity. Marcu, Gender and sex-related differences in normal tissue effects induced by platinum compounds, Pharmaceuticals 15: 255 (2022). Finally, complete blood counts did not have any significant differences between the complexes and controls, and therefore, results are not shown.

[0387] Cyanide exposure can be difficult to detect in patients and raise concerns about the safety of therapeutic interventions. Therefore, assessing the risk of toxicity (e.g., AKI) associated with platinum complexes is important. Sprague-Dawley rats are well recognized as a sensitive model for detecting platinum-induced nephrotoxicity. Perse & Veceric, Cisplatin-induced rodent model of kidney injury: characteristics and challenges, Biomedical Research Int'l 2018: 1462802 (2018). At doses five times higher than the effective dose in mice, compounds 6 and 12 produced AKI symptoms after 5 days, consistent with observations in cisplatin-treated Sprague-Dawley rats. Brenner et al., Comparison of cobinamide to hydroxocobalamin in reversing cyanide physiologic effects in rabbits using diffuse optical spectroscopy monitoring, J Biomedical Optics 15(1): 017001 (2010). Interestingly, the carboxamides in compounds 9 and 13 significantly attenuate the AKI observed in the rat model. While the correlation between Pt reactivity and toxicity has been previously discussed, this study demonstrates that platinum complexes can slowly convert to less toxic forms, creating agents with improved safety.

[0388] Ligands such as methionine with thioether functional groups have been used to ameliorate the nephrotoxic effects of cisplatin in rats (Jones et al. (1989), supra; Jones et al. (1991), supra; Basinger et al. (1990), supra). Thioethers and other ligands that bind strongly to platinum have been proposed to function as antioxidants (Stankovic et al. (2020), supra). For example, sulfhydryls supplied by glutathione or anionic sulfur ions (e.g., thiolates and WR-2721) have been used to mitigate cisplatin-induced nephrotoxicity, but the efficacy of platinum anticancer drugs has been impaired in these cases (Jones et al. (1991), supra). The most promising antioxidants, such as dithiocarbamates and WR-2721, have shown reductions in renal platinum levels. Jones et al., Relative effectiveness of some compounds for the control of cisplatin-induced nephrotoxicity, Toxicology 68: 227-247 (1991); Volarevic et al. (2019), supra. In the acute cyanide scavenging setting, the use of fast-acting platinum rescue agents as a single bolus IM dose offers benefits. These comparisons of compounds 6, 9, 12, and 13 provide insight into the potential balance needed to achieve safety and efficacy for S,N-chelated Pt(II) complexes with a therapeutic index of 5.

[0389] [Example 14] Pharmacokinetics of the intramuscular formulation A proof-of-concept study was conducted to assess the similarities and potential differences in pharmacokinetic parameters between compounds 6 and 9 in rats. The data obtained will be used to estimate the dose required upon allometric scaling to achieve efficacy.

[0390] The study design involved two groups of five rats (two males and three females), each treated with 45 μmol / kg of either Compound 6 or 9. Each compound was administered IM to mimic the efficacy model and product concept. Plasma levels of total Pt in the animals were monitored over 48 hours (Figure 51), with 250 μL of blood sampled at each time interval and replaced with phosphate-buffered saline using a Culex™ stress-free autosampler unit. Samples were collected at 0.5, 10, 20, 30, 45 minutes, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and 48 hours post-dose. Upon sample collection, the blood samples were immediately centrifuged, and the resulting plasma was stored at -80°C in separate vials for each time point.

[0391] These data were evaluated for differences in absorption rate, elimination rate, and overall exposure according to AUC values (Table 12). Both complexes appeared to reach maximum concentrations within 7-9 minutes after IM administration, which is consistent with a highly diffusible agent capable of rapid absorption. However, the dose-normalized maximum concentration in plasma for Compound 6 was 0.01.

[0392]

number

[0393] [Table 13]

[0394] The inset in Figure 51 highlights the biphasic pharmacokinetic profile observed for compound 6, suggesting a dominant distribution phase within the first 2 hours. In contrast, compound 9 appeared to have a steady-state pharmacokinetic phase, with distribution events not kinetically distinct from the elimination rate. Despite these possible differences, the AUC and clearance rates revealed a lack of statistically significant differences between the two compounds. The extrapolated clearance rates and terminal elimination half-lives for compounds 6 and 9 were also not statistically different. These elimination rates appeared to be faster than those reported for other platinum drug complexes in rats. Wang et al., Pharmacokinetics and tissue distribution of novel traditional Chinese medicine—platinum anticancer agents in rats, J Inorganic Biochemistry 101: 909-917 (2007). Taken together, these results highlight that compounds 6 and 9 have utility as cyanide scavenger agents with rapid absorption by IM administration, but also point to different pharmacokinetic outcomes despite their relatively minor structural differences.

[0395] [Example 15] Blood-brain barrier (BBB) permeability studies Cyanide can also be a potent neurotoxin, and therefore, the distribution of compounds 6 or 9 into the brain may help elucidate their potential for mitigating neurotoxicity. To assess potential brain distribution, the permeability of compounds 6 and 9 was assessed across an in vitro blood-brain barrier (BBB) triculture model as shown in Table 13 and described in Lubin & Knipp, "Design of experiment based optimization of an in vitro direct contact triculture blood brain barrier model for permeability screening," Pharmacy & Pharmacology Int'l J 9: DOI: 10.15406 / ppij.2021.09.00340 (2021)."

[0396] Briefly, permeability was examined in the apical (A; blood-facing) to basolateral (B; neuron-facing) direction and in the basolateral to apical (B to A) direction to determine the relative brain parenchymal exposure and efflux ratio (P) for compound 6 (Met2PtCl2(+2NaCl)) and compound 9 (Met(NH2)2PtCl2(+2NaCl)). app,B→A / P app,A→B For each permeability coefficient determination, 100 μM of each platinum complex was added to the Ca 2+ and Mn 2+ The compounds were dissolved in Hank's balanced salt solution (HBSS) containing 1000 mM NaCl. The osmolality of the sample solutions was between 240 and 260 mmol / kg. The efflux ratios for compounds 6 and 9 were 1.10 and 0.98, respectively, confirming that the permeation rates were controlled by passive diffusion.

[0397] Permeation rates were determined by loading the complexes apically for AB permeability and basolaterally for BA permeability. The receiver chamber contained 100 μL sample aliquots that were collected at 0, 15, 30, 45, 60, 120, and 180 min and analyzed by HPLC. Analysis was performed at 220 nm with an Agilent Zobax Eclipse XD8 C18 column.

[0398] [Table 14]

[0399] The permeability coefficients compare favorably to rates determined for established markers with higher in vivo brain distribution and penetration across the BBB. Kulczar et al., Development of a direct contact astrocyte-human cerebral microvessel endothelial cells blood-brain barrier coculture model, J Pharmacy & Pharmacology 69: 1684-1696 (2017). The BBB permeation rates confirm that both complexes can scavenge cyanide in the brain parenchyma.

[0400] [Example 16] Formulation: Tunable Ligands The selection process for Pt(II) compounds / agents and their formulations involves testing established in vivo high-throughput models of cyanide poisoning. Among the agents identified in Table 1 above, the use of thiosulfate ligands appears to be detrimental to the agent's utility, despite being recommended to counteract cisplatin toxicity. Curiously, thiosulfate is also a known scavenger of cyanide.

[0401] The agents in Table 1 are options for use as ligands in conjunction with one or more compounds herein. None of the actives in Table 1 exhibited lethality to zebrafish when tested up to 250 μM (5-fold higher than cyanide), and in addition, they exhibited increased potency compared to HCP-AKN and cisplatin-AKN. The compounds in Table 1 (excluding the thiosulfate ligand) represent candidates for resynthesis and retesting in pig kidney cell and zebrafish studies before proceeding to nephrotoxicity testing.

[0402] The process for formulating Pt(II) agents begins with either PtCl2 or Na2PtCl4H2O. Each offers different advantages in terms of elemental composition and purity of the final product. Initial reaction conditions require solubilization of Pt in water by reaction with a bidentate ligand (such as those described herein or similar bidentate ligands). To provide a concentration sufficient to meet the requirements of an autoinjector for IM administration in the field, the water solubility criteria are targeted at 200 mM or 50 mg / mL. The ligand to Pt ratio is carefully selected to allow for the isolation of mono- or bis-addition products. Preparation of mixed-ligand materials, such as MetPt(taurine)2 (Table 1), was accomplished by isolating the intermediate mono-addition product (MetPtCl2) followed by reaction with a second orthogonal ligand (taurine). Depending on the starting material and isolation, additional methods are known for isolating materials that vary in salt form.

[0403] Ligand design principles have been guided in the past by the use of methyl sulfides linked to primary amine groups via either carbon 2 or carbon 3. The synthesis of the present ligands is consistent with this approach. For example, a dimer of methionine bridged via the S-methyl group has been prepared (identified in Table 1 as (BridgedMet2)Pt). New ligand variants include, but are not limited to, carboxamide variants of carboxylates that alter the titratable group of the molecule; alterations in the electron-donating or electron-withdrawing characteristics of the S-Me group; and flexibility in the carbon chain bridging the sulfur and nitrogen atoms. Combinations of different ligands at the Pt(II) center are being pursued to incorporate agents such as taurine, which is reported to have renal sparing activity.

[0404] Elemental composition is determined by a combination of ICP-MS and NMR. Solutions of known weight per volume are prepared. These are analyzed by ICP-MS to determine platinum content, and by various nuclei (e.g., NMR) for ligand and salt content. 1 H, 13 C. 35 Cl, 23 Analyses are performed by NMR (Na). All quantification is performed by comparison to known standards. Batch production reproducibility is a key factor in these studies.

[0405] Methods have been developed to assess the cyanide reactivity and stoichiometry of each Pt(II) complex. Reactions between the platinum complex and cyanide are initiated, and samples are analyzed by UV-vis spectroscopy, HPLC, and NMR. Real-time reaction kinetics can be measured by UV by measuring absorbance at 255 nm over time. HPLC confirms the expected product, PtCN42-, and provides a second method for observing real-time reaction kinetics in the event of ligand interference at 255 nm. NMR is used to monitor the reaction using 13C-KCN. The extent of reaction is analyzed by quantifying the product formed and comparing the results to the amount of starting platinum complex utilized. Measuring the extent of reaction over days or weeks provides a measure of "functional stability" for each formulated platinum complex. Stability testing of aged formulations follows the same methodology. These are stored ambient samples or accelerated by storage at 37°C for 3 days. The samples are expected to maintain greater than 85% capacity for trapping cyanide in solution.

[0406] [Example 17] Zebrafish cyanide protection This assay is now well established and is used for the initial evaluation of cyanide protection of each agent or formulation. The primary metric to be determined is the EC 100 The cyanide concentration used in this screen is 50 μM. The cutoff for the performance of the Pt(II) agent is Pt(CN)4 2- Based on optimal conversion to , the cyanide concentration will be set at 1 / 4 of the cyanide concentration (i.e., 12.5 μM).

[0407] In parallel, each candidate's LD 100 is determined in non-cyanide treated zebrafish. Cut-off LD 100is 250 μM or 5× the cyanide dose. Briefly, zebrafish larvae (6 days post-fertilization) are loaded into 96-well plates. Approximately 480 larvae per 96-well plate for each of vehicle, positive and negative controls, and a 10-point dose-response curve per drug.

[0408] Use a 10-point dose-response analysis to screen compounds (0.4-500 µM).

[0409] In control animals, KCN is added at a dose of 50 μM, which induces 100% mortality within 1 hour. After addition of cyanide, the plates are sealed with adhesive PCR plate foil and incubated at 28°C.

[0410] The minimum effective dose to rescue 100% of the larvae is 4 hours after treatment (EC 100 ) is reported. For assessment of compound toxicity, larvae are treated with the compound for 24 hours and viability is assessed by observing heart rate and tactile response. The dose causing 100% lethality is reported (LD 100 ).

[0411] All new active candidate Pt(II) agents are retested on days 1 and 7 as pre-formulated materials with established pH, buffer, and ionic strength. 100 The results of any changes in are correlated with cyanide reactivity stability studies to ensure that the cutoff criteria in Figure 53 are met.

[0412] The use of sulfide ligands enhances the rate of cyanide loading, which can accelerate sulfhydryl loading, other biomatrix components, and even water. This unpredictable speciation poses a challenge comparable to in vitro analytical methods. Therefore, the development potential of formulated active ingredients can be assessed by tracking the efficacy of cyanide rescue using an in vivo zebrafish model.

[0413] [Example 18] Study of the nephrotoxic potential of Pt(II) agents Drug-induced nephrotoxicity is an important consideration when developing platinum-based therapeutics, making it important to de-risk novel platinum entities early in the development pipeline. Potential adverse events include glomerular injury (inadequate filtration of macromolecules leading to proteinuria and hypoalbuminemia), renal tubular injury (lack of renal tubular reabsorption resulting in hypophosphatemia and hypokalemia), crystal nephropathy (characterized by crystalline deposits in the urine), and renal inflammation (reducing renal blood flow and glomerular filtration).

[0414] Nephrotoxicity is the most common adverse event associated with platinum drugs, particularly cisplatin. Cisplatin is known to damage the kidney, causing tubular injury and inflammation. Due to their similarity to cisplatin, assessing this toxicity is crucial for developing platinum compounds as cyanide antidotes. In accordance with the 3R principle, well-established in vitro models are utilized to assess the nephrotoxic potential of compounds prior to animal studies, and the zebrafish model is utilized to act as a bridge between in vitro and in vivo mammalian models. Successful completion of these studies allows for the assessment of nephrotoxicity posed by the compounds of the present invention while minimizing the number of rats used in definitive toxicity studies.

[0415] Zebrafish have conserved renal physiology compared to mammals and are an established model for assessing nephrotoxicity. Juvenile zebrafish larvae as young as 4 days postfertilization possess functional pronephros structures that run transversely along the length of the larva. Zebrafish (larval and adult) also exhibit similar cisplatin-induced toxicity, including neurotoxicity, ototoxicity, and renal damage.

[0416] LLC-PK1 cells (derived from porcine proximal tubules) can differentiate between cisplatin and oxaliplatin toxicity in transwell permeability assays. These responses to cisplatin occur at drug concentrations within the range of nephrotoxic blood levels in rat models. Fluorescent dextran is used to assess monolayer permeability. Cisplatin (30 μM), but not oxaliplatin (30 μM), disrupts the barrier within these cells, allowing fluorescent dextran to leak into the external chamber. The effect of our candidate Pt(II) agents on barrier integrity is assessed by measuring the fluorescent signal in the external chamber. Any concentrations that disrupt the LLC-PK1 barrier are interpreted in the context of drug levels seen in the blood during pharmacokinetic evaluation. Toxic doses in rats are predicted to closely resemble those that disrupt polarized LLC-PK1 layers, as previously shown with cisplatin.

[0417] Briefly, LLC-PK1 cells are grown to confluence on a transwell membrane. Before the start of the assay, the barrier integrity is determined by measuring transepithelial electrical resistance using an epithelial volt-ohmmeter (WPI), and then treated with three doses of compound. Cisplatin (30 μM) is used as a positive control, and oxalaplatin (30 μM) is used as a negative control. Fluorescent dextran is used to assess barrier integrity. The fluorescent signal in the medium in the outer chamber is measured using a spectrophotometer. In addition, cell viability is assessed using CellTiter-Glo® assay.

[0418] Second, the cytotoxicity in this cell line is evaluated. The concentration required to kill cells is expected to be much higher than the concentration required to destroy the barrier. Compounds are ranked from least to most toxic based on these in vitro assays.

[0419] The toxicity of platinum compounds has characteristics such as effects on blood flow and activation of immune system.The zebrafish model faithfully captures these characteristics, and as a lower vertebrate, is in line with the NIH 3R rule.The zebrafish model is used to assess the nephrotoxicity of the platinum compounds of the present invention and evaluate their effects on renal function in vivo.

[0420] Previously, an assay for directly measuring glomerular filtration in zebrafish has been developed. Control compounds (such as cisplatin, carboplatin, and oxaliplatin) are used in each assay. The effect of candidate Pt(II) cyanide antidotes on glomerular filtration is measured using fluorescent dextran. Compounds known to increase BUN and CREA in rats are expected to decrease glomerular filtration in zebrafish. The ranking of toxicity in this zebrafish assay from least to most toxic is compared to that of the LLC-PK1 cell assay.

[0421] Zebrafish larvae (5 days postfertilization) were exposed to the compounds for 4 to 24 hours. Cisplatin (30 μM) served as a positive control, and oxaliplatin (30 μM) served as a negative control. They were then anesthetized and microinjected with 10 kDa FITC-dextran into the heart. Z-stack images were acquired for each animal at 1, 6, and 24 hours postinjection. To quantify dextran clearance, maximum intensity projections of the image stacks were created, and the fluorescent signal in the same region of the dorsal aorta and posterior cardinal vein for each animal was analyzed using Image J to quantify the fluorescence intensity over time. Animal number: 25 larvae / group × 5 groups (vehicle, positive control, and three doses of platinum complex) = 125 larvae / assay.

[0422] The rat is an accepted model for testing nephrotoxicity. For each study, animals (6 male / 6 female Sprague-Dawley) were bred to the EC2000 dose observed in the mouse cyanide inhalation model. 100Treatment is performed at 1x and 5x the normal serum BUN (allometrically scaled) via IP administration. Blood chemistries are assessed on days 1 and 7 post-treatment. Increases in both BUN and CREA are interpreted as decreased glomerular filtration. In isolation, each of these biomarkers can be nonspecific. The absence of hypoalbuminemia, hypophosphatemia, or hypokalemia confirms glomerular injury and loss of tubular reabsorption, which may or may not result from tubular cell death. As with cisplatin, these observations would be consistent with a potential inflammatory effect. Inflammation caused by the direct and secondary effects of platinum agents can reduce glomerular filtration (and result in higher serum BUN and creatinine) independently of hypoalbuminemia, hypophosphatemia, and hypokalemia.

[0423] [Example 19] Efficacy studies in a mouse model of cyanide inhalation The top Pt(II) candidates and / or their new formulations will be advanced into murine studies based on the criteria outlined in FIG.

[0424] A rigorous model was developed to simulate a realistic cyanide exposure scenario. Mice were exposed to HCN gas, injected intramuscularly with an antidote, and then re-exposed to HCN. The model assumed that emergency medical personnel would require approximately 15 minutes to arrive at the disaster site and another 25 minutes to treat the cyanide-exposed individuals and evacuate the contaminated area. A custom-built, sealed chamber was used in a chemical fume hood to minimize the risk of cyanide exposure to laboratory personnel while allowing visual monitoring of the animals. The chamber was preheated to 30°C, and the mice were placed in the chamber. Liquid isoflurane was injected to achieve a concentration of 2%.

[0425] The IACUC requires that the chamber be heated for the mice, which additionally aids in vaporizing the isoflurane and cyanide gas. The mice are anesthetized within 2 minutes, at which point KCN is injected into the beaker containing HCl and a magnetic stir bar. The device is mounted on top of a magnetic stir plate, so that the KCN rapidly mixes with the acid, producing HCN. A circulating fan within the chamber ensures rapid equilibration, with the HCN concentration reaching a steady state within 5 minutes and remaining constant for up to 1 hour. Precise control of gas concentrations over a wide range can be achieved.

[0426] These studies have two animal groups: vehicle control and Pt(II) candidate treatment. The outcome is survival, and the criterion for inclusion is 90% or greater survival in a 100% lethal assay.

[0427] Number of animals: Sample size was determined using a chi-squared test with alpha set at 0.05 and power set at 0.9. 100% lethality is expected in untreated mice. Aiming for at least 90% survival in treated animals, a sample size of 11 was calculated for each group. A corresponding number of control saline-treated mice is used in each group, yielding 24 mice [2 groups × 6 animals / group × 2 sexes]. Comparisons are made between treated and untreated animals of the same group and sex. Survival curves are generated and analyzed using the log-rank test. Clinical evaluation of mice is assessed dichotomously as either normal or abnormal and using a standard t-test.

[0428] [Example 20] Evaluation of the metabolite glyoxylate using Pt(II)-based scavenger agents The metabolite glyoxylate is evaluated using platinum(II)-based scavenger agents. Glyoxylate, or other agents that mitigate the effects of cyanide, do not eliminate cyanide from the body. At the same time, using the metal as a scavenger for cyanide carries the risk of toxicity (i.e., the kidneys for Pt(II)). Therefore, combining a chelator with a metabolic modulator achieves unparalleled efficacy, extending the therapeutic window by more than 10% (Figure 54).

[0429] The combination of a Pt(II) cyanide scavenger and glyoxylate rescues cyanide-intoxicated zebrafish and mice (Figure 54). In zebrafish and mice, the combination of glyoxylate with a Pt complex protected animals from lethal cyanide exposure at doses where each single agent was ineffective. 0% survival was observed with PCP or glyoxylate alone at the listed doses.

[0430] In zebrafish, glyoxylate stimulates EC 100 = 32 μM, and doses below 15 μM were ineffective, while PCP had an EC 100 = 62 μM, with doses of 32 μM or less being ineffective. In combination studies, 15 μM PCP plus 8 μM glyoxylate rescued 100% of the zebrafish. Consistent findings were observed in mice (Figure 55). Glyoxylate (60 mg / kg; n=6) or PCP (31.5 mg / kg; n=6) administered alone resulted in 16% survival, while the combination of the two agents resulted in 66% (n=6).

[0431] In a separate study, to monitor cyanohydrin formation 13 C NMR was used to evaluate the chelating affinity of glyoxylate for cyanide (Figure 56). In addition, several keto metabolites were included for comparison. The pyruvate equilibrium constant with cyanide was calculated as K a = 11mM-1 or K D= 90 μM. A competition experiment between pyruvate and glyoxylate (or any other keto metabolite) was used to directly assess the dissociation constant for cyanide (Figure 56). These results lay the groundwork for selective Pt(II) chelation by cyanide (higher affinity > 104) in the presence of glyoxylate, ensuring that the metabolite is available for metabolic salvage.

[0432] These studies are based initially on a published Pt(II) complex bearing two methionine ligands (Met2Pt). Preliminary data indicate that Met2Pt exhibits a 5×EC 100 The results indicate that rats exhibited elevated CREA and BUN levels when dosed at 1000 mg / kg (not shown). Reducing the effective Pt(II) dose by half widened the therapeutic window to 5.

[0433] Preliminary NMR data ( 1 H, 13 C. 195 The Pt(II) data indicate that the 3:1 glyoxylate / Met2Pt sample remained unchanged over 3 days (not shown). These preliminary data demonstrate the feasibility of co-formulating glyoxylate with reactive Pt(II) complexes.

[0434] Combination drug testing in the zebrafish cyanide assay will initially be performed in the presence of a subeffective dose of 8 μM glyoxylate. This level of glyoxylate represents one-fifth of the total cyanide. Theoretically, the Pt(II) complex must be present at at least 12.5 μM to consume all of the cyanide under these conditions. However, the minimum amount of cyanide scavenging required to keep zebrafish viable is currently unknown. Therefore, testing approaches will aim to reduce the overall burden of the administered metal by finding the optimal ratio with glyoxylate.

[0435] Platinum complexes are tested at doses ranging from 0.4 to 12.5 μM. When combined with glyoxylate, the EC 100 Platinum compounds exhibit increasing potency as a ≥2 change in NADH / NAD ratio and / or a 2-fold improvement in nicotinamide adenine dinucleotide (NADH) / NAD ratio. Fixation of cyanide to cytochrome c oxidase prevents its reoxidation by oxygen, thus causing electron congestion in the electron transport chain. Complex I is then trapped in a reduced state and therefore cannot reduce NADH to regenerate NAD+, which is required for the tricarboxylic acid (TCA) cycle. As the NADH / NAD+ ratio increases, negative feedback inhibition of the TCA cycle ensues, which shifts cells from aerobic to anaerobic metabolism.

[0436] The NADH / NAD+ ratio will be measured in lysates from cyanide-treated zebrafish larvae (6 days post-fertilization) using an enzymatic assay (Abcam ab65348) and compared to cyanide-treated animals with the leading candidate and vehicle-treated animals (no cyanide). Additionally, as a second confirmation, the lactate:pyruvate ratio will also be assessed in zebrafish lysates, as it is in near equilibrium with the NADH:NAD+ ratio.

[0437] Finally, the cystine / cysteine ratio was measured in zebrafish lysates as a surrogate marker of extracellular redox stress, which is enhanced in cyanide-poisoned pigs treated with glyoxylate. Targeted mass spectrometry was used to assess these four metabolites using a Sciex 4000 QTRAP triple quadrupole mass spectrometer in positive ion mode (cystine, cysteine) and an Agilent 6490 QQQ triple quadrupole mass spectrometer in negative ion mode (pyruvate, lactate). The combination, which exhibited a two-fold improvement in NADH:NAD+ and pyruvate / lactate in addition to restoring the cystine / cysteine ratio to baseline, was carried forward to mouse studies.

[0438] For the mouse study, there were three groups: vehicle control, platinum complex (EC 50), and agent combinations. For each group, a corresponding number of control saline-treated mice are required, yielding 36 mice [3 groups x 6 animals / group x 2 sexes]. The target concentration for glyoxylate is an ineffective dose of 40 mg / kg (1 / 3 EC 100 ) and the Pt(II) complex is EC 50 The current dose of glyoxylate used in large animal porcine models corresponds to 3 molar equivalents to the cyanide dose.

[0439] These doses will be adjusted as information from zebrafish efficacy and nephrotoxicity studies emerge. For example, if the glyoxylate:Pt(II) ratio in the combination is refined in zebrafish kidney function assays to reduce or eliminate signs of kidney dysfunction, the efficacy of that ratio in a cyanide toxicity model will be retested before proceeding to a mouse model.

[0440] [Example 21] Efficacy and safety studies of Pt(II)-based agents alone and in combination with the metabolite glyoxylate in a porcine model for cyanide poisoning The FDA requires efficacy studies in at least two mammalian animal models to obtain FDA approval through animal regulations. The pig model allows for the collection of unique information not captured in mouse or zebrafish models, including continuous hemodynamic data, clinical laboratory values, and serial blood sampling for metabolite profiling and PK studies. The EC of the identified front-runner candidate was 80 Determine the EC in fixed ratio combinations with glyoxylate 80 This large animal model provides a robust test for a transportable cyanide countermeasure delivered as a single intramuscular injection.

[0441] Acute cyanide poisoning leads to hypoventilation and apnea, and patients presenting with respiratory failure are at increased risk of mortality. Therefore, the primary model is a spontaneously breathing animal, the well-established non-ventilated intravenous infusion porcine model. a Since cyanide exists almost exclusively as HCN at physiological pH, with a pH of 9.2, infusion of cyanide salts produces HCN, the form of cyanide absorbed through the lungs or stomach. Cyanide infusion models produce the same end products as inhalation or ingestion models but have the advantage of knowing the exact amount of cyanide the animal receives. Furthermore, the porcine model mimics the physiological effects of cyanide poisoning observed in humans (apnea and cardiovascular collapse). Additionally, pigs also provide a good model for evaluating pharmacokinetics and efficacy due to their large size and the fact that their circulatory system is similar to that of humans.

[0442] An evaluation of the metabolic effects of glyoxylate compared to cyanide scavengers in pigs was conducted. Preliminary data established that glyoxylate exhibits evidence of redox metabolic effects that reverse the effects of cyanide exposure in a pig model (83% survival at 10 mg / kg IM, n=6; Figure 57).

[0443] Next, we measured the circulating lactate:pyruvate ratio, which is approximately in equilibrium with the intracellular NADH:NAD ratio. At the end of the cyanide infusion, plasma lactate:pyruvate decreased by 36% compared to baseline levels in pigs (Figure 58A, gray box; p=0.01).

[0444] Elevated lactate levels suggest a defect in mitochondrial oxidative phosphorylation during cyanide exposure. After glyoxylate administration, the lactate:pyruvate ratio rapidly increased from 0.66 ± 0.14 to 1.99 ± 0.39 at 7.5 min postinjection (Figure 58A; p = 0.006), indicating rapid lactate production or pyruvate consumption in response to glyoxylate. The lactate:pyruvate ratio then declined slightly below baseline (0.82 ± 0.29) by 45 min postinjection. To assess the specificity of these findings, the lactate:pyruvate ratio of glyoxylate was compared to that of a known cyanide chelator (HCP formulated with DMSO).

[0445] Although HCP (20 mg / kg IM) restored lactate:pyruvate to baseline levels 7.5 min after treatment, glyoxylate demonstrated a distinct effect on lactate:pyruvate (Figure 58A). The sharp rise in the lactate:pyruvate ratio between t = 0 and t = 15 min observed in glyoxylate-treated pigs did not occur in HCP-treated animals (1.99 ± 0.39 vs. 1.15 ± 0.28, p = 0.0418). The effects of glyoxylate on both pyruvate consumption and lactate production, combined with the lactate:pyruvate ratio, suggest that glyoxylate administration in cyanide-intoxicated animals alters metabolic pathways in a manner distinct from known cyanide scavengers. These findings also suggest that glyoxylate administration temporarily shifts the intracellular NADH / NAD+ balance.

[0446] The plasma Cys:CySS ratio is an established biomarker for extracellular redox balance. During cyanide infusion, the extracellular redox environment shifted toward an oxidized state, as evidenced by a decrease in plasma Cys:CySS. At the end of the cyanide infusion, Cys:CySS decreased by 27% (p=0.04) compared to baseline (Figure 58B). After IM injection of glyoxylate, the Cys:CySS ratio increased from 0.73 ± 0.27 to a peak ratio of 2.11 ± 0.78 at 60 minutes postinjection (Figure 58B; p=0.01).

[0447] These results indicate that the extracellular redox environment was shifted to a reduced state in glyoxylate-treated animals. In contrast to glyoxylate, HCP did not improve the cyanide-induced shift to an oxidized state (Figure 58B). After HCP administration, Cys:CySS continued to remain below baseline until the end of the study (0.56 ± 0.09 vs. 1.00). The overall results suggest that cyanide chelation alone is not sufficient to ameliorate cyanide-induced oxidative stress in the extracellular compartment.

[0448] These data demonstrate the unique beneficial biological effects of glyoxylate compared to cyanide chelators, independent of scavenging. However, as a single agent, the efficacy of glyoxylate may be suboptimal. The current IM dosing levels required for glyoxylate are greater than the desired volume for an autoinjector. In addition, EC 80 The molar equivalent of glyoxylate required to achieve this is more than three times the total cyanide injected. At this point, the risk of large amounts of oxalate formation from glyoxylate is unclear. However, large amounts of crystalline oxalate have been observed in pig urine. Therefore, it would be beneficial to offer a combination therapy of glyoxylate and the platinum complex candidates herein.

[0449] Preliminary results indicate that Pt-sulfide bidentate complexes exhibit potential cyanide countermeasure utility in the same animal model. Preliminary results with HCP formulated in DMSO also support potential efficacy in the large animal porcine model. To further leverage these observations, an additional top Pt(II) complex candidate (Example 16) will be advanced through the evaluation process for testing in the porcine model. The workflow and target performance criteria are outlined in Figure 59.

[0450] The target dose of Pt is justified as being one-quarter of the total cyanide dose in the pig model. This is because the Pt(CN)4 2- This represents the optimal scenario for capture as a cyanide scavenger. A product concept for field-ready countermeasures is the use of a pre-filled, ready-to-use autoinjector. Current autoinjectors are approximately 3 mL, but the availability of new technology for volumes up to 5 mL is anticipated. Empirical testing in a pig model with other cyanide scavenger agents has established the time it takes for the animals to regain breathing. Using the same methodology, peak blood level concentrations of the active scavenger are predicted as a key criterion for reproducible performance (Figure 60).

[0451] Candidate agents emerging in Examples 16-19 will be tested in pigs after meeting stringent qualification requirements in mouse models. Scale-up synthesis and full-batch characterization of candidate formulations will be completed one month prior to planned animal studies, as described in Example 20. Attempts to minimize ionic strength and pH effects of the formulations are ongoing.

[0452] The dose of the most potent platinum complex that rescues 80% of animals in a model with >80% lethality in control-treated animals is established by starting with the effective dose determined in mouse studies and scaling it to pigs using allometric dose scaling. The platinum complex is administered intramuscularly to two animals per dose for up to three doses (six pigs). Once the optimal dose is determined, a full efficacy study is conducted using male and female pigs (20 pigs). Pigs are exposed to cyanide via intravenous infusion of potassium cyanide until apnea (cessation of breathing) occurs. At 5 minutes after apnea, treatment with either the platinum complex or control occurs, and the cyanide infusion is discontinued. After treatment, animals are monitored for 90 minutes. Outcomes include survival, return of breathing, normalization of cardiovascular parameters, and normalization of blood gases (pH, arterial oxygenation) and lactate.

[0453] The most potent platinum complex dose that rescued 80% of animals in a model with >80% lethality in control-treated animals was 3.5 mg / kg glyoxylate (1 / 3 EC 100 The EC was established in combination with platinum complexes administered at effective doses determined in the previously described study in two pigs, and the dose was adjusted for up to three doses (six pigs). 80 Once the optimal dose of the combination therapy is determined, a full efficacy study will be conducted using male and female pigs (20 pigs). Pigs will be exposed to cyanide via intravenous infusion of potassium cyanide until apnea (cessation of breathing) occurs. At 5 minutes of apnea, treatment with either the platinum complex or control will be administered and the cyanide infusion will be discontinued. After treatment, animals will be monitored for 90 minutes. Outcomes will include survival, return of breathing, normalization of cardiovascular parameters, and normalization of blood gases (pH, arterial oxygenation) and lactate.

[0454] Yorkshire-cross pigs weighing 50 kg are used for these studies. After the study, the pigs are anesthetized with intramuscular ketamine (10 mg / kg), intubated, and maintained under sedation with 1-2% inhaled isoflurane while breathing room air spontaneously. The pigs undergo both full cardiovascular and respiratory monitoring. Potassium cyanide is infused intravenously at 0.17 mg / kg until apnea (<6 breaths per minute) occurs. At a predefined time after apnea, the cyanide infusion is discontinued and saline or a first-line drug is administered intramuscularly. Arterial blood gases, blood chemistries, and blood lactate are monitored every 20 minutes. Serial blood samples are collected for metabolite profiling and PK studies. In surviving animals, muscle biopsies are obtained to assess muscle integrity at the injection site.

[0455] Because cyanide exposure alters cardiovascular function, resulting in altered hemodynamics and possibly drug distribution, the pharmacokinetics and toxicity of the compounds herein will be evaluated in non-cyanide-exposed pigs. The pharmacokinetics of the platinum complex will be assessed independently using the doses used in the efficacy studies described above, administered via intramuscular injection. The pharmacokinetics of the combined platinum complex and glyoxylate administered intramuscularly at the doses used in the efficacy studies described above will then be assessed. Drug concentrations and blood chemistries (including liver and renal function) will be measured at baseline and at 5, 15, and 30 minutes, 1, 2, 4, and 8 hours, and 1, 3, and 7 days post-injection. A baseline blood sample will be obtained for each study. We plan to have five animals in each group, two males and three females, resulting in a total of 10 pigs for these studies. Plasma derived from the blood samples was split into two arms of bioanalysis (Figure 59), first processed for platinum metal analysis using ICP-MS, the second arm of the bioanalysis is metabolic profiling using an established HPLC-MS platform.

[0456] The log-rank test is used to compare survival in the control and treatment arms; to account for the potential effect of gender, a log-rank test stratified by gender is used to confirm differences in survival. A sample size of 10 animals in the control and treatment arms yields 90% power (alpha 0.05) to distinguish 80% and 20% survival for the treatment and control arms, respectively. Metabolite data are Bonferroni corrected. For pharmacokinetic studies, mean values of blood concentration curves are plotted for each group. For PK analysis, the data are first analyzed using a non-compartmental approach, which provides estimates of clearance, half-life, volume of distribution, mean residence time, maximum drug concentration, and time to maximum concentration. Following this analysis, the data are evaluated by compartmental modeling (single and multi-compartment models) to select the model that best fits the data.

Claims

1. A compound having the structure of formula (I): 【Chemical 1】 or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof [In the formula, Pt is platinum(II); Each L １ and L ２ is a ligand, and each L １ forms a bidentate ligand, and each L ２ form bidentate ligands, each bidentate ligand comprising N, S, or both N and S coordinated to said platinum(II), at least one of said ligands being a leaving group, and at least two of said ligands directly bonded to said platinum(II) each independently comprising an alkyl, a carboxamide, an amine, an aminosulfide, a carboxylate, a carboxyester, a carbonyl, or a thioether comprising any combination of the foregoing; each n is independently from about 1 to 5].

2. A compound having the structure of formula (II): 【Chemistry 2】 or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof [In the formula, Pt is platinum(II); Each L １ and L ２ is a ligand, and each L １ forms a bidentate ligand, and each L ２ form bidentate ligands, each bidentate ligand comprising N, S, or both N and S coordinated to said platinum(II), at least one of said ligands being a leaving group, and at least two of said ligands each independently comprising an alkyl, a carboxamide, a carboxyester, an amine, an aminosulfide, a carboxylate, a carbonyl, or a thioether comprising any combination of the foregoing; R １ is C １～３ Alkyl, C １～３ Haloalkyl, C ２～４ Alkenyl, C ２～４ Alkynyl, and C ６～１０ aryl or absent; each n is independently from about 1 to 5].

3. A compound having the structure of formula (III): 【Chemistry 3】 or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof [In the formula, Pt is platinum(II); Each L １ and L ２ is a ligand, and each L １ forms a bidentate ligand, and each L ２ form bidentate ligands, each bidentate ligand comprising N, S, or both N and S coordinated to said platinum(II), at least one of said ligands being a leaving group, and at least two of said ligands directly bonded to said platinum(II) comprising an alkyl, carboxamide, amine, aminosulfide, carboxylate, carbonyl, or thioether comprising any combination of the foregoing; Each R １ is C １～３ Alkyl, C １～３ Haloalkyl, C ２～４ Alkenyl, C ２～４ Alkynyl, and C ６～１０ aryl or absent; each n is independently from about 1 to 5].

4. Each L １ and L ２ 4. The compound of claim 1, wherein is a leaving group.

5. Each R １ 4. The compound of claim 2 or 3, wherein is absent.

6. The compound of claim 1 comprising a cis configuration.

7. The compound of claim 1 comprising a trans configuration.

8. 8. The compound of any one of claims 1 to 3, 6 and 7, wherein a first bidentate ligand comprises a sulfide and a second bidentate ligand comprises an amide.

9. 8. The compound of any one of claims 1 to 3, 6 and 7, wherein at least one thioester ligand comprises an amino sulfide.

10. 8. The compound of any one of claims 1 to 3, 6 and 7, wherein the thioester ligands each comprise an amino sulfide.

11. 8. The compound of any one of claims 1 to 3, 6 and 7, wherein the bidentate ligands independently comprise 5- or 6-membered bidentate ligands.

12. The compound has the following structure: 【Chemistry 4】 or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of any of the foregoing structures.

13. The following structure, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof: 【Chemistry 5】 2. The compound of claim 1, comprising:

14. The following structure, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer thereof: 【Chemistry 6】 2. The compound of claim 1, comprising:

15. 4. The compound of claim 1, wherein at least one of the ligands comprises a methyl thioether group.

16. 4. The compound of any one of claims 1 to 3, wherein at least one of the thioether ligands comprises methionine or S-methylcysteine, optionally containing one or more amidated carboxylates.

17. 7. The compound of any one of claims 1 to 3, 5 and 6, wherein the bidentate ligands independently comprise 5- or 6-membered bidentate ligands, and at least one of the bidentate ligands comprises a carboxylate or carboxamide substituent.

18. R １ is C １～３ 4. The compound of claim 2 or 3, wherein the compound is alkyl.

19. HCP-AKN, Cisplatin-AKN, (SalylCys) ２ Pt, (SMe penicillamine) ２ P, (cilastatin) ２ Pt, or (bridged Met ２ ) Pt, MetPt (taurine) ２ 4. The compound of claim 1, wherein

20. A compound according to any one of claims 1 to 19, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of a compound according to any one of claims 1 to 19; a pharmaceutically acceptable carrier and / or excipient; A pharmaceutical composition comprising:

21. 21. The pharmaceutical composition of claim 20, further comprising a pharmaceutically acceptable excipient.

22. 21. The pharmaceutical composition of claim 20, which is suitable for intramuscular injection.

23. 26. Use of a compound of any one of claims 1 to 19, a pharmaceutical salt, N-oxide, solvate, tautomer or stereoisomer of a compound of any one of claims 1 to 19, or a pharmaceutical composition of any one of claims 20 to 22 in the manufacture of a medicament for the treatment of a disease or condition in a subject.

24. 24. The use of claim 23, wherein the disease or condition is cyanide poisoning or exposure.

25. 24. The use of claim 23, wherein the medicament is formulated for intramuscular administration.

26. 24. The use of claim 23, wherein the medicament is formulated in a single bolus dosage.

27. 24. The use of claim 23, wherein the medicament is formulated at a pH of about 5 or greater.

28. 24. The use of claim 23, wherein the medicament is stored at a pH of about 5 or less.

29. 1. A method of treating cyanide poisoning or exposure in a subject, comprising administering a therapeutically effective amount of: A compound according to any one of claims 1 to 19, A pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of a compound of any one of claims 1 to 19; or A pharmaceutical composition according to any one of claims 20 to 22. administering to said subject a first therapy comprising:

30. 31. The method of claim 30, wherein the administering step comprises intramuscular injection.

31. 32. The method of claim 30 or 31, wherein administering the therapeutically effective amount of the first therapy comprises administering a single dose.

32. further comprising administering a second therapy to the subject, wherein the second therapy comprises: a therapeutically effective amount of one or more of glyoxylate, hydroxocobalamin, methemoglobin, riboflavin, methotrexate, 4-dimethylaminophenol (4-DMAP), dicobalt edetate, glucose, activated charcoal, and ethylenediaminetetraacetic acid (EDTA) disodium cobalt; intravenous isotonic fluids, and / or Oxygen therapy 31. The method of claim 30, comprising administering to the subject:

33. 33. The method of claim 32, wherein the first and second therapies are administered sequentially and / or alternatingly with one another.

34. 33. The method of claim 32, wherein the first and second therapies are administered concurrently.

35. 33. The method of claim 32, wherein the second therapy is glyoxylate and the therapeutically effective amount of the first therapy is about 3.0 to 5.5 mg / kg (based on the subject's body weight).

36. 36. The method of claim 35, wherein the therapeutically effective amount of the first therapy is about 3.5 mg / kg (based on the subject's body weight).

37. 31. The method of claim 30, wherein the therapeutically effective amount is about 3.0 to 5.5 mg / kg (based on the subject's body weight).

38. 31. The method of claim 30, wherein at least one ligand of the compound, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of the compound, or pharmaceutical composition comprises one or more carboxamide substituted amino acid ligands.

39. 31. The method of claim 30, wherein the administering step comprises intramuscularly injecting the compound, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of the compound, or a pharmaceutical composition into the subject, and wherein the compound or pharmaceutical composition reaches a peak concentration in the subject at or about 7 to 9 minutes after administration (e.g., at or about 7 minutes to 9 minutes, at or about 7 minutes to 9 minutes, or at or about 7 minutes to 9 minutes).

40. 39. The method of claim 38, wherein after being administered intramuscularly to the subject, the compound, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of the compound, or pharmaceutical composition undergoes pH-induced isomerization resulting in a cyanide scavenging rate that is reduced relative to the cyanide scavenging rate of the compound, a pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of the compound, or pharmaceutical composition within 1 hour of administration to the subject.

41. 1. A combination therapy for treating cyanide poisoning or exposure in a subject, comprising: To the subject, a therapeutically effective amount of a cyanide chelator; a therapeutically effective amount of an agent for ameliorating cyanide-induced oxidative stress in said subject; A combination therapy comprising administering

42. 42. The combination therapy of claim 41, wherein the cyanide chelator comprises a platinum(II) thioether comprising a bidentate ligand.

43. the cyanide chelating agent is A compound according to any one of claims 1 to 19, A pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of a compound of any one of claims 1 to 19. A compound having the following structure: or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer: 【Chemistry 7】 or a pharmaceutical composition according to any one of claims 20 to 22.

43. The combination therapy of claim 41 or 42, comprising:

44. 44. The combination therapy of claim 42 or 43, wherein the agent for ameliorating cyanide-induced oxidative stress in the subject is glyoxylate or an analogue or functional fragment thereof.

45. 44. The combination therapy of claim 42 or 43, wherein the agent for ameliorating cyanide-induced oxidative stress in the subject comprises a ketone or aldehyde compound selected from the group consisting of glyoxylate, glyceraldehyde, acetaldehyde, dihydroxyacetone, pyruvate, methylglyoxal, and alpha-ketoglutarate.

46. 1. A kit for treating cyanide poisoning or exposure, comprising: A compound according to any one of claims 1 to 19; A pharmaceutically acceptable salt, N-oxide, solvate, tautomer or stereoisomer of a compound of any one of claims 1 to 19; A compound having the following structure: or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer: 【Chemistry 8】 and, and a pharmaceutically acceptable carrier and / or excipient.

10. A kit comprising a drug injection device with one or more fluid chambers pre-filled with a first formulation comprising:

47. 47. The kit of claim 46, wherein the first formulation comprises a targeted effective dose of the compound, or a pharmaceutically acceptable salt, N-oxide, solvate, tautomer, or stereoisomer, for intramuscular injection.

48. 47. The kit of claim 46, wherein the pre-filled fluid chamber is a syringe or cartridge.

49. 49. The kit of any one of claims 46 to 48, wherein the formulation has a pH value of 5 or less.

50. 49. The kit of any one of claims 46 to 48, wherein the drug injection device is an autoinjector or a hand-held injector.

51. 51. The kit of any one of claims 46-50, further comprising one or more fluid chambers pre-filled with a second formulation comprising a therapeutically effective amount of an agent for ameliorating cyanide-induced oxidative stress in the subject and a pharmaceutically acceptable carrier and / or excipient.

52. 52. The kit of claim 51, wherein the agent for ameliorating cyanide-induced oxidative stress in the subject is glyoxylate or an analog or functional fragment thereof.

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