Methods for inactivating pathogens, microorganisms, and parasites

Abstract

This invention provides a method for inactivating or reducing pathogens from a sample. [Solution] (i) Compound having structure I: JPEG2026090275000051.jpg46169 The method comprises the steps of: (ii) processing a sample; (ii) incubating the sample from the previous step for a time sufficient to inactivate or reduce pathogens from the sample; (iii) forming a first sample to be treated by removing or reducing compounds having structure I in the sample from the previous step by treatment with a solid phase agent; and (iv) forming a second sample to be treated by treating the first sample from the previous step with one or more neutralizing agents that eliminate or further reduce the toxicity of compounds having structure I.

Description

[Technical Field]

[0001] The present invention relates to compositions and methods for use in industry and research for the inactivation or reduction of pathogens, microorganisms, or parasites in pharmaceuticals, biologics, medical devices, and cosmetics. More specifically, the present invention provides compositions and methods for inactivating and / or reducing pathogens, microorganisms, or parasites (e.g., contaminants) in samples, culture media, compositions, utility products, devices, surfaces, or organisms by eliminating or reducing residual alkylating agent compounds and / or their by-products following treatment with an alkylating agent compound. [Background technology]

[0002] In general, existing pathogens and infectious disease organisms, as well as newly emerging and other undesirable organisms (e.g., contaminants), including structures such as biofilms or biocontaminations, pose significant problems in various fields including pharmaceuticals, manufacturing, drug production, biologics, cosmetics, food, medical devices, research, and other industries. Therefore, it is important to inactivate pathogens or undesirable organisms in a wide variety of samples, such as organisms, or products and compositions, such as food, drugs, plants, blood or blood products, bodily fluids, culture media of eukaryotic or prokaryotic origin, vaccines or vaccine formulations, cosmetics, biologics and pharmaceutical compositions, or household, industrial or medical instruments, devices or practical items, such as fluid conduits, heat exchangers or aquatic vessels, or their surfaces.

[0003] Currently, there are no widely applicable techniques for reducing common pathogens, undesirable microorganisms, or parasites to inactivate organisms in samples, compositions, and practical applications. Some amphiphilic quaternary ammonium salts are highly versatile disinfectants, particularly at high concentrations, but they are inactive against non-enveloped viruses. Small reactive molecules, such as chlorine gas, sodium hypochlorite, ethylene oxide, methyl bromide, formaldehyde, or ozone, are broadly defined antimicrobial agents and toxic to all living organisms; however, their reactivity, especially towards proteins, prevents their widespread use in biological products, intravenous fluids, and in vivo applications. Furthermore, their chemical reactivity often makes them unsuitable for many applications.

[0004] Targeting and inactivating the nucleic acids of pathogens is a universal technique for preventing pathogen replication and infectivity and can be applied to all types of pathogens—viruses, bacteria, fungi, prions, protozoa, and other parasites or undesirable organisms. Several existing methods utilize this technique by using intercalators, such as methylene blue, psoralen derivatives (U.S. Patents 6,455,286 and 6,133,460), and riboflavin (U.S. Patent 7,985,588), which selectively bind to nucleic acids and damage them upon photoactivation, thereby providing broad antipathogen activity.For example, Estcourt et al., Jory et al., Magron et al., and Yonemura et al. describe the inactivation of pathogens in translucent blood components such as plasma and platelets by using photosensitized compounds (Estcourt LJ, Malouf R, Hopewell S, Trivellara M, Doree C, Stanworth SJ, Murphy MF), Pathogen-reduced platelets for the prevention of bleeding. Cochrane Database Syst Rev.2017;7:CD009072,doi:10.1002 / 14651858.CD009072.pub3,PubMed PMID:28756627, Jori G, Brown SB. Photosensitized inactivation of microorganisms.Photochem.Photobiol.Sci.2004;3(5):403-5,doi:10.1039 / b311904c.PubMed PMID:15122355, Magron A, Laugier J, Provost P, Boilard E. Pathogen reduction technologies: The pros and cons for platelet transfusion.Platelets.2018;29(1):2-8,doi:10.1080 / 09537104.2017.1306046, PubMed PMID:28523956, Yonemura S, Doane S, Keil S, Goodrich R, Pidcoke H, Cardoso M. Improving the safety of whole blood-derived transfusion products with a riboflavin-based pathogen reduction technology.Blood Transfus.2017;15(4):357-64,doi:10.2450 / 2017.0320-16, PubMed PMID:28665269).A significant drawback of these methods is the need for photoactivation, which limits their applications to translucent components only, preventing their use in important biological products such as whole blood or red blood cell preparations.

[0005] Alkylating agents, which inactivate pathogens or other contaminants by alkylating nucleic acids, can be used to inactivate pathogens without requiring photoactivation. The challenge of this approach is to develop compounds that effectively penetrate the cell walls, membranes, and envelopes of pathogens and possess sufficient selectivity to avoid modification of biopharmaceutical proteins. Even representative of the most selective inactivators for alkylating pathogens, such as PEN110 (N-(2-aminoethyl)aziridine) and the alkylating intercalator S303, exhibit insufficient specificity for nucleic acids and residual reactivity to other biological compounds (e.g., proteins). This could lead to the formation of nascent antigens if such alkylating agents are used to process blood products for intravenous fluids (Sobral PM et al., Viral inactivation in hemotherapy: systematic review on inactivators with action on nucleic acids. Rev Bras Hematol. Hemoter. 2012;34(3):231-235, doi:10.5581 / 1516-8484.20120056, PubMed PMID:23049426; Conlan MG et al., Antibody formation to S-303-treated RBCS in the setting of chronic RBC transfusion. Blood 2004;104(11):382). Other monoaziridine-polyamine complexes as bactericides are disclosed in U.S. Patent No. 6,617,157, and intercalators modified with alkylating moieties for selective targeting of pathogen nucleic acids are disclosed in U.S. Patents No. 6,410,219 and No. 5,691,132. The drawbacks of the disclosed structures and methods are that they do not acquire the required nucleic acid selectivity and do not avoid protein modification.

[0006] U.S. Patent No. 10,173,976, the disclosure of which is incorporated herein by reference, describes compositions and compounds having two or more aziridinyl groups linked together via a polyamine construct, which have a highly selective affinity for nucleic acids, a low tendency to modify proteins, and can inactivate pathogens, prokaryotic or eukaryotic nucleic acids (e.g., DNA and / or RNA), or prion-related nucleic acids in a sample with high selectivity.

[0007] A disadvantage of this and other common alkylating agents that target nucleic acids for use as pathogen inactivators is that residual alkylating agent compounds (e.g., in or on organisms, compositions, samples, devices, instruments, or practical items) may be toxic and can cause harm immediately after pathogen inactivation or during subsequent use. This disadvantage can be addressed by removing the antipathogenic agent after pathogen inactivation, or by inactivating it, i.e., by converting it into a less harmful or harmless substance.

[0008] U.S. Patent No. 7,293,985, the disclosure of which is incorporated by reference, describes the use of a dipeptide containing a thiol, preferably glutathione, or cysteine ​​residue, for inactivating in vitro a pathogen inactivator compound containing a nucleic acid intercalator linked to a mustard-type alkylating agent, the mustard group of which can react in situ to form an electrophile. The drawback of this method is that it does not result in sufficient inactivation of this type of nucleic acid-targeting alkylating agent, and when blood treated in this way is transfused to humans, neoplastic antigens and autoimmune side effects occur (Conlan MG et al., Antibody formation to S-303-treated RBCS in the setting of chronic RBC transfusion. Blood 2004;104(11):382).

[0009] U.S. Patent Application No. 20040137419, the disclosure of which is incorporated herein by reference, describes a method for removing positively charged antibacterial compounds, in particular PEN110, N-(2-aminoethyl)aziridine, from a treated composition by the use of a cation exchange resin.

[0010] U.S. Patent No. 6,544,727, the disclosure of which is incorporated herein by reference, describes a method and apparatus for removing psoralens and psoralen photoproducts formed after photoirradiation from blood products. The method comprises contacting psoralens and the irradiated blood product with a resin capable of adsorbing psoralens and psoralen photoproducts.

[0011] In this field, there is a need for improved pathogen inactivation methods applicable to a wide range of fields and applications, specifically, methods for pathogen inactivation that preserve proteins and other substances in the treated sample, and methods that leave little to no toxic compounds in the treated sample. [Overview of the project]

[0012] In one embodiment, the present invention provides compositions and methods for inactivating and / or reducing pathogens, microorganisms, infectious substances, such as prions, or parasites (e.g., contaminants) in a sample (including biological samples, culture media, compositions, utility items, apparatus, surfaces, organisms, etc.) by eliminating or reducing residual alkylating compound and / or its by-products following treatment with an alkylating compound. Elimination or reduction of residual alkylating compound may be carried out by treatment with a solid-phase agent that reacts with or isolates the alkylating compound, or by treatment with a solution of a neutralizing compound that eliminates or reduces the toxicity or other undesirable properties of the alkylating agent, preferably by eliminating its alkylating properties, and optionally subsequently by removing the products of the neutralization of the alkylating compound and / or excess neutralizing compound with a solid-phase agent that isolates them.

[0013] In one embodiment, the present invention relates to a method for inactivating or reducing pathogens, microorganisms, infectious substances or parasites (e.g., contaminants) in a sample, comprising (i) a compound having structure I or a plurality of compounds:

[0014] [ka]

[0015] [During the ceremony, Each R1 is independently selected from H, Cl, F, alkyl groups, CH3, CH2CH3, CH(CH3)2, alkenyl groups, phenyl groups, alkyloxy groups, acyloxy groups, or other substituted alkyl groups. Each R2 independently represents H, alkyl group, CH3, CH2CH3, CH(CH3)2, alkenyl group, phenyl group, cycloalkyl group, alkyloxy group, or substituted alkyl, alkenyl, cycloalkyl or phenyl group, or part of structure II:

[0016] [ka]

[0017] Selected from, Each R3 is independently selected from H, Cl, F, alkyl groups, CH3, CH2CH3, CH(CH3)2, alkenyl groups, phenyl groups, alkyloxy groups, acyloxy groups, or other substituted alkyl groups. n is independently 3, 4, or 5 in each occurrence. m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 in each occurrence. or treatment of the sample with a chemically acceptable salt, hydrate or solvate thereof, and (ii) Removal or reduction of any remaining compound(s) having structure I by treating with a solid phase agent that either reacts with the compound or sequesters it, or by treating with a solution of a neutralizing compound that eliminates or reduces the toxicity or other undesirable properties of the compound having structure I, preferably by eliminating its alkylating properties, and in some cases subsequently by removing the product of the neutralization of the compound having structure I and / or any excess neutralizing compound with a solid phase agent that sequesters them. The present invention provides a method that includes such a method.

[0018] The compound of structure I contains at least two aziridine groups linked by a polyamine construct that binds to nucleic acids with high affinity and inactivates them efficiently by alkylation. In addition, the compound permeates viral envelopes and / or capsids with high efficiency, is actively taken up by bacterial and eukaryotic polyamine transporters, and shows a low tendency to bind to and modify proteins. Since the compound of structure I is cytotoxic to eukaryotic cells, it needs to be detoxified or removed from the sample, composition, product or organism being treated.

[0019] In one embodiment, the method of the present invention represents converting a remaining compound of structure I into a less toxic or non-toxic compound by reaction with a neutralizing compound, thereby eliminating the alkylation properties of the compound of structure I, for example, by ring-opening of the aziridine ring. The neutralizing compound is a nucleophilic compound, such as a thiosulfate, thiophosphate, thiourea, thiocarboxylic acid, dithiocarboxylic acid, thiocarbonate O-ester, dithiocarbonate O-ester, or mercaptan, i.e., a thiol (preferably with a pK of 6-8). a It has or has a mercapto, i.e., a thiol group sp 2 or partial sp 2 It is a mercaptan (or thiol) bonded to a hybridized carbon atom.

[0020] In some cases, the products of the neutralization (also referred to as inactivation) of the compounds of Structure I, or the remaining neutralizing (inactivating) agent compounds, may themselves have an undesirable effect on the treated sample or its future use. In another embodiment, the method involves the use of a solid-phase agent that is insoluble in the treated medium and that either chemically reacts and covalently bonds with the products of neutralization and / or excess neutralizing agent compound(s), or absorbs or sequesters them, followed by removal of the solid-phase agent to remove and / or reduce the products of neutralization and / or neutralizing agent compound(s). The solid-phase agent may be a thiosulfate group (-S-SO3 - Na + ) that is functionalized with a mercaptan-type or thiol-type neutralizing agent compound and sequesters it, or a cation-exchange resin or anion-exchange resin that is a solid-phase agent that isolates the cationic or anionic neutralizing agent compound of neutralization by ion exchange, or an absorption solid-phase agent such as activated carbon that absorbs polyamines or sulfur-containing organic moieties with high affinity.

[0021] In another embodiment of the method, after treatment of a pathogen-containing sample with a compound of Structure I, the remaining compound is removed by treatment with a solid-phase agent containing a reactive group that reacts and covalently bonds with the compound(s) of Structure I, followed by removal of the solid-phase agent by filtration or other means. Examples of such reactive groups are thiosulfate, -OS(O)(O - )S - ; thiosulfonate -S(O)(O - )S - ; mercapto or thiol group, substituted mercapto or thiol group, thiourea, thiocarboxylic acid or dithiocarboxylic acid, thiocarbonic acid or dithiocarbonic acid O-ester, thiophosphonate, or thiophosphate. The pK of the thiol group aThe coefficient may be less than 9, more preferably less than 8. In another embodiment, the solid phase agent contains not only reactive groups but also other groups that enhance the reactivity of the compound of structure I without reacting with it, by protonation, by non-covalent bonding with it to increase its local concentration, or by enhancing the reactivity of the reactive group. In yet another embodiment, the solid phase agent contains a non-reactive hydrophilic group, such as polyethylene glycol, which improves its wettability in an aqueous medium and reduces its undesirable effect on components of the medium being treated.

[0022] Another embodiment characterizes the solid phase as a cation exchange resin that forms multiple ion pairs with the remaining structure I compound and thereby retains it with high efficiency.

[0023] Some embodiments provide a method for inactivating pathogens in vivo in animals or humans, in which the compound of structure I is preferably formulated and applied to the animal or human, and the neutralization or removal of the compound of structure I is carried out in vitro in a body fluid such as plasma or blood, which is then returned to the original animal or human (infusion). In another embodiment, both treatment with the compound of structure I and its removal or neutralization, as well as the possible removal of the neutralization product and neutralizing compound, are carried out in vitro in a body fluid of an animal or human, such as blood or plasma, which is preferably collected by apheresis, and then returned to the animal or human.

[0024] Furthermore, closed systems used in accordance with methods for pathogen inactivation of whole blood, red blood cells, or other blood products intended for intravenous fluid administration are also described herein. [Brief explanation of the drawing]

[0025] [Figure 1] This shows the interaction of a compound of structure I with a solid phase agent that has a nucleophilic thiol group bonded via linker L and an attached anionic sulfo group directly bonded to the polymer P matrix. [Figure 2]This describes a closed-loop whole blood processing system for whole blood collection, in which pathogen inactivation is achieved by a compound of structure I formulated together with an anticoagulant solution in a blood collection bag, and for the removal of any remaining compound of structure I by passing the blood to be processed through a cartridge containing a solid phase agent. [Figure 3] This describes a closed-loop whole blood processing system for whole blood collection, in which pathogen inactivation is achieved by a solid formulation of a structure I compound pre-filled in a blood collection bag, and for the removal of any remaining structure I compounds by passing the blood to be processed through a cartridge containing a solid phase agent. [Figure 4] This invention presents a closed-loop whole blood processing system for whole blood collection, in which pathogen inactivation is achieved by a liquid formulation of a compound of structure I, and for neutralization of residual compounds by a liquid formulation of an inactivating agent. [Figure 5] This invention describes a closed-loop whole blood processing system for whole blood collection, in which pathogen inactivation is achieved by a liquid formulation of a compound of structure I, and for the removal of any remaining compounds of structure I by passing the blood to be processed through a cartridge containing a solid phase agent. [Figure 6] This invention presents a closed-loop whole blood processing system for whole blood collection, inactivation of pathogens by a liquid formulation of a compound of structure I, neutralization of residual compounds by a liquid formulation of an inactivator, and removal of the neutralization products of the compound of structure I by a solid-phase agent. [Figure 7] This describes a closed-loop whole blood processing system for whole blood collection, inactivation of pathogens by a liquid formulation of a structure I compound, removal of residual structure I compounds by a solid phase agent, leukocyte filtration, and separation of red blood cell concentrate (RBCC) and plasma from the leukocyte-depleted blood. [Figure 8] This invention describes a closed-loop whole blood processing system for whole blood collection, leukocyte filtration in which pathogen inactivation is achieved by a liquid formulation of a structure I compound, removal of residual structure I compounds by a solid phase agent, and separation of red blood cell concentrate (RBCC) and plasma from the blood to be processed. [Figure 9]This invention describes a closed-loop whole blood processing system for whole blood collection, pathogen inactivation with a liquid formulation of structure I compounds, two-step removal of residual structure I compounds as free beads or with a solid phase agent pre-filled in a semipermeable material, leukocyte filtration, and separation of erythrocyte concentrate (RBCC) and plasma from leukocyte-depleted blood. [Figure 10] This invention describes a closed-loop whole blood processing system for whole blood collection, pathogen inactivation with a solid formulation of a compound of structure I, and neutralization of residual compounds with a liquid formulation of an inactivator. [Figure 11] The diagram shows a container containing a solid formulation of a compound of structure I, where the container for the solvent used to dissolve the formulation is connected by a destructible seal, and the container containing the sample to be processed is connected by another destructible seal. [Figure 12] This shows a closed system for sterile pre-humidification of a solid phase agent filled in a cartridge. [Figure 13] This describes a closed system for rinsing solid phase agents before their use. The system is incorporated into a closed system for sample processing under sterile conditions. [Figure 14] The HPLC analysis of 10 μM 21-mer oligodeoxyribonucleotides (5' ATA CCT CAT GGT AAT CCT GTT 3') incubated with 200 μM compound X in PBS (pH 6.7) for 0 hours (top) and 6 hours (bottom) at 37°C is shown. [Figure 15] The mass spectrometry of a 100 μM 23-mer oligonucleotide in PBS is shown before (upper spectrum) and 6 minutes after (lower spectrum) the addition of compound X (100 μM). The observed ions (m / z 1845.22 and 1933.54) have a charge state of -4 and correspond to neutral molecules with masses of 7384.9 Da (oligonucleotide, calculated mass 7384.0 Da) and 7738.2 Da (covalent adduct of oligonucleotide with compound X, calculated mass 7737.3 Da). [Figure 16]The ESI+ mass spectrometry results for 8 μM cytochrome C after incubation at 40°C for 30 hours with compound X (top, 1 mM; middle, 100 μM; bottom, without compound X, control) are shown. The MS peaks, from right to left, correspond to the 7x, 8x, 9x, and 10x positively charged molecular ions of cytochrome C. [Figure 17] This shows that the anti-F protein mAbs bound to compounds VI and X inactivated respiratory syncytial virus (RSV). A: Binding of mAbs to untreated (Ctr) and RSV inactivated with 100 μM of compound VI or compound X (all incubated at 40°C for 4 hours). B: Binding of mAb D25 to untreated (Ctr) and RSV inactivated with 100 or 500 μM of compound VI (all incubated at room temperature for 6 hours). [Figure 18] The kinetics of the neutralization of compound X by ethyl 2-mercapto in PBS at room temperature are shown. The concentration of compound X decreases with a first-order rate constant of 0.022 min⁻¹, and the concentration of intermediate Q1 XXI decreases with a first-order rate constant of 0.026 min⁻¹. [Figure 19] The logarithmic plot of the concentration of compound VI during incubation with 1 mM sodium thiosulfate is shown. [Figure 20] The plots show the rate of neutralization of compound X. A shows the rate of neutralization of compound X, as well as the rates of formation of compounds XXIV and XXV. B shows a logarithmic plot of the concentration of compound X, which reveals a linear dependence and demonstrates a first-order reaction kinetics with a first-order rate constant K = -0.0416 min⁻¹, corresponding to the half-life of compound X T1 / 2 = 16.6 minutes. [Figure 21] The left panel shows the mass chromatogram of the LC-MS analysis of the neutralization of compound X with thiophenol after incubation for 100 seconds. The right panel shows the mass spectra of the peaks corresponding to compound X and its neutralization products XXVI and XXVII. The analysis reveals that compound X is neutralized to a considerable extent after 100 seconds. [Figure 22A]This shows the effects of dummy-treated and compound VI-treated serum on the growth of four different cell lines in a 48-well plate over a period of 6-7 days. A. Porcine PT cells; B. Human A172 cells; C. Human MCF-7 cells; D. Bovine BTT cells grown in FBS-containing medium; E. Bovine BTT cells grown in HS-containing medium. The T0 column represents the cell count at seeding. The first of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with untreated control serum; the second of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with dummy-treated serum; and the third of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with compound VI-treated serum. Each time point represents the mean of the three wells. Error bars represent the standard deviation. [Figure 22B] This shows the effects of dummy-treated and compound VI-treated serum on the growth of four different cell lines in a 48-well plate over a period of 6-7 days. A. Porcine PT cells; B. Human A172 cells; C. Human MCF-7 cells; D. Bovine BTT cells grown in FBS-containing medium; E. Bovine BTT cells grown in HS-containing medium. The T0 column represents the cell count at seeding. The first of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with untreated control serum; the second of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with dummy-treated serum; and the third of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with compound VI-treated serum. Each time point represents the mean of the three wells. Error bars represent the standard deviation. [Figure 22C]This shows the effects of dummy-treated and compound VI-treated serum on the growth of four different cell lines in a 48-well plate over a period of 6-7 days. A. Porcine PT cells; B. Human A172 cells; C. Human MCF-7 cells; D. Bovine BTT cells grown in FBS-containing medium; E. Bovine BTT cells grown in HS-containing medium. The T0 column represents the cell count at seeding. The first of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with untreated control serum; the second of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with dummy-treated serum; and the third of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with compound VI-treated serum. Each time point represents the mean of the three wells. Error bars represent the standard deviation. [Figure 22D] This shows the effects of dummy-treated and compound VI-treated serum on the growth of four different cell lines in a 48-well plate over a period of 6-7 days. A. Porcine PT cells; B. Human A172 cells; C. Human MCF-7 cells; D. Bovine BTT cells grown in FBS-containing medium; E. Bovine BTT cells grown in HS-containing medium. The T0 column represents the cell count at seeding. The first of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with untreated control serum; the second of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with dummy-treated serum; and the third of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with compound VI-treated serum. Each time point represents the mean of the three wells. Error bars represent the standard deviation. [Figure 22E]This shows the effects of dummy-treated and compound VI-treated serum on the growth of four different cell lines in a 48-well plate over a period of 6-7 days. A. Porcine PT cells; B. Human A172 cells; C. Human MCF-7 cells; D. Bovine BTT cells grown in FBS-containing medium; E. Bovine BTT cells grown in HS-containing medium. The T0 column represents the cell count at seeding. The first of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with untreated control serum; the second of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with dummy-treated serum; and the third of three columns (for days 1-7) represents the number of cells in the well containing medium supplemented with compound VI-treated serum. Each time point represents the mean of the three wells. Error bars represent the standard deviation. [Modes for carrying out the invention]

[0026] As used herein, the term “sample” means culture media, compositions, products, apparatus, practical items, or organisms, which may be prokaryotes, monocellular or multicellular eukaryotes, plants, animals, blood or blood products, body fluids, culture media of eukaryotic or prokaryotic origin, vaccine formulations, biologics or biological preparations, clinical samples, biopsy materials, research samples, cosmetics, pharmaceutical compositions, consumables, equipment, underwater fluid conduits, pipes, hoses, heat exchangers or surface vessels, and their surfaces.

[0027] The terms neutralizer, neutralizer compound, or neutralizer agent, when used in the context of a compound of structure I, generally refer to a molecule that can react with the aziridinyl group of a compound of structure I in a sample to open its ring.

[0028] As used in the context of the methods described herein, the term “solid-phase agent” is defined as a solid used to remove from a sample a compound of structure I that is insoluble in the sample medium, or a product of the inactivation of a compound of structure I, or a product of the chemical transformation or decomposition of a compound of structure I, or a neutralizing agent.

[0029] As used herein, the term “contaminant” refers to viruses, bacteria or any other microorganism, prions, or pathogens including fungi, protozoa, unicellular or multicellular parasites, eukaryotes including but not limited to parasitic helminths, schistosomiasis or nematodes or their eggs, unicellular or multicellular eukaryotes, unicellular or multicellular algae, and crustaceans, or any other undesirable organism or infectious material. As used herein, the term “contaminant” may also refer to undesirable biological structures including, but not limited to, bacterial biofilms or other microbial biofilms, lichens, foulings or biofouling deposits.

[0030] The present invention relates to treatment with a compound of structure I, and the subsequent remaining compound of structure I:

[0031] [ka]

[0032] [During the ceremony, Each R1 is independently selected from H, CH3, CH2CH3, CH(CH3)2, Cl, F, alkyl group, alkenyl group, phenyl group, alkyloxy group, acyloxy group, or substituted alkyl group. Each R2 independently represents H, CH3, CH2CH3, CH(CH3)2, alkyl group, alkenyl group, phenyl group, cycloalkyl group, alkyloxy group, or substituted alkyl, substituted alkenyl, substituted cycloalkyl or substituted phenyl group, or part of structure II:

[0033] [ka]

[0034] Selected from, Each R3 is independently selected from H, CH3, CH2CH3, CH(CH3)2, Cl, F, alkyl group, alkenyl group, phenyl group, alkyloxy group, acyloxy group, or other substituted alkyl groups. Each instance of n is independently 3, 4, or 5. Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 in each occurrence. The present invention provides a method for inactivating / reducing contaminants in a sample by removing or neutralizing (deactivating) a chemically acceptable salt, hydrate, or solvate thereof.

[0035] In some embodiments, the compound of structure I has structure IA:

[0036] [ka]

[0037] [During the ceremony, Each R2 independently represents H, alkyl group, CH3, CH2CH3, CH(CH3)2, alkenyl group, phenyl group, cycloalkyl group, alkyloxy group, or substituted alkyl, alkenyl, cycloalkyl, phenyl group, or part of structure IIA:

[0038] [ka]

[0039] Selected from, Each R3 is independently selected from H, Cl, F, alkyl group, CH3, CH2CH3, CH(CH3)2, alkenyl group, phenyl group, alkyloxy group, acyloxy group, or substituted alkyl group. Each instance of 'a' is independently selected from 1, 2, or 3. Each instance of b is independently selected from 0, 1, 2, 3, 4, 5, or 6. It may have.

[0040] In some embodiments, the compound of structure I has structure IB:

[0041] [ka]

[0042] [During the ceremony, Each R2 is independently selected from H, CH3, CH2CH3, or CH(CH3)2 each time it appears. Each R3 is independently selected from H, CH3, CH2CH3, or CH(CH3)2 each time it appears. Each instance of 'a' is independently selected from 1, 2, or 3. [b is selected from 0, 1, 2, 3, 4, 5, or 6] It may have.

[0043] The term "alkyl" refers to the radical of a saturated aliphatic group, including linear and branched alkyl groups. In preferred embodiments, linear or branched alkyl groups have fewer than six carbon atoms in their main chain (e.g., C1-C6 for linear types, C3-C6 for branched types). Preferred alkyl groups include CH3, CH2CH3, CH2CH2CH3, or CH(CH3)2.

[0044] The term "substituted alkyl" refers to alkyl groups listed above that are independently substituted with 1 to 3 substituents selected from the group consisting of F, Cl, OH, OCH3, OCH2CH3, OCH(CH3)2, OC(CH3)3, OC6H5, and OCOCH3.

[0045] The term "cycloalkyl" refers to a saturated carbocyclic group having 3 to 6 carbon atoms in the ring. Preferred cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0046] The term "alkenyl group" refers to radicals of unsaturated aliphatic groups, including linear and branched alkenyl groups, that have 1 to 3 double bonds. In preferred embodiments, linear or branched alkenyls have fewer than 6 carbon atoms in their main chain (e.g., C2-C6 for linear alkenyls and C3-C6 for branched alkenyls).

[0047] The term "substituted alkenyl" refers to the alkenyl groups shown above that are independently substituted with 1 to 3 substituents selected from the group consisting of F, Cl, OH, OCH3, OCH2CH3, OCH(CH3)2, OC(CH3)3, OC6H5, and OCOCH3.

[0048] The term "substituted phenyl" refers to a phenyl group that is substituted with one to three substituents independently selected from the group consisting of F, Cl, OH, OCH3, OCH2CH3, OCH(CH3)2, OC(CH3)3, OC6H5, and OCOCH3.

[0049] The term "alkyloxy group" refers to an alkyl group defined above, which is bonded by an oxygen atom. Representative alkyloxy groups include methoxy, ethoxy, propyloxy, and tert-butoxy.

[0050] The term "acyloxy group" refers to a group having the structure -O-(C=O)-R, where R is the alkyl group or substituted alkyl group shown above.

[0051] As used herein, each expression, such as alkyl, m, n, R1, R2, R3, etc., is intended to be independent of its definition anywhere else in any given structure if it appears more than once in that structure.

[0052] It should be understood that the terms "substituted" or "substituted with" implicitly include the condition that such substitutions are subject to the acceptable valencies of the substituted atom and substituent, and that the substitution results in a stable compound, such as one that does not spontaneously undergo transformations by rearrangement, cyclization, elimination, etc.

[0053] As described above, in certain embodiments, the compound of structure I exists as a salt. Preferred salts are relatively non-toxic inorganic and organic acid addition salts of the compound of structure I. These salts may be prepared in situ in the administration vehicle, or separately by reacting a purified compound of structure I in the form of a free base with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Typical salts include hydrogen bromide, hydrogen chloride, sulfates, bisulfates, phosphates, perchlorates, tetrafluoroborates, nitrates, acetates, valers, oleates, palmitates, stearates, laurates, benzoates, lactates, phosphates, tosylates, citrates, maleates, fumarates, succinates, tartrates, napthylates, methanesulfonates, glucoheptonates, lactobionates, and laurylsulfonates (see, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19). Low nucleophilicity of the anion is preferable, such as sulfates, perchlorates, methanesulfonates, or tetrafluoroborates.

[0054] Compounds of structure I have polyamine properties, possessing two or more aziridinyl groups at their terminals. These compounds have multiple aliphatic nitrogen atoms, each capable of being positively charged in vitro or in vivo. Due to their polycationic nature and appropriate spacing between positive charges, the compounds selectively bind to polyanionic nucleic acids, preferably alkylating them at the guanine N7 position. This results in crosslinking, effectively inactivating the pathogen genome, eliminating the pathogen's infectivity, or killing the organism.

[0055] Compounds having structure I can be synthesized by the methods disclosed herein. The following schemes, for example, the synthesis of compositions and compounds, are shown for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art will readily recognize different chemical methods and synthesis schemes for compounds of structure I.

[0056] The synthesis method for the compound of structure I is shown in the following scheme.

[0057] Scheme 1 shows the method for preparing compound IV:

[0058] [ka]

[0059] Scheme 2 shows the method for preparing compound VI:

[0060] [ka]

[0061] Scheme 3 shows the method for preparing compound X:

[0062] [ka]

[0063] Scheme 4 shows the method for preparing compound VI:

[0064] [ka]

[0065] Scheme 5 shows the method for preparing compound VI:

[0066] [ka]

[0067] Generally, compounds of structure I are viscous oils and readily soluble in water, aqueous buffers, and organic solvents. They can be converted to salt forms when treated with acid. When a solution containing it in a nonpolar aprotic solvent such as ether is treated with a stoichiometric amount of acid anhydride, preferably at a low temperature, the salt can precipitate and be isolated by filtration. In some embodiments of the present invention, the salt form is used in place of the free base in oily form for long-term storage.

[0068] A solution of the free base of a compound of structure I is alkaline and can absorb carbon dioxide from the atmosphere, which can reduce the stability of the solution and accelerate its hydrolysis or other decomposition. The free base of a compound of structure I can be stabilized by the addition of a small amount of a basic compound, such as sodium hydroxide. For example, a glycerol solution of compound X is significantly stabilized for long-term storage by the addition of 0.1% sodium hydroxide.

[0069] Compounds of structure I can be converted into solid solutions by rapid solidification through cooling of a solution containing the compound in which it is solid at room temperature. For example, if compound VI is added to molten polyethylene glycol in an amount of 3% or less, and the resulting solution is rapidly cooled, preferably in a thin film, a solid solution of compound VI is formed. This solution has significantly higher storage stability than compound VI in its original form. The stability of the solid solution can be further enhanced by the addition of a small amount of strong base, such as sodium hydroxide. A preferred solid for preparing a solid solution of a compound of structure I has a melting point higher than 40°C and lower than 120°C, is readily soluble in aqueous media, is chemically neutral, and does not have adverse effects on the sample being processed or its intended use.

[0070] The experiments and data of the inventors shown in the examples of the present invention demonstrate that representative compounds of structure I rapidly form covalent adducts with RNA and DNA oligonucleotides, inactivating a variety of high-titer pathogens (enveloped and non-enveloped, DNA and RNA viruses, G+ and G- bacteria, mycoplasmas, fungi and protozoa) at low concentrations (100-500 μM) and various temperatures (20-40°C) in various media such as growth media, whole blood, concentrated red blood cells, plasma and serum.

[0071] According to the method of the present invention, contaminants in a sample are treated with a compound of structure I in its original form, or with a composition containing one or more compounds of structure I. In this case, the composition may be formulated as a liquid, solution, gel, solid, powder, or particles, or it may be encapsulated, dissolved, dispersed, pulverized, micronized or converted into nanoparticles, or it may be in other formulation forms, or a combination thereof. The solvent for the composition of compounds of structure I may be water, an aqueous buffer or aqueous salt solution, an organic solvent, for example, but not limited to, dimethyl sulfoxide, dimethylacetamide, ethanol, isopropanol, acetone, polyethylene glycol(s) of various molecular weights, glycerol, propylene glycol, benzyl alcohol or mixtures thereof, liquefied gas, or mixtures thereof. The solvent may contain various organic or inorganic additives, stabilizers, activators or auxiliaries.

[0072] In embodiments of the present invention, a sample containing contaminants is treated with one or more compounds of structure I at a temperature of 0 to 100°C, preferably 10 to 60°C, more preferably 20 to 40°C, for a period of 30 seconds to 72 hours, preferably 20 minutes to 24 hours, more preferably 60 minutes to 8 hours, at a pH of 1 to 14, preferably 4 to 9, more preferably 6 to 8, and at a concentration of 10 nM to 10 mM, preferably 1 μM to 1 mM, more preferably 100 μM to 500 μM.

[0073] The contaminant inactivation effect of a compound(s) of structure I increases with increasing concentration, dose or volume, treatment time, and temperature. On the other hand, the potential undesirable effects on the sample being treated may also increase with compound concentration, dose or volume, treatment time, and temperature. Users of the method can determine the optimal concentration, dose or volume, treatment time, and temperature of the compound(s) of structure I based on the type and characteristics of the medium being treated, the nature and type of pathogens or undesirable organisms present therein, and the desired level of their inactivation. For example, a temperature-stable utility, such as a biofouling heat exchanger, can be treated at a high temperature, e.g., above 60°C, for a long period, e.g., 24 hours or more. On the other hand, the optimal treatment temperature for a delicate sample, such as a platelet concentrate, may be room temperature, and the treatment time may be limited to 1-2 hours or less, while for a heat-resistant sample, such as heat-treated animal serum, the optimal temperature may be above 40°C with a treatment time of 1-6 hours. Users may experimentally determine the optimal concentration, dose, or amount, processing time, and temperature of the compound(s) of structure I, using the methods disclosed herein and similar methods known to those skilled in the art.

[0074] The optimal treatment parameters (concentration, time, temperature) may depend not only on the characteristics of the sample being treated and the nature of any pathogens or other undesirable organisms present within it, but also on the desired degree of inactivation / reduction, which can be determined by the intended use of the sample. For example, if the sample being treated is animal serum intended for use as a supplement to cell growth media, the required level of virus capable of infecting cells may be less than one infectious particle per dose used, which may require a reduction / inactivation level greater than 9 logarithmically. On the other hand, if the product being treated is industrial piping intended to prevent biofilm formation or biocontamination, a microbial reduction of 1 or 2 logarithmically may be sufficient.

[0075] The present invention provides a method for sterilizing a sample to be treated by 50% to 100% or less, by selecting one compound of structure I and processing parameters (concentration / dosage / volume, time, temperature, pH, formulation composition), thereby inactivating one pathogen or undesirable organism, and in some cases all pathogens or undesirable organisms present in the sample.

[0076] On the other hand, the structure, mechanism of action, and the inventors' experiments suggest that the compound of structure I is cytotoxic and must be removed or its cytotoxicity eliminated for the safe use of the treated sample or for the safety of the treated organism.

[0077] In some embodiments of the present invention, the alkylation properties of a compound of structure I, and therefore the cytotoxicity resulting from such alkylation properties, are used to target a sample containing residual compounds of structure I with nucleophilic small molecules or ions, for example, but not limited to, thiosulfates, preferably sodium thiosulfate; thiophosphates, preferably sodium thiophosphate; thioureas or substituted thioureas, for example monomethyl-, N,N- or N,N'-dimethyl-, trimethyl- or tetramethylthiourea; thiocarboxylic acids, for example thioacetic acid (CH3C(O)SH), thiopropionic acid, thiooxalic acid, thiomalonic acid, thiosuccinic acid; dithiocarboxylic acids, for example dithioacetic acid (CH3C(S)SH); thiocarbonate O-esters, for example ethyl thiocarbonate; dithiocarbonate O-esters, for example ethyl dithiocarbonate; or mercaptans, i.e., thiols, for example, but not limited to However, these can be reduced or removed by treatment with 2-mercaptoethanol, 3-mercaptopropane-1,2-diol (1-thioglycerol), 2-thioglycerol, 1,2- or 1,3-dithioglycerol, 2-aminoethanethiol, 2-(methylamino)ethanethiol, 2-(dimethylamino)ethanethiol, 2-mercapto-N,N,N-trimethylethaneaminium salt, (methylsulfonyl)methanethiol, (ethylsulfonyl)methanethiol, sulfonyl dimethanethiol, thioglycolic acid (HSCH2CO2H), 2-mercaptosuccinic acid, aromatic or heterocyclic thiols, such as thiophenol, furan-2-thiol, 2-thiopyridine, 1H-imidazole-2-thiol, 1H-imidazole-5-thiol, thiobarbituric acid, thiosalicylic acid, or 4-mercaptobenzoic acid. Some examples of preferred thiol compounds are shown below:

[0078] [ka]

[0079] As shown in the examples, nucleophilic small molecules react with compounds having structure I by opening their aziridine ring, and thus eliminating their ability to alkylate nucleic acids. The rate of this reaction depends on temperature, pH, and concentration, as well as the nucleophilicity of the nucleophilic small molecule.

[0080] The nucleophilicity of thiols increases significantly with deprotonation, and therefore their nucleophilicity is mainly due to the deprotonated anionic form of the thiol (Danehy, JP; Noel, CJ. The Relative Nucleophilic Character of Several Mercaptans toward Ethylene Oxide. Journal of the American Chemical Society 1960, 82, 2511-2515). In general, the nucleophilicity of anionic nucleophiles of the same class, especially thiol nucleophiles, increases with their basicity, that is, with higher pK. a Nucleophiles having a lower pK a It will have a more nucleophilic anionic form compared to nucleophiles that have a higher pK. In addition, the concentration of the deprotonated (anionic) form of the nucleophile will be the same as the pK of the nucleophile. a The pK of the nucleophile decreases as the difference between it and the pH of the culture medium increases, that is, the pK of the nucleophile. a It decreases as the pH of the medium rises above the pH of the medium.

[0081] In some embodiments of the present invention, a preferred thiol-type neutralizing agent for the compound of structure I is a pK close to the pH of the medium in which inactivation occurs. a Therefore, if neutralization occurs at or near pH 7, the preferred thiol-type neutralizing agent has a pK close to 7. a This has the property that the nucleophilicity of the anionic form of the neutralizing agent increases with increasing basicity, and that when the concentration of the anionic form exceeds the pH of the medium, its pK a The best compromise is found between the decrease as the concentration increases. This teaching is based on the fact that the half-life of a representative compound of structure I having formula X is 10 mM thiophenol (pK aIn contrast to the half-life of the same compound under the same conditions, which was measured to be less than 1 minute in the presence of glutathione (pK of the SH group) (pK = 6.52), the half-life of the same compound was measured to be less than 1 minute in the presence of glutathione (pK of the SH group). a This is indicated by the inventors' experiment, which took 450 minutes in the presence of (=8.75).

[0082] In another embodiment of the present invention, preferred thiol-type neutralizing agents of the compound of structure I are those that are part of a double bond or aromatic ring system or are complete or partial sp 2 It has a thiol group directly bonded to a carbon atom that has a hybridization of the type.

[0083] In yet another embodiment, a preferred thiol-type neutralizing agent of the compound of structure I has at least one electron-accepting group, for example, a sulfone group (-S(O2)-R), a sulfoxide group (-S(O)-R), an ester group (-C(O)OR), or an amide group (-C(O)NH2, -C(O)NHR, -C(O)NR2), where R is any alkyl or substituted alkyl group, and the electron-accepting group is bonded to the carbon atom to which the SH group is bonded.

[0084] In some embodiments of the present invention, a compound(s) of structure I remaining on a sample, composition, surface, apparatus, or organism is neutralized by contacting it with a neutralizing agent compound(s) or a solution containing the neutralizing agent compound(s) in a suitable solvent(s), such as water, aqueous buffer or salt solution, organic solvent(s), such as dimethyl sulfoxide, dimethylacetamide, ethanol, isopropanol, acetone, polyethylene glycol(s) of various molecular weights, glycerol, propylene glycol, benzyl alcohol, or a mixture thereof, for a period of time required to achieve the desired neutralization or degree of neutralization, preferably less than 72 hours, more preferably less than 24 hours, even more preferably less than 8 hours, and even more preferably less than 4 hours, at a temperature of 0 to 100°C, preferably 10 to 60°C, even more preferably 20 to 40°C, and at a pH of 1 to 14, preferably 4 to 9, even more preferably 6 to 8. The concentration of the neutralizing agent compound in the sample to be treated may be 1 M or less, preferably 0.1 M or less, and more preferably 10 mM or less.

[0085] It is understood that the optimal conditions for the fastest and most efficient neutralization of a compound of structure I remaining in the medium being treated vary and depend on the type of medium and the type of neutralizing compound, and that their rational selection and experimental optimization can be achieved by those skilled in the art using experimental methods disclosed herein or similar to those disclosed herein.

[0086] The desirable degree of neutralization, or reduction in the amount of remaining structure I compound(s), is less than 50%, preferably more than 2 times, more preferably more than 10 times, i.e., more than 1 logarithmically, more preferably more than 2 logarithmically, even more preferably at least 3 logarithmically, even more preferably at least 4 logarithmicly, even more preferably at least 5 logarithmicly, even more preferably at least 6 logarithmicly, even more preferably at least 7 logarithmicly, even more preferably at least 8 logarithmicly, even more preferably at least 9 logarithmicly, even more preferably at least 10 logarithmicly or more.

[0087] In some embodiments of the present invention, the neutralization products of compounds having structure I, i.e., the products of their reaction with neutralizing compound(s), or the products of the reaction between compounds having structure I and components of the sample to be treated, may have properties undesirable for the intended use. In other cases, the neutralizing compound may have properties undesirable. In all of these cases, the neutralization products or reaction products, or the neutralizing compound, may be removed from the sample to be treated or their amounts reduced by treatment with a solid phase agent insoluble in the medium to be treated, which chemically reacts with and covalently bonds, absorbs, or otherwise sequesters the neutralization or reaction products of the compounds having structure I and / or the neutralizing compound(s). After treatment, the solid phase agent may be removed from the medium to be treated by filtration, centrifugation, sedimentation, or other suitable physical means. Alternatively, the solid phase agent may be brought into contact with the medium to be treated via a membrane, bag, or other suitable physical barrier that allows the neutralization product or reaction product of the compound(s) of structure I together with the components of the sample to be treated, or that allows the neutralizing compound(s) to permeate but not the solid phase agent.

[0088] The solid phase may be a microporous, macroporous, or gel-like porous organic polymer, or any highly porous solid of organic or inorganic type, such as, but not limited to, amorphous carbon, activated carbon, charcoal, silica gel, titania, or zirconia, or it may be a nonporous solid with small particle size exhibiting high dispersibility, i.e., a high surface-to-volume ratio. The solid phase may also be of a mixed type, such as solid nonporous particles covered with a layer of porous material.

[0089] The organic polymer is preferably crosslinked and may be a polystyrene polymer, or a polyacrylate polymer, or a polymethacrylate polymer, or a polyurethane polymer, or a polyamide polymer, or a dextran polymer, for example, but not limited to Sephadex®, or an agarose polymer, for example, but not limited to Sepharose®, or a cellulose polymer, or a modified cellulose polymer, for example, but not limited to carboxymethylcellulose or diethylaminoethylcellulose or methylcellulose, or other polysaccharides, or any other linear, branched or crosslinked homopolymer or heteropolymer or block copolymer having an iso-type or atactic-type configuration or other stereoregularity, or any other suitable macromolecule insoluble in the medium being treated.

[0090] For treatment in aqueous media, hydrophilic organic polymers, or polymers that can be wetted, expanded, or swollen in aqueous media, are highly preferred.

[0091] In some embodiments, the solid phase agent chemically reacts or covalently bonds with the neutralization or reaction product of the compound(s) of structure I, and / or the neutralizing compound(s). For example, epoxy-modified resins, e.g., epoxy-modified polyacrylate resins, e.g., Lifetech® ECR8215M, or epoxy-modified agarose resins, e.g., Praesto® Epoxy300, both manufactured by Purolite Ltd, Bala Cynwyd, PA, USA, readily react with nucleophilic compounds, particularly those used as neutralizing agents for the compounds of structure I in this disclosure, e.g., sodium thiosulfate, disclosed by Axen et al. in Preparation of modified agarose gels containing thiol groups, Acta Chem. Scand. B 1975, 29, 471. In this reaction, the nucleophilic neutralizing agent opens the epoxy ring and covalently bonds to the polymer molecule. Another example is a polymer functionalized with a functional group containing an electrophilic sulfur atom, such as S-methanesulfonate (PSS(O2)CH3, where P represents the polymer molecule) or S-thiosulfate ester (PSS(O2)CH3) - M + (In the formula, P represents a polymer molecule and M represents a metal cation) The reaction is: (PSS(O2)O - M + + RSH → PS-SR + M + SO3 2- Accordingly, thiols, for example, compounds of the thiol type with structure I, readily react with neutralizing agents to form a disulfide bond between the thiol-type neutralizing agent and the polymer. Polymers of that kind, their preparation and reactions are disclosed by Roth and Theato in RSC Polymer Chemistry, Ser. 6 (2013): Thiol-X in Polymer and Material Science, Chapter 4: Thiol-Thiosulfonate Chemistry in Polymer Science, pages 76-94 and the references cited therein, all of which are incorporated herein by reference. If the sample to be treated contains proteins or other macromolecules that can react with the electrophilic functional groups of the solid phase agent, the solid phase agent is brought into contact with the matrix via a semipermeable membrane, for example, a dialysis membrane with a cutoff of 1000-10000 Da, which allows small molecules to permeate but not macromolecules.

[0092] In another embodiment, the solid phase absorbs the neutralization product, decomposition product, or reaction product with matrix components of the compound(s) of structure I and / or the neutralizing compound(s). Examples of such solid phases include activated carbon or charcoal, which absorb polyamine-type compounds with high affinity (Cohen, SS, A Guide to the Polyamines, Oxford Univ. Press, 1988) and also absorb sulfur-containing organic compounds, such as thiol-type neutralizing agents, such as, but not limited to, thiophenol, thioanisole, furan-2-thiol, thiosalicylic acid, 4-thiobenzoic acid, dithioacetic acid, or thioglycolic acid, with high affinity.

[0093] In another embodiment, the solid phase absorbs the neutralization or reaction products of the compound(s) of structure I by forming multiple ion pairs with them. The compound(s) of structure I, the neutralization products, and the decomposition or reaction products with matrix components have multiple (more than three) aliphatic nitrogen atoms, which are protonated at neutral or acidic pH. As a result, the compounds become polycationic, meaning they have three or more positive charges at neutral, near-neutral, or acidic pH.

[0094] Solid phase agents containing multiple negatively charged groups can form multiple ion pairs with polycationic compounds and absorb them through electrostatic interactions. Such solid phase agents may be cation exchange resins, such as strong cation exchange resins, preferably containing sulfo or sulfate groups, or weak cation exchange resins, preferably containing carboxyl groups. Examples of such cation exchange resins include Dowex® 50X2-200, Dow Chemicals' Amberlite® IR-120, or Purolite's NRW160.

[0095] The exchangeable cations bound to the cation exchange resin are selected to be suitable for or not adversely affect the sample or its application, with sodium being preferred for biological materials. The ion exchange capacity of the resin should be at least 0.01 meq / ml, preferably at least 0.1 meq / ml, and more preferably at least 1 meq / ml.

[0096] Numerous types of cation exchange resins exist, based on various polymer species, degrees of crosslinking, degree of functionalization, porosity, purity, and degree of exudative release. Those skilled in the art can select an ion exchange resin that is compatible with the medium to be treated, does not exhibit harmful effects, and has a high degree of functionalization and retention of neutralized compounds.

[0097] In another embodiment of the present invention, any excess of an anionic neutralizing agent of the compound of structure I, such as thiosulfate, thiophosphate, thiocarboxylic acid, thioacetate, thioglycolate, thiol acetate, dithiocarboxylic acid salt, 2-mercaptoacetate, 2-mercaptosuccinate, 2-mercaptopropionate, thiosalicylic acid, or 4-mercaptobenzoic acid, is removed from the sample or medium by the use of a solid phase agent covalently bonded to multiple cationic groups, such as an anion exchange resin. The anion exchanger may be weak, but is preferably a strong anion exchanger, and is bonded to a suitable anionic group that is compatible with and does not adversely affect the sample and its properties, such as a primary, secondary, or tertiary amine group or a quaternary ammonium group forming an ion pair with a chloride, sulfate, succinate, lactate, or other cationic group, though not limited to these.

[0098] In one embodiment of the present invention, after inactivating contaminants by treatment with a compound of structure I, the remaining compound(s) of structure I are removed from the sample by treatment with a solid-phase agent that reacts with and covalently bonds with the compound(s) of structure I. The solid-phase agent may contain a reactive group that reacts with the aziridine ring(s) of the compound(s) of structure I to open their rings. The solid-phase agent has a general structure XVII:

[0099] [ka]

[0100] It has, in the formula, Q is a reactive group that undergoes chemical reactions and covalent bonding with one or more compounds of structure I. P is a solid-phase matrix, which may be a microporous, macroporous, or gel-like porous organic polymer, or it may be any highly porous solid of organic or inorganic type, for example, amorphous carbon, activated carbon, charcoal, silica gel, titania, zirconia, etc., or it may be a non-porous solid with small particle size exhibiting high dispersibility, i.e., a high surface-to-volume ratio, or it may be a mixed type, for example, solid non-porous particles covered with a layer of porous material.

[0101] The organic polymer is preferably crosslinked and may be a polystyrene polymer, or a polyacrylate polymer, or a polymethacrylate polymer, or a polyurethane polymer, or a polyamide polymer, or a dextran polymer, for example, but not limited to Sephadex®, or an agarose polymer, for example, but not limited to Sepharose®, or a cellulose polymer, or a modified cellulose polymer, for example, but not limited to carboxymethylcellulose or diethylaminoethylcellulose or methylcellulose, or other polysaccharides, or any other linear, branched or crosslinked homopolymer or heteropolymer or block copolymer having an iso-type or atactic-type configuration or other stereoregularity, or any other suitable macromolecule insoluble in the medium being treated.

[0102] For treatment in aqueous media, hydrophilic organic polymers, or polymers that can be wetted, expanded, or swollen in aqueous media, are highly preferred.

[0103] The reactive group Q is preferably a nucleophilic group, for example, but not limited to, thiosulfate -OS(O)(O - )S - , or thiosulfonate -S(O)(O - )S -, or mercapto, i.e., thiol groups -SH, -CH2SH, -CH2CH2SH, -CF2CH2SH, -OCH2CH2SH, -NH2CH2CH2SH, -NH(Me)CH2CH2SH, -N(Me2)CH2CH2SH, -COCH2SH, -S(O2)CH2SH, thiourea -NHC(S)NH2, or substituted thiourea groups, thiocarboxylic acid -C(O)S - , dithiocarboxylic acid -C(S)S - , thiocarbonate O-ester -OC(O)S - Dithiocarbonate O-ester or xanthaute -OC(S)S - These are thiophosphonates -PO(OH)SH, thiophosphates -OPO(OH)SH, o-, m-, or p-thiophenyl groups -C6H4SH, thiosalilate groups, m-, or p-thiobenzoate groups -O2CC6H4SH, or salts thereof.

[0104] In preferred embodiments, Q is a double bond, or an aromatic ring structure, or a complete or partial sp 2 It is an -SH group directly linked to a hybridized carbon atom.

[0105] In another preferred embodiment, the -SH group is -S - and H + pK of dissociation to a The value is less than 10, preferably less than 9, and most preferably less than 8.

[0106] In another embodiment, the solid phase agent has the general structure XVIII:

[0107] [ka]

[0108] It has, in the formula, P and Q are as shown in XVII, and L is a linker or branched linker linking group Q to the solid-phase matrix P. L can be linear, branched, or dendrimer, and may contain one or more Q groups bonded thereto. Examples of L are divalent atoms, or linearly linked groups of atoms which may be the same or different as matrix P and one of more groups Q, and which may or may not be bonded to other atoms or groups of atoms. Specific examples of L may be oxygen or sulfur atoms, imino (NH) groups, methylene, ethylene, propylene, ethoxyethylene groups, oligo or polyoxyethylene, oligo or polyester, or polyamide-type linkers. Particularly preferred are polyethylene oxide-type linkers having a length of 2 to 10,000 monomer units, preferably 8 to 200 monomer units.

[0109] In another embodiment, the solid phase agent contains not only a nucleophilic group Q but also an accessory group K, which is shown in general structures XIX and XX. Group K does not react or covalently bond with the compound(s) of structure I. Instead, they assist in the reaction of group Q with the compound(s) of structure I.

[0110] [ka]

[0111] The function of group K is not limited, but it enhances the nucleophilicity of group Q through so-called neighboring effect or neighboring electron pair effect, or enhances the deprotonation of nucleophilic group Q, resulting in an anionic group Q with greater nucleophilicity. -This can be achieved by increasing the number of nucleophiles, by forming hydrogen bonds with the nucleophilic group Q, by interacting with the transition state formed between the compound(s) of structure I and the nucleophilic group Q and lowering its energy, by forming non-covalent bonds or ion pairs with the compound(s) of structure I, thereby increasing their local concentration, or by protonating the aziridine nitrogen of the compound(s) of structure I or forming a complex with it, thereby increasing their reactivity.

[0112] The following shows a reaction between an example of a compound with structure I and an example of a solid phase agent with structure XVIII:

[0113] [ka]

[0114] Figure 1 shows the interaction between a representative compound of structure I and a solid phase agent having a nucleophilic thiol group linked by linker L and an associated anionic sulfo group directly bonded to the polymer P matrix. Compounds having structure I are bound by multiple electrostatic interactions with sulfo groups and attracted to the vicinity of the nucleophilic SH group. This nucleophilic SH group attacks the carbon atom of the aziridine ring, which is protonated and activated, causing it to open and the neutralization product of the structure I compound to bond to the solid phase agent.

[0115] In another embodiment, the accessory group K of structures XIX and XX is a hydrophilic group that functions to enhance the wettability or swelling of the polymer matrix P in an aqueous environment. Often, samples containing pathogens can have a high water content. Such examples include blood, blood products or components, other body fluids, interstitial fluid, cell growth cultures or media, vaccine products or intermediates, or other biopharmaceuticals. Many polymers are hydrophobic and, therefore, without proper modification, can exclude aqueous fluids from their pore spaces, meaning they cannot be wetted or swell in such environments, thus preventing the reaction of reactive groups Q with compounds of structure I. By introducing a sufficient number of hydrophilic accessory groups K, the wettability inside the porous solid phase can be enhanced, thereby allowing the reactive groups Q to access aqueous solutions containing compounds of structure I. Examples of such hydrophilic groups, but are not limited to, sulfo or sulfonyl groups as depicted in Figure 1, or carboxylic acid groups, which have the additional advantage of being able to bond to polycationic compounds of structure I by ion pairing. Other such hydrophilic groups may be hydroxyl groups or polyol groups, such as 2-hydroxyethyloxy (HOCH2CH2O), 2,3-dihydroxypropyloxy (HOCH2CH(OH)CH2O-), or oligo and polyethylene glycol moieties having various monomer unit numbers.

[0116] Polymer matrices P of solid-phase agents having structures XVII-XX can have undesirable effects on certain components of certain samples. For example, the surfaces of many polymers, such as polystyrene, polyurethane, polymethacrylate, and polyamide, can bind to proteins from biopharmaceuticals and biological fluids, or disrupt their conformation, structure, and / or activity, activate coagulation cascade factors and platelets, or induce immune responses. These problems can be mitigated or eliminated by modifying such polymers with bindings of ethylene glycol oligomers or polymers of sufficient length and density. This technique, sometimes referred to as "pegylation" by those skilled in the art, has been applied to many biopolymers, most often therapeutic proteins, and polymers that come into contact with biological fluids in vivo or in vitro, as described by Harris MJ (Ed.) in *Poly(Ethylene Glycol) Chemistry. Biotechnical and Biomedical Applications*, Plenum Press, New York and London, 1992 and the references cited therein.

[0117] According to one embodiment of the present invention, the solid phase agent is divinylbenzene crosslinked polystyrene modified with the above-mentioned nucleophilic reactive group Q and a polar group which is an ethylene glycol oligomer or polyethylene glycol with a molecular weight of 150 to 100,000 Da, preferably 2,000 to 40,000 Da, more preferably 4,000 to 20,000 Da, and a density of one or fewer groups per monomer unit.

[0118] In another embodiment, the polymer is an acrylate or methacrylate polymer containing a nucleophilic reactive group Q and a polar group which is a polyol, for example, but not limited to 2-hydroxyethyl, 2,3-dihydroxypropyl, di-, tri-, tetra-, penta- or oligo- or polyethylene glycol, wherein the polar group is bonded to the C-1 i.e., carbonyl group of the acrylate or methacrylate polymer at a density sufficient to acquire desired hydrophilicity or other beneficial properties, which may include, but not limited to, non-immunogenicity, non-thrombogenicity, or non-binding or affinity to proteins or receptors, or to other components of the sample or composition or body fluid being treated.

[0119] In another embodiment, residual compounds of structure I are removed by treating a sample with a solid phase agent that is bonded to multiple anionic groups and electrostatically binds to the compound(s) of structure I by forming multiple ion-pair interactions with the positively charged nitrogen atom of the compound(s) of structure I. Such solid phase agents and such methods are disclosed herein with respect to the removal of products during the neutralization of compounds of structure I. Since compounds of structure I are polycationic near neutral, neutral, or acidic pH, the same methods and solid phase agents can be used for the removal of residual compounds of structure I from samples, culture media, compositions, utility products, or biological materials.

[0120] In another embodiment of the present invention, any remaining compounds of structure I are removed from the sample to be treated by contact with a solid phase agent that absorbs compounds of structure I. Such solid phase agents include, but are not limited to, activated carbon, charcoal, amorphous carbon, amorphous silica, silica gel, amorphous alumina, titania or zirconia, or other solid phase agents having affinity and ability to absorb compounds of structure I. The solid phase agent used to absorb compounds of structure I preferably has a high surface area-to-mass ratio, which can be achieved by using either a porous, microporous or nanoporous solid or a highly dispersible nonporous solid. Porous absorbing solid phase agents can be formed as powders, bulk solids, or particles of various diameters and shapes ranging from microns to 10 mm in diameter. Preferred particle sizes are 50 μm to 5 mm, more preferably 0.1 to 0.5 mm, which allows for sufficiently short diffusion of the compound to be absorbed into the bulk or particles, as well as sufficiently high rates of filtration and sedimentation for the removal of the particles.

[0121] In another embodiment, contact between the absorbing solid phase and the medium to be treated may occur not directly, but via a semipermeable barrier that allows the passage of the compound to be absorbed while preventing the passage of medium components, such as proteins or other macromolecules, that do not wish to interact with the solid phase. Examples of such semipermeable barriers include modified cellulose membranes or other dialysis membranes that allow the diffusion of compound(s) of structure I and have a molecular weight cutoff that prevents the diffusion of molecules with larger molecular weights, such as biopolymers.

[0122] In one embodiment of the method described herein, the method is used for the inactivation of viruses, which may be enveloped, non-enveloped, DNA or RNA viruses, retroviruses, bacteriophages, or any other viruses. Examples of such viruses include, but are not limited to, hepatitis B (HBV), hepatitis C (HCV), human immunodeficiency virus (HIV, types 1 and 2), malaria, syphilis, brucellosis, leptospirosis, arbovirus infections (e.g., Colorado tick fever), relapsing fever, Chagas disease (Trypanosoma cruzi), West Nile virus (WNV), human T-lymphotropic virus type 1, and viral hemorrhagic fevers (e.g., Ebola virus and Marburg virus).

[0123] In one embodiment of the method described herein, the method is used for the inactivation of prokaryotes, such as archaea, or bacteria, including Gram-positive and Gram-negative bacteria, spore-forming bacteria and bacterial spores, or mycoplasmas. Examples of pathogenic bacteria and antimicrobial-resistant bacteria that can be treated by the methods provided herein include, but are not limited to, Clostridium difficile (C. difficile), Enterobacteriaceae (CRE) bacteria, Neisseria gonorrhoeae, Campylobacter, Acinetobacter, fluconazole-resistant Candida, Extended-Spread-Effective-Bacteriaceae (ESBL), tuberculosis (TB), drug-resistant Salmonella serotype Typhi, vancomycin-resistant Enterococcus (VRE), multidrug-resistant Pseudomonas Aeruginosa, drug-resistant non-typhoid Salmonella, drug-resistant Streptococcus Pneumoniae, drug-resistant Sigella, methicillin-resistant Staphylococcus Aureus (MRSA), vancomycin-resistant Staphylococcus Aureus, erythromycin-resistant Group A Streptococcus, and clindamycin-resistant Group B Streptococcus.

[0124] In another embodiment, a method is used to inactivate eukaryotes, unicellular or multicellular eukaryotes, including but not limited to fungi, protozoa, parasitic helminths, schistosomiasis or nematodes, or their eggs, unicellular or multicellular algae, and crustaceans.

[0125] The methods provided herein may be used for the treatment of undesirable biological structures, including but not limited to bacterial biofilms or other microbial biofilms, lichens, deposits, or biofouling deposits.

[0126] The method of the present invention can be used not only to inactivate pathogenic microorganisms, but also to inactivate non-pathogenic cells, such as leukocytes, in a sample to be treated, where their presence is undesirable, for example, in intravenous blood or blood products.

[0127] The methods provided herein can be used not only for the inactivation of viruses, prokaryotes, and eukaryotes, but also for the inactivation of other infectious agents such as prions, where their pathogenic activity or infectivity depends on the presence or activity of nucleic acids, particularly ribonucleic acids, as disclosed by Botsios, S. and Manuelidis, L. in “CJD and Scrapie Require Agent-Associated Nucleic Acids for Infection”, J. Cell Biochem., 2016, 117, 1947-58, and by Supattapone, S. in “Synthesis of high titer infectious prions with cofactor molecules”, J. Biol. Chem., 2014, 289, 19850-4.

[0128] The methods provided herein may be used for processing samples, compositions, culture media, practical items, or biological organisms. Samples may be human or animal blood, leukocyte-depleted blood, whole blood, blood products, e.g., plasma, serum, red blood cells or red blood cell concentrates, platelets or platelet concentrates, serum or plasma components, factors or enzymes, blood and blood components intended for intravenous administration, apheresis blood components, body fluids, animal serum, e.g., serum used as a cell culture additive, culture media of eukaryotic or prokaryotic origin, vaccines, vaccine formulations, microbial suspensions for the preparation of whole-pathogen-killing vaccines, cosmetic and pharmaceutical compositions, beverages, food; or practical items, instruments, devices or their surfaces; or biological organisms, e.g., animals, mammals or human organisms and parts thereof, e.g., biological samples and biopsy materials. The methods may be used for processing biological products, including but not limited to antibodies, immunoglobulins, hormones, enzymes, growth factors, coagulation factors, albumin, or complement system components. Practical items may include, but are not limited to, medical or veterinary devices, such as disposable devices and equipment. Practical items also include, but are not limited to, industrial or household equipment, electrical appliances, equipment, mechanisms, machinery or materials, or any other articles in which the presence of pathogens or other organisms is undesirable or needs to be controlled. Practical items also include, but are not limited to, pipes, ducts, hoses, pipelines, vents, heat exchangers, sewer pipes, channels or other conduits for any fluid or gas, or any surface in contact with aqueous fluids, such as marine vessels, screens or filters, in cases where the presence of pathogens, microorganisms or other organisms is undesirable or needs to be controlled, such as in cases of biological contamination.

[0129] Methods for inactivating pathogens may be carried out in intravenous blood or blood products, in which case treatment with the compound(s) of structure I, and subsequent removal, inactivation, and removal of the product or inactivator and / or inactivator, shall be carried out in a sterile, partially or completely closed system.

[0130] In some embodiments, as shown in Figure 2, the compound of structure I is filled into a blood collection bag together with an anticoagulant solution.

[0131] In other embodiments, as shown in Figure 3, the compound of structure I, formulated as a liquid or solid formulation, is filled into a separate blood bag.

[0132] In other embodiments, as shown in Figures 4–9, the compound of structure I, formulated as a liquid or solid formulation, is pre-filled in a small container attached to a blood collection or blood processing bag and separated therefrom by a destructible sealant.

[0133] In another embodiment, as shown in Figure 10, the compound of structure I is filled into a capsule that is connected to a container of solution by a destructible sealant and to a blood processing bag by another destructible sealant.

[0134] In some embodiments, as shown in Figures 4 and 6, the neutralizing agent solution or liquid formulation may be contained in a container attached to the blood processing bag by a destructible seal, or it may be directly contained in the blood bag for neutralization. A solid phase agent for removing any remaining structure I compound or the product of its neutralization or the neutralizing agent may be contained in a cartridge connected to the processing and containment bag by a destructible seal, as shown in Figures 2, 3, 5, 6, 7 and 8, or it may be contained in the blood bag in the form of free beads or in a semipermeable container (bag), as shown in Figure 9.

[0135] The method using the whole blood integrated closed processing system shown in Figure 2 is as follows: Step 1 - Blood is collected using a phlebotomy needle and placed into a collection bag containing an anticoagulant and a compound of structure I; Step 2 - Blood is incubated to inactivate pathogens; Step 3 - The blood to be processed is passed through a cartridge containing a solid phase agent to remove any remaining compounds of structure I, and the purified blood is collected into a purified blood bag.

[0136] The method using the whole blood integrated closed processing system shown in Figure 3 is as follows: Step 1 - Blood is collected using a phlebotomy needle and placed into a collection bag containing an anticoagulant; Step 2 - The anticoagulated whole blood is transferred to a processing bag containing a solid formulation of a compound of structure I, mixed, and incubated for pathogen inactivation; Step 3 - The blood to be processed is passed through a cartridge containing a solid phase agent to remove any remaining compounds of structure I, and the purified blood is collected into a purified blood bag.

[0137] The method using the whole blood integrated closed-loop system shown in Figure 4 is as follows: Step 1 - Blood is collected using a phlebotomy needle and placed into a bag containing an anticoagulant; Step 2 - The capsule containing the liquid formulation of the compound of structure I is opened and the formulation is added to the blood; Step 3 - The blood is incubated with the compound of structure I; Step 4 - The capsule is broken open, the liquid formulation of the inactivator is added, mixed, and incubated for neutralization of the compound of structure I.

[0138] The method using the whole blood integrated closed processing system shown in Figure 5 is as follows: Step 1 - Blood is collected using a phlebotomy needle and placed into a collection bag containing an anticoagulant; Step 2 - The capsule containing the liquid formulation of the compound of structure I is opened and the formulation is added to the blood; Step 3 - The blood is mixed with and incubated with the compound of structure I; Step 4 - The blood to be processed is passed through a cartridge containing a solid phase agent to remove any remaining compound of structure I, and the purified blood is collected into a purified blood bag.

[0139] The method using the whole blood integrated closed-loop system shown in Figure 6 is as follows: Step 1 - Blood is collected using a phlebotomy needle and placed into a bag containing an anticoagulant; Step 2 - The capsule containing the liquid formulation of the compound of structure I is opened and the formulation is added to the blood; Step 3 - The blood is incubated with the compound of structure I; Step 4 - The capsule is broken, the liquid formulation of the inactivator is added and mixed, and incubated for neutralization of the compound of structure I; Step 5 - The neutralization product of the compound of structure I is removed by passing the blood to be processed through a cartridge containing a solid phase agent.

[0140] The method using the whole blood integrated closed-loop system shown in Figure 7 is as follows: Step 1 - Blood is collected using a phlebotomy needle and placed into a bag containing an anticoagulant; Step 2 - The capsule containing the liquid formulation of the compound of structure I is opened and the formulation is added to the blood; Step 3 - The blood is incubated with the compound of structure I; Step 4 - Any remaining compound of structure I is removed, and the blood to be processed is filtered for leukocytes by passing it through a cartridge containing a solid phase agent and a leukocyte filter; Step 5 - The RBCC bag containing the purified and leukocyte-removed blood is centrifuged; Step 6 - The separated plasma is transferred to a plasma bag; Step 7 - The preservative solution is transferred to red blood cells and mixed to prepare a concentrated blood cell solution.

[0141] The method using the whole blood integrated closed-loop system shown in Figure 8 is as follows: Step 1 - Blood is collected using a phlebotomy needle and placed into a bag containing an anticoagulant; Step 2 - Whole blood is leukocyte-removed by filtering it with a leukocyte filter and placing it into an LF blood bag; Step 3 - The capsule containing the liquid formulation of the compound of structure I is opened and the formulation is added to the leukocyte-filtered blood in the LF blood bag; Step 4 - The blood is mixed and incubated with the compound of structure I; Step 5 - Any remaining compound of structure I is removed by passing the blood to be processed through a cartridge containing a solid phase agent; Step 6 - The RBCC bag containing the purified and leukocyte-removed blood is centrifuged; Step 7 - The separated plasma is transferred to a plasma bag; Step 8 - The preservative solution is transferred to red blood cells and mixed to prepare a blood cell concentrate.

[0142] In some embodiments of the present invention, the reduction of the remaining structure I compound to a desired level may not be achieved with a single treatment with the solid phase agent. In such cases, two or more subsequent treatments with the solid phase agent may be required, as shown in Figure 9.

[0143] The method using the whole blood integrated closed processing system shown in Figure 9 is as follows: Step 1 - Collect blood with a phlebotomy needle and place it in a bag containing an anticoagulant; Step 2 - Break the seal of a capsule containing a liquid formulation of the compound of structure I and add the formulation to the blood; Step 3 - Mix and incubate the blood with the compound of structure I; Step 4 - Remove any remaining compound of structure I by transferring the blood to be processed to a first bag containing a solid phase agent (either as fluid beads or filled in a semipermeable bag) and incubating it; Step 5 - After the first removal step Step 6 - The blood is transferred to a second bag containing a solid phase agent (either as fluid beads or filled in a semipermeable bag) and incubated to perform a second removal of any remaining structure I compounds; Step 7 - The blood to be treated is filtered by passing it through a leukocyte filter and sending it to an RBCC bag; Step 8 - The RBCC bag containing the purified and leukocyte-removed blood is centrifuged; Step 9 - The separated plasma is transferred to a plasma bag; Step 10 - The preservative solution is transferred to red blood cells and mixed to prepare a concentrated blood cell solution.

[0144] The method using the whole blood integrated processing system shown in Figure 10 is as follows: Step 1 - Collect blood using a phlebotomy needle and place it in a bag containing an anticoagulant; Step 2 - Break the seal of the capsule containing the compound of structure I and dissolve the compound of structure I in the solvent from the solvent bag; Step 3 - Add the solution of the compound of structure I to the collected blood, mix, and incubate; Step 4 - Add the neutralizing agent solution and incubate to neutralize any remaining compound of structure I.

[0145] Figure 11 shows another example of a container for a solid formulation of a compound of structure I, in which the container for the solvent for dissolving the formulation is connected by a destructible seal, and the container containing the sample to be processed is connected by another destructible seal.

[0146] In another embodiment, the solid phase agent is filled into a cartridge, stored in a dry state within the cartridge, and pre-moistened and / or rinsed with a liquid composition suitable for the sample to be treated and its method of use before use. As an example, Figure 12 shows a closed system containing a cartridge filled with dry solid phase agent, housed between two filter media. The cartridge is connected to a container containing a wetting medium by a breakable seal, and to a container for the purified sample by another breakable seal. The wetting medium container is connected to a container for treating the sample with a compound(s) of structure I by a breakable seal. Breaking the seal between the cartridge and the container with the wetting medium, and transferring the medium into the cartridge, results in wetting of the solid phase agent. Breaking the remaining seal allows the sample to pass through the wetting solid phase agent.

[0147] In another embodiment, the solid phase agent is rinsed under sterile conditions before use. Such rinsing may be important in minimizing or eliminating exudative substances that may accumulate in the solid phase agent during storage. The washing is preferably carried out with a composition that is compatible with the solid phase agent, the sample to be treated, and its intended use. Figure 13 shows a closed system in which the rinsing of the solid phase agent filled in a cartridge is performed with a solvent in a container connected to the solid phase agent cartridge by a breakable sealant. The washing medium is then collected in another integrated container after the sealant between the cartridge and the container is broken. The two breakable sealants are then resealed with appropriate clips or resealing devices, such as T-Seal (Terumo tube sealing device). By breaking the remaining sealant, the sample to be treated can pass through the washed solid phase agent.

[0148] In some embodiments, the solid phase agent is contained within a cartridge / column, with one or both ends of the cartridge / column sandwiched by a permeable barrier. The barrier allows the sample to pass through the cartridge but not the solid phase agent. Examples of such barriers, but not limited to, are filters / screens, discs made of sintered material, meshes, sieves or fabrics, or any other porous material, or materials with openings or channels whose diameter is smaller than the diameter of the solid phase agent particles. Such barriers are shown by dashed lines in Figures 12 and 13.

[0149] In another embodiment, the closed system of the present disclosure for inactivating pathogens by means of sterilization is sterilized by UV or gamma irradiation, heat treatment, high or low pH solvent treatment, or other chemical treatment, such as treatment with ethylene oxide, ozone, bleach, glutaraldehyde, formaldehyde, hydrogen peroxide, peracetic acid or silver compounds, or by other methods known to those skilled in the art. Liquid formulations of the compounds of structure I and their neutralizing agents may be sterilized by filtration, UV or gamma irradiation, heat treatment, or other methods known to those skilled in the art. Solid phase agents may be sterilized by UV or gamma irradiation, heat treatment, high or low pH solvent treatment, or chemical treatment before or after filling into cartridges or other containers or semipermeable bags, and before or after incorporating into a closed system.

[0150] The examples of pathogen reduction closed systems shown in Figures 2-13 are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0151] In some embodiments, the pathogen(s) are present in a living organism, which may be an animal, mammal, such as a primate, rodent, marine mammal, or any wild or domesticated animal, or a human. In these embodiments, treatment with the compound of structure I is carried out in vivo. This in vivo treatment may be carried out intravenously, orally, topically, rectally, subcutaneously, intramuscularly, by inhalation, or a combination thereof, and the treatment may be carried out by single, multiple, or sequential doses in a dose(s) sufficient to achieve the desired reduction of the pathogen(s). Such in vivo treatment may be followed or combined with in vivo treatment with an inactivator of the compound of structure I, such as, but not limited to, sodium thiosulfate.

[0152] In other embodiments, the treatment of an organism with a compound of structure I is performed in vivo, and the neutralization and / or removal of the compound(s) of structure I, or the removal of the products of their neutralization or degradation, is performed in vitro by treating the organism's bodily fluids, such as blood or plasma, and then returning them to the original organism (infusion). Such in vitro treatment may be performed by periodic removal, treatment, and infusion of a portion of the bodily fluids in several stages, or by continuous withdrawal, treatment, and infusion. In the latter case, the use of an apheresis process and continuous treatment of apheresis plasma is preferred. The neutralization or removal of a compound of structure I may be performed by passing the compound(s) through a cartridge containing a solid phase agent for isolating the compound(s), or by mixing it with a solution of a neutralizing agent and then incubating it, and optionally then passing it through a cartridge having a solid phase agent for isolating the neutralization products and / or the neutralizing agent.

[0153] In other embodiments, the treatment of a pathogen-containing organism is carried out by in vitro treatment of the organism's body fluids. This treatment may be carried out by periodic removal, treatment, and infusion of a portion of the body fluids each time; or by continuous withdrawal, treatment, and infusion. In the latter case, the use of an apheresis process and continuous treatment of apheresis plasma is preferred. The in vitro treatment is carried out by adding an appropriate amount of a formulation of a structure I compound(s) to the body fluids, and incubation, and preferably subsequently, treatment for the removal or neutralization of any remaining structure I compound(s), and / or optional treatment for the removal of inactivation or degradation products of the structure I compound(s), and subsequently infusion of the purified body fluid into the original organism. Treatment for the removal or neutralization of the structure I compound(s) and / or the removal of their neutralization products is carried out as described above for in vivo treatment with the structure I compound(s).

[0154] In a preferred embodiment of a method for treating an organism with a compound(s) of structure I, either intra vivo or in vitro, at least one pathogen present in the organism and targeted for inactivation by the treatment is resistant to one or more anti-pathogen treatments. [Examples]

[0155] Example 1

[0156] Compound VI, N 1 ,N 4 -Bis(3-(aziridine-1-yl)propyl)-N 1 ,N 4 Synthesis of dimethylbutane-1,4-diamine.

[0157] A. Synthesis of aziridine: 58.4 g (0.503 mol) of 2-chloroethylamine hydrochloride was dissolved in 100 ml of water. The solution was added dropwise to a solution containing 56.5 g of sodium hydroxide in 20 ml of water while stirring. After stirring at 50°C for a further 2.5 hours, the aziridine was purified by distillation under partial vacuum. Solid NaOH was added to the distillate in several batches with vigorous stirring and cooled to a temperature of 0-8°C. The mixture was stirred at this temperature for 30 minutes. The solid NaOH was removed by decanting the liquid, and the upper layer was separated to obtain 22.5 g of wet aziridine. This material was dried by decanting powdered KOH in small amounts, with each small addition followed by decantation until the KOH maintained a dry appearance. The obtained dried aziridine was stored at -20°C under a KOH palette. Yield 16.02 g, 74% clear liquid.

[0158] B. Synthesis of 2-(1-aziridinyl)propanal-methylacetal, IV: 6.65 g, 7.93 ml, 0.120 mol of acrolein was added to 100 ml of MeOH. Ar was passed through the solution and cooled under Ar in a dry ice bath. 4.99 g, 6.00 ml, 0.124 mol of aziridine was added dropwise while stirring. The dry ice bath was removed and the reaction mixture was left at room temperature. The solution of 2-(1-aziridinyl)propanal-methylacetal, IV obtained in this way was sealed and stored under Ar at -20°C. 1 H NMR(300MHz,CD3OD)δ:4.66(t,J=5.54Hz,1H),3.36(s,3H),2.30-2.44(m,2H),1.79-1.93(m,2H),1.76-1.79(m,2H),1.30-1.33(m,2H). 13 C NMR (75MHz, CD3OD) δ:97.9,57.5,36.5,26.6.

[0159] C. N 1 ,N 4 -Bis(3-(aziridine-1-yl)propyl)-N 1 ,N 4Synthesis of N,N'-dimethylbutane-1,4-diamine VI: A methanol solution of compound IV from step B was cooled in an ice bath. 5.85 g, 50.4 mmol of N,N'-dimethylbutane-1,4-diamine was added dropwise with stirring. The bath was removed, and after 30 minutes, 10 g of sodium borohydride was added in several batches with stirring while cooling at -4 to +4°C. The mixture was allowed to stand at room temperature for 4 hours, then worked out with water, extracted with ether, and the product was purified by silica gel chromatography. The fraction containing the product was concentrated by evaporation, and the residue was subjected to vacuum distillation to obtain 3.84 g of compound VI as a pale yellow oil. 1 H NMR(300MHz,C6D6)δ:2.43(t,J=7.2Hz,4H),2.30(m,4H),2.13(t+s,J=6.7Hz,10H),1.75(m,4H),1.55(m,4H),1.51(m,4H),0.79(m,4H). 13 ¹³C NMR (75MHz, C6D6) δ: 60.74, 58.55, 56.49, 42.52, 28.77, 27.50, 26.11. MS (Electrospray, positive mode) m / z: 283.1, calculated values ​​[M+H] + 283.2.

[0160] Example 2

[0161] Synthesis of compound XVI, 3-(aziridine-1-yl)-N-(3-(aziridine-1-yl)propyl)-N-methylpropan-1-amine.

[0162] Compound XVI was synthesized as in Example 1 using 4.35 ml of a 40% aqueous solution of 3.91 g of methylamine instead of N,N'-dimethylputrescine. After vacuum fractionation, 2.48 g of compound XVI was obtained as a light-colored oil. 1 H NMR(500MHz,C6D6)δ:2.43(t,J=7.0Hz,4H),2.12(s,3H),2.11(t,J=7.0Hz,4H),1.74(m,4H),1.54(m,4H),1.51(m,4H),0.77(m,4H). 13¹³C NMR (75MHz, C6D6) δ: 60.00, 55.72, 41.78, 28.01, 27.50, 26.79. MS (Electrospray, positive mode) m / z: 198.1, calculated value [M+H] + 189.2.

[0163] Example 3

[0164] Compound X, N 1 -(3-(aziridine-l-yl)propyl)-N 4 -(3-((3-(aziridine-1-yl)propyl)(methyl)amino)-propyl)-N 1 ,N 4 Synthesis of dimethylbutane-1,4-diamine.

[0165] A. N 1 ,N 5 ,N 10 - Synthesis of trimethylspermidine: 5.70 g, 6.16 ml, 39.3 mmol of spermidine is mixed with 61.1 g, 66.6 ml, 0.824 mol of ethyl formate. The mixture is refluxed for 30 hours, then evaporated and concentrated under vacuum to obtain N2. 1 ,N 5 ,N 10 - 9.32 g of triformylspermidine was obtained as oil. 9.00 g of lithium aluminum hydride was added to 300 ml of dry tetrahydrofuran. N I ,N 5 ,N IO -9.00 g of triformylspermidine was added dropwise under argon with stirring. The reaction mixture was refluxed for 4 hours and then cooled to room temperature. 22 ml of water was added dropwise while cooling and efficient mechanical stirring (foaming), followed by 90 ml of 50% potassium hydroxide aqueous solution. After vigorous stirring for 1 hour, 150 ml of tetrahydrofuran was added and the mixture was separated into layers. The lower layer was extracted with 150 ml of tetrahydrofuran, and the extract was combined with the upper layer. The combined organic layers were concentrated by evaporation under vacuum, the residue was dissolved in 75 ml of diethyl ether, and dried overnight over solid potassium hydroxide. The dried ether solution was concentrated by evaporation, and the residue was subjected to vacuum fractionation to obtain 5.30 g of N2. 1 ,N 5 ,N10 -Trimethylspermidine was obtained. ¹H NMR (300MHz, C6D6) δ: 2.53 (t, J=6.7Hz, 2H), 2.45 (t, J=6.6, 2H), 2.22-2.35 (m, 4H), 2.30 (s, 3H), 2.28 (s, 3H), 2.12 (s, 3H), 1.58 (m, 2H), 1.46 (m, 4H). 13 ¹³C NMR (75MHz, C6D6) δ: 58.28, 56.52, 52.44, 51.03, 42.21, 36.83, 28.21, 28.19, 25.67. MS (Electrospray, positive mode) m / z: 188.1, calculated value [M+H] + 188.2.

[0166] B. Synthesis of Compound X: According to Example 1, 3.71 g, 4.43 ml, 67 mmol of acrolein, 56 ml of methanol, 2.79 g, 3.35 ml, 69 mmol of aziridine, and 5.30 g, 28.1 mmol of N,N'-dimethylputrescine were used instead of N,N'-dimethylputrescine. 1 ,N 5 ,N 10 Compound X was synthesized using trimethylspermidine and 5.58 g of sodium borohydride. After workup and vacuum fractionation, 2.99 g of compound X was obtained as a grayish-white oil. 1 H NMR(300MHz,C6D6)δ:2.39-2.45(m,4H),2.32-2.38(m,4H),2.27-2.31(m,4H),2.14(s,6H),2.13(s,3H),2.10-2.1 5(m,4H),1.73(quintet,J=7.0Hz,4H),1.58-1.68(m,2H),1.54-1.56(m,4H),1.47-1.53(m,4H),0.80-0.82(m,4H). 13 ¹³C NMR (75MHz, C6D6) δ: 60.74, 58.59, 58.55, 56.60, 56.56, 56.51, 56.48, 42.65, 42.54, 28.76, 27.50, 26.43, 26.14, 26.10. MS (Electrospray, positive mode) m / z: 354.1, calculated values ​​[M+H] + 354.3.

[0167] Example 4

[0168] Compound XIV, N 1 , N 4 -Di(3-((3-(aziridin-1-yl)propyl)-(methyl)amino)propyl)-N 1 , N 4 -Dimethylbutane-1,4-diamine synthesis

[0169] A. N 1 , N 5 , N 10 , N 14 -Tetramethylspermine synthesis: N 1 , N 5 , N 10 , N 14 -Tetramethylspermine was prepared as 1.59 g of a pale gray oil by reducing 1.60 g, 7.86 mmol of spermine according to Example 3, followed by treatment with 2.00 g of lithium aluminum hydride in 50 ml of dry tetrahydrofuran, work-up with water, and vacuum fractional distillation. H NMR (300 MHz, C6D6) δ: 2.53 (t, J = 6.7 Hz, 4H), 2.34 (t, J = 6.9, 4H), 3.30 (s, 6H), 2.28 (m, 4H), 2.13 (s, 6H), 1.59 (quintet, J = 6.8 Hz, 4H), 1.50 (m, 4H), 0.87 (bs, 2H). 13 C NMR (75 MHz, C6D6) δ: 57.92, 56.25, 50.75, 41.94, 36.53, 27.88, 25.37. MS (electrospray, positive mode) m / z: 258.1, calculated value [M + H] + 258.3.

[0170] B. Synthesis of Compound XIV: According to Example 1, 10 mmol of 3-(aziridin-1-yl)propanal in 9 ml of methanol and 0.80 g, 3.1 mmol of N instead of N,N'-dimethylputrescine 1 , N 5 , N 10 , N 14Compound XIV was synthesized using tetramethylspermine and 0.77 g of sodium borohydride. After work-treatment with water, vacuum fractionation, and purification by silica gel chromatography, 0.398 g of compound XIV was obtained as a grayish-white oil. 1 H NMR(300MHz,C6D6)δ:2.44(t,J=7.0Hz,4H),2.34-2.40(m,8H),2.31(m,4H),2.15(s,6H),2.14(s,6H),2.11-2.16(m ,4H),1.75(quintet,J=7.0Hz,4H),1.65(quintet,J=7.4Hz,4H),1.53(m,4H),1.47-1.53(m,4H),0.79-0.81(m,4H). 13 ¹³C NMR (75MHz, C6D6) δ: 60.77, 58.64, 56.64, 56.60, 56.54, 42.65, 28.78, 27.51, 26.46, 26.18. MS (Electrospray, positive mode) m / z: 425.2, calculated value [M+H] + 425.4.

[0171] Example 5

[0172] Reactivity to nucleic acids

[0173] The reactivity to nucleic acids was tracked by reacting 10 μM of 21-mer synthetic oligodeoxyribonucleotide-5' ATA CCT CAT GGT AAT CCT GTT-3', which contains all four nucleic acid bases in its sequence, with 200 μM of compound X in PBS (pH 6.7) at 37°C. Figure 14 shows the HPLC analysis of the incubation mixture after incubation at 37°C for 0 hours (upper chromatogram) and 6 hours (lower chromatogram). The peaks corresponding to the oligonucleotide decreased, and compounds with higher retention times appeared, clearly demonstrating the formation of a covalent adduct between compound X and the oligonucleotide.

[0174] The reaction of 100 μM of a 23-mer synthetic oligonucleotide (UGG ACU CCG AUA ACG GAG UAU GU) with 100 μM of compound X in PBS at pH 7 and room temperature was examined by mass spectrometry. The results are shown in Figure 15. The upper panel shows the mass spectrum of the oligonucleotide before treatment, and the lower panel shows the mass spectrum of the reactant 6 minutes after the addition of compound X. In the upper panel, the peak at 1845.22 m / z is due to the oligonucleotide ion (M-4H) / 4 with a charge state of -4, corresponding to a neutral molecule with a mass of 7384.9 Da (calculated oligonucleotide mass of 7384.0 Da). In the lower spectrum, an additional peak appears 6 minutes after incubation with compound X, with an m / z of 1933.54, corresponding to a neutral molecule with a mass of 7738.2 Da. The molecular weight of the covalent monoadduct of compound X and oligonucleotide is 7737.3 Da.

[0175] Example 6

[0176] Lack of reactivity of the compound in structure I with cytochrome C.

[0177] Inactivating pathogens in blood products using alkylating agent molecules has potentially harmful side effects—their reaction with proteins can produce nascent antigens, i.e., haptens. To assess the hapten potential of compounds of structure I, their ability to modify cytochrome C was tested. Cytochrome C (MW12384Da) was chosen as a model protein because it contains many nucleophilic side-chain amino acids that are potential targets for alkylation by compounds of structure I: 19 Lys, 2 Cys, 3 Asp, 9 Glu, 3 His, and 4 Tyr. A phosphate-buffered aqueous solution of 0.1 mg / mL (8 μM) of cytochrome C was incubated with 0 (control), 0.1, 1, and 10 mM of compound VI or X at pH 7.0 for 30 hours at 40°C. At 1, 4, and 30 hours, aliquots of the incubation mixture were directly injected into an LCQ Advantage mass spectrometer (Thermo-Finnigan, San Jose, CA) in positive ionization mode using electrospray mass spectrometry to analyze the formation of covalent adducts between the protein and the test compounds. The results clearly showed the absence of covalent adducts between both test compounds VI and X and cytochrome C at all concentrations and time points (see Figure 16 for representative mass spectra).

[0178] Example 7

[0179] Lack of reactivity of structure I compounds with viral surface proteins.

[0180] The potential of compounds of structure I to modify pathogen proteins was evaluated using respiratory syncytial virus (RSV) as a model pathogen, and RSV fusion (F) was selected for testing for modification. The F protein is a large (574 amino acid) surface glycoprotein associated with the viral envelope, which plays a crucial role in host recognition and viral insertion. This protein was selected due to its high susceptibility and instability, the availability of monoclonal antibodies specific to various antigenic epitopes, and its sensitivity to conformational changes of the F protein. Sucrose gradient-purified RSV was treated with compound VI and compound X at concentrations of 100 μM each for 4 hours at 40°C. The remaining compounds VI and X were neutralized as described in Example 16. Controls included pseudo-treated RSV incubated at 40°C for 4 hours and untreated virus kept at 4°C. The ELISA assay was performed according to the procedure described in Schmidt et al, J Virol. 2014;88(l7):10165-76.doi:10.1128 / JVI.01250-14.PubMed PMID:24965456. Eight 1:2 serial dilutions with PBS were seeded in three replicates into a 96-well plate (50 μl / well) and incubated overnight at 4°C. The wells were washed with PBS and blocked with PBS / 1% BSA. Anti-protein F antibody was added, the mixture incubated for 2 hours, then washed, and the anti-mouse IgG HRP complex was added. After another wash, TMB substrate and sulfuric acid were added, and readings were performed using an ELISA reader SPECTRAmax PLETS (Molecular Devices, Sunnyvale, CA). Figure 17 shows the results of ELISA determination of the binding affinity of anti-F antibody to RSV treated with compounds VI and X. These experiments revealed that treatment with compounds VI and X under conditions that completely inactivate the virus did not alter the degree of recognition of the F protein by highly specific and conformationally sensitive monoclonal antibodies, demonstrating that no modification of the F protein occurred as a result of the treatment.

[0181] Example 8

[0182] Bacterial inactivation in bacterial growth media by compounds VI, X, and XIV

[0183] Panels of G+ and G- bacteria were inactivated in their respective sugar-making media using compounds VI, X, and XIV. All cells were grown to metaphase logarithmic growth in the corresponding media, harvested by centrifugation, resuspended in Ringer's solution (RS), and treated with 100 μM of compounds VI, X, and XIV at room temperature, with these compounds added to the suspension as 100-fold concentrated solutions in RS. Only RS was provided as a control. At the end of incubation, unreacted compounds VI, X, and XIV were neutralized with 10 mM sodium thiophosphate during incubation at room temperature for 30 minutes. Live cells were counted using serial dilutions by standard agar plate counting. Table 1 summarizes typical results of 1 hour of treatment of E. coli, P. fluorescens, Y. enterocolitica, B. cereus, S. aureus, and S. epidermidis with compounds VI, X, and XIV. Clearly, a reduction in viable cells was observed in all three compounds, even after 1 hour of treatment. Compounds X and XIV showed significantly higher titers than compound VI. Two species were inactivated below the detection limit (1.00 Log) in these two compounds. 10 CFU / mL).

[0184] [Table 1]

[0185] Example 9

[0186] Virus inactivation by compound VI and compound X

[0187] Porcine parvovirus (PPV) was inactivated using compounds VI and X. Compounds VI and X were used at 100 μM in RS (pH 6.9) at room temperature, with a 10% virus contamination rate. The remaining compounds were inactivated by incubation with 10 mM Na2S2O3 at room temperature for 2 hours. A standard limiting dilution assay acceptable for PPV-positive porcine testicular cells was used to obtain log 10 TCID 50 The viral titer, expressed in units of / mL, was determined. After incubating indicator cells for 6 days, the infected wells were counted by visual inspection under a microscope. To confirm the results, secondary infection was performed using the prepared medium from the initial plate wells as a sample.

[0188] Human respiratory syncytial virus (RSV) was inactivated using compound VI or compound X. For this purpose, sucrose gradient-purified viruses were treated with 100 μM of compound VI and compound X at room temperature. Aliquots were taken at 1, 4, and 6 hours of incubation and inactivated with 10 mM sodium thiophosphate for 30 minutes at room temperature. Viral titers were determined using standard 10x serial dilutions in a modified plaque assay. No significant change in RSV infectivity was observed in the simulated-treated viruses even after 6 hours of incubation at room temperature (titer reductions ranged from 0.11 to 0.36 log in different experiments). 10 (It was within the range of PFU / mL)

[0189] Bovine viral diarrhea virus (BVDV) was inactivated using compounds VI and X. The protocol used for PPV inactivation was adopted for BVDV inactivation, except that bovine turbinate cells were used as the indicator cells.

[0190] The experimental results are shown in Table 2. A reduction in viral titer of 5 to 7 logarithms was observed, and after incubation with compound VI for 6 hours, all viruses were killed to below the detection limit.

[0191] [Table 2]

[0192] Example 10

[0193] Bacterial inactivation by compounds VI and X in whole blood (WB), leukocyte-depleted blood (LB), and packed red blood cells (RBCC)

[0194] Two psychrophilic bacteria, the G - species Y. enterocolitica and P. fluorescens, and two G+ bacteria, S. epidermidis and B. cereus, which are both known blood contaminants, were used in this test. Approximately 0.1% bacterial suspension stock solution prepared with RS was admixed to all blood samples, and left at room temperature for 30 minutes for equilibration. Fresh overnight-grown bacterial cultures were used for each admixture. Compounds VI and X were added to the admixed blood at final concentrations of 100, 250, and 500 μM. The control sample (Ctr) was given only the solvent. Incubation was carried out at room temperature for 6 hours, and then 100x sodium thiosulfate, an inactivator, was added and further incubated at room temperature for 2 hours. After incubation and inactivation, aliquots for serial dilution and plate-drop counting were taken, and a bacterial growth-promoting solution (containing tryptone, peptone, yeast extract, and casamino acids) was added to the remaining volume. The growth / no-growth results were confirmed by streaking on agar plates.

[0195] Table 3 represents the results of typical inactivation experiments in WB, LB, and RBCC, respectively.

[0196]

Table 3

[0197] Example 11

[0198] Viral inactivation by compounds VI and X in whole blood (WB), leukocyte-depleted blood (LB), and packed red blood cells (RBCC)

[0199] All blood samples were mixed with approximately 20% of the virus stock solution prepared by RS and left at room temperature for 30 minutes to equilibrate. Virus inactivation tests using BVDV or PPV were performed in the same manner as the bacterial inactivation protocol in Example 10. T0 and 6 hours after incubation were performed (Log 10 TCID 50 The viral titer (expressed in / mL) was determined as described in Example 9. The results of BVDV and PPV inactivation are shown in Table 4.

[0200] [Table 4]

[0201] Example 12

[0202] Inactivation of RSV by compound XVI

[0203] Inactivation of RSV with compound XVI at different concentrations at room temperature and 40°C was carried out as described in Example 9. The results are shown in Table 5.

[0204] [Table 5]

[0205] Example 13

[0206] Inactivation of BVDV and PPV by compound VI in thermoactivated fetal bovine serum (FBS)

[0207] Aliquots of FBS were mixed with 5% (vol / vol) of BVDV and PPV stock solution and equilibrated at room temperature for 60 minutes. 10 mM of compound VI in phosphate buffer (pH 6.9) was added to the mixed FBS to a final concentration of 100 μM, and all aliquots were treated as described in Table 6.

[0208] [Table 6]

[0209] The virus-contaminated serum samples were treated with 100 μM of Compound VI at 40 ± 1 °C for 60 minutes. Aliquots from all samples (Controls 1-4 and treated samples) were serially diluted (1:5 or 1:10) with serum-free DMEM, and 25 μL from each dilution was seeded in triplicate onto their respective indicator cells in 96-well plates. The plates were incubated at 37 °C for 60 minutes in a 5% CO2 incubator to allow virus adsorption. Undiluted samples were additionally used to infect host cells in 24-well plates or 10-cm Petri dishes in order to increase the detection limit. After adsorption, all wells were filled with DMEM / 5% FBS without aspiration of the 25 μL dilution, and the plates were further incubated in a CO2 incubator at 37 °C for 6 - 7 days. The development of cytopathic effects due to the virus in each well was detected by visual inspection, and used to calculate the respective virus titer expressed as Log 10 TCID 50 / mL. The detection limit was 0.2 infectious particles per mL. In some cases, to confirm the results of the assay, the supernatant from the inoculated wells was collected after 6 - 7 days and used to infect fresh cells in 24-well plates.

[0210] The experimental results shown in Table 7 demonstrate that treatment with Compound VI effectively inactivated both BVDV and PPV below the detection limit of the assay.

[0211]

Table 7

[0212] Example 14

[0213] Compound of Structure I as an inactivator of protozoa and fungi

[0214] In fresh human red blood cells, the bloodborne parasites Plasmodium falciparum 3D7 and Babesia divergens Rouen were inactivated at physiological temperature for 24 hours. Compound XIV at a concentration of 250 μM exhibited strong antiparasitic activity, reducing the number of live Plasmodium organisms by approximately 7 logarithms and Babesia by 8 logarithms. Inactivation of Candida albicans, a representative pathogenic fungus, by more than 6 logarithms, and inactivation of Tetrahymena thermophila, a model organism of ciliated protozoa, by 3 logarithms were achieved with 250 μM of compound XIV in their respective growth media.

[0215] Example 15

[0216] Neutralization of compound X with 2-mercaptoethyl acetate

[0217] A 100 μM solution of compound X in phosphate-buffered saline was incubated with 10 mM ethyl 2-mercaptoacetate at room temperature. The change in compound X concentration, as well as the formation of the neutralization intermediate compound (XXI) and the final neutralization compound XXII, were determined by LC-MS analysis of the mixture. The neutralization reaction scheme is shown below. The peak areas of compound X, the intermediate neutralization product Q1 (compound XXI), and the final neutralization product Q2 (compound XXII) are shown in Table 8 and Figure 18 below.

[0218] [ka]

[0219] [Table 8]

[0220] Example 16

[0221] Neutralization of residual compound VI with sodium thiosulfate

[0222] Tests of the reaction between sodium thiosulfate and the compound of structure I showed that Na2S2O3 rapidly reacts with the aziridine group of the compound, opening the ring and converting it to a biologically well-tolerated thiosulfate ester that is expected to undergo rapid renal excretion. The reaction rate of 100 μM of compound VI and 1 mM Na2S2O3 in PBS was determined by LC-MS analysis of the reaction mixture (Figure 19). The reaction followed first-order kinetics, with a rate constant of 0.00614 min at 6°C. -1 , 0.0379 min at 25℃ -1 Therefore, at this reaction rate, the half-life of compound VI at 25°C in the presence of 10 mM Na2S2O3 is 1.83 minutes, and the concentration of compound VI remaining after 2 hours is 5.5 × 10⁻⁶. -19 This would result in M. LC-MS analysis of the reaction product confirmed that it is compound XXIII, formed by the reaction of bisthiosulfate (compound VI) with two molecules of Na2S2O3.

[0223] [ka]

[0224] Example 17

[0225] Neutralization of compound X by methyl thiosalicylate

[0226] To 178 μL of phosphate-buffered saline, 2 μL of a 10 mM methanol solution of compound X and 20 μL of a 100 mM methanol solution of methyl thiosalicylate were added, resulting in final concentrations of 100 μM for the inactivator and 10 mM for methyl salicylate. This solution was analyzed by liquid chromatography-mass spectrometry for changes in the concentration of compound X and the formation of covalent adducts (compounds XXIV and XXV) between compound X and methyl thiosalicylate, as schematically shown herein.

[0227] [ka]

[0228] The results shown graphically in Figure 20A indicate a decrease in the concentration of compound X due to the formation of intermediate compound XXIV, which is further converted to compound XXV. The rate of neutralization of compound VI can be determined by plotting the logarithm of the concentration of compound X, determined by the peak area, against the incubation time. This plot, shown in Figure 20B, reveals a linear dependence of compound X's T 1 / 2 Corresponding to a half-life of 16.6 minutes, the primary rate constant is K = -0.0416 min -1 This presents a first-order reaction kinetic theory.

[0229] Example 18

[0230] Neutralization of structure I compounds by thiophenol

[0231] To 178 μL of phosphate-buffered saline, 2 μL of a 10 mM methanol solution of compound X and 20 μL of a 100 mM methanol solution of thiophenol were added, resulting in final concentrations of 100 μM for the inactivator and 10 mM for the thiophenol. This solution was analyzed by liquid chromatography-mass spectrometry for changes in the concentration of compound X and the formation of compounds XXVI and XXVII, which are covalent adducts of compound X and thiophenol as schematically shown herein.

[0232] [ka]

[0233] Figure 21 shows the results of LC-MS analysis of compound X and thiophenol at various time points. The left panel of Figure 21 shows the total ion current mass chromatogram of the LC-MS analysis, where the peaks correspond to compounds X, XXVI, and XXVII. The right panel shows the mass spectra of the corresponding peaks. The analysis reveals that compound X is neutralized to a considerable extent after 1 minute 40 seconds (100 seconds), with the peak area ratios of compounds X, XXVI, and XXVII being 21:52:27, respectively. The ratios of these peaks after 10 minutes are 3:29:68, and after 20 minutes they are 0.5:16:83.5, suggesting rapid conversion from compound X to the covalent monoadducts and diadducts, XXVI and XXVII.

[0234] Example 19

[0235] Preparation of solid phase agent XXVIII having a thiosulfonate functional group, and its use for neutralizing compound VI.

[0236] Sulfonyl chloride-functionalized divinylbenzene crosslinked polystyrene resin (Sigma-Aldrich catalog number 498211-5g) was mixed with 5 ml of 2 M sodium hydrogen sulfide solution (prepared by saturating an aqueous solution of sodium sulfide nonahydrate with hydrogen sulfide) under argon. The mixture was sonicated for approximately 3 minutes, then stirred at 55°C for 4 hours. The resin was then filtered and washed three times with degassed water, three times with degassed methanol, and twice with degassed ether. The resin was dried under an argon stream and then under vacuum. 1.039 g of dried resin (compound XXVIII), which is thiosulfonate-functionalized polystyrene / divinylbenzene resin, was obtained. Aliquots of compound XXVIII were added to a PBS solution of compound VI in 100 μM. LCMS analysis demonstrated a time-dependent decrease in the concentration of compound VI in the mixture.

[0237] [ka]

[0238] The reaction scheme for the preparation of solid phase agent XXVIII, as well as the reaction of neutralization and covalent sequestration of compound VI by solid phase agent XXVIII, which involves the formation of compound XXIX (e.g., a covalent adduct of compound VI and solid phase agent XXIX), are described herein.

[0239] Example 20

[0240] Preparation of a mercaptophenyl-functionalized methacrylate resin-based solid phase agent XXX, and its use for neutralization and covalent sequestration of compound VI.

[0241] 400 mg of 4-mercaptophenylacetic acid (Sigma-Aldrich catalog number 653152-5G) was dissolved in 2 ml of dimethyl sulfoxide, and the solution was left at room temperature overnight. The formed dimethyl sulfide was removed under a vacuum of 10 torr, and the excess dimethyl sulfoxide was removed overnight at 45°C under a vacuum of 0.05 torr. This yielded a quantitative yield of 4-mercaptophenylacetic acid disulfide as a waxy, yellowish solid.

[0242] 300 mg of aminoethyl group-functionalized methacrylate resin (Purolite Ltd, Llantrisant, Wales, UK, product number D6195, trademark Chromalite MAM2, 0.5 mmol of amino groups and 68% moisture per 1 ml of wet resin) was dried under vacuum at 35°C by three evaporations from 2 ml of dry N,N-dimethylformamide. The dried resin was suspended in 1 ml of dry N,N-dimethylformamide, and a solution of 370 mg of 4-mercaptophenylacetic acid disulfide in 1 ml of dry tetrahydrofuran was added to this suspension. 172 mg of benzotriazole-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate was added to this suspension under stirring, followed by the dropwise addition of 172 μL of N,N-diisopropylethylamine, and the reaction mixture was sealed under argon. After 24 hours, a solution of 330 mg of dithiothreitol in 1 ml of deionized and degassed water was added with stirring. After 10 minutes, the resin was recovered by vacuum filtration and repeatedly washed with degassed acetonitrile, tetrahydrofuran, methanol, and 0.2 mM diethylenetriaminepentaacetic acid (DPTA). The mixture was then purged with argon to obtain 314 mg of wet mercaptophenyl-functionalized methacrylate resin. The amount of mercapto groups introduced into the product compound XXX was determined using Eelmann's procedure (Riener, CK; Kada, G.; Gruber, HJ, Anal. Bioanal. Chem., 2002, 373, 266-76) and was 0.21 mmol per gram of wet resin. The water content was 71%. Aliquots of compound XXX were added to a PBS solution of 100 μM compound VI. LC-MS analysis of this mixture demonstrated a time-dependent decrease in compound VI in the mixture.

[0243] [ka]

[0244] The above reaction scheme describes the synthesis of solid phase agent XXX and the reaction between it and compound VI, which involves the formation of compound XXXI (for example, a covalent adduct XXXI between compound VI and solid phase agent XXX).

[0245] Example 21

[0246] Preparation of thiophenol-functionalized polyethylene glycol-grafted polystyrene-divinylbenzene resin XXXII, and its use for neutralizing compound VI.

[0247] 900 mg of 4-mercaptophenylacetic acid was added to a solution of 1.60 g of triphenylmethyl chloride in 50 ml of anhydrous dichloromethane. The mixture was stirred at room temperature under argon for 3 hours. 30 ml of water was added, and the mixture was stirred for 5 minutes. The dichloromethane layer was separated, dried over sodium sulfate, and concentrated by evaporation under vacuum to obtain 2.3 g of crude product as a white solid. This material was purified by silica gel chromatography on a gradient from chloroform to chloroform / methanol 10:1 to obtain 1.62 g of 74% 2-(4-(triphenylmethylthio)phenyl)acetic acid.

[0248] 200 mg of Tentagel S NH2 resin (Rapp Polymere GmbH, Tuebingen, Germany, product number S30132, divinylbenzene crosslinked polystyrene resin grafted with polyethylene glycol having amino group terminus) was swollen in 5 ml of dry N,N-dimethylformamide for several hours, after which the excess solvent was removed by pipette. 151 mg of benzotriazole-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 119 mg of 2-(4-(triphenylmethylthio)phenyl)acetic acid, and 44 mg of anhydrous 1-hydroxybenzotriazole were dissolved in 1.2 mL of dry N,N-dimethylformamide. 75 mg of diisopropylethylamine, 101 μL was added with stirring, and after 1 minute, the resulting solution was added to the swollen resin. After shaking at room temperature for 2 hours, the resin was filtered off, washed with 3 × 2 mL of N,N-dimethylformamide, and then dried under an argon stream. The resin was suspended in 2 mL of a tetrahydrofuran solution of 2.5% triisopropylsilane and 2.5% water. After 2 minutes, the resin was filtered under argon, and deprotection was repeated. The resin was then filtered under argon, washed three times with 3 mL of degassed acetonitrile, and dried under an argon stream to obtain 203 mg of TentaGel S resin (e.g., compound XXXII) containing mercaptophenyl groups. The amount of mercapto group introduced was determined using Eilmann's procedure and was 0.12 mmol per gram of dry resin. Aliquots of compound XXXII were added to a PBS solution of compound VI in 100 μM. LC-MS analysis demonstrated a time-dependent decrease in compound VI in the mixture.

[0249] [ka]

[0250] The above reaction scheme describes the synthesis of solid phase agent XXXII and the reaction between it and compound VI, which involves the formation of compound XXXIII (for example, a covalent adduct XXXIII between compound VI and solid phase agent XXXII).

[0251] Example 22

[0252] Preparation and use of solid phase agents that bind to one or more compounds of structure I, and their neutralization or decomposition products by ion pair formation.

[0253] H + 500g of Purolite NRW160 polystyrene-divinylbenzene crosslinked resin functionalized with sulfonic acid groups of the form is prepared according to the following steps: Na + Transfer to morphology: The beads were washed with 3 volumes of saturated NaCl solution on a vacuum filter and under a sterile hood, followed by washing with 2 volumes of 1M NaOH. After sterilization with NaOH, the beads were washed with sterile deionized water until the pH of the washing solution became neutral. The beads were incubated with 2 volumes of methanol for 15 minutes, and after removing the methanol, they were rinsed again with 3 volumes of sterile deionized water. Finally, after incubation with methanol (2 volumes), the alcohol was removed by filtration, and the beads were dried under vacuum.

[0254] 50 mg of dried beads were added to 1 mL of phosphate-buffered saline solution containing 100 μM compound X. LC-MS analysis of this mixture showed that the concentration of compound X in the supernatant was reduced to less than 30 nM.

[0255] Example 23

[0256] Preparation of a solid phase cartridge

[0257] Empty polypropylene cartridges measuring 5×50mm, 20×120mm, and 20×200mm (diameter × length, mm, catalog numbers PF-DLE-F0004, PF-DLE-F0025, and PF-DLE-F0040, Interchim, Montlucon Cedex, France), fitted with a polypropylene filter at the bottom, were filled with solid phase agents. For dry solid phase agents, the cartridges were filled to 2 / 3 of their capacity to accommodate bead swelling when wet. Another polypropylene filter disc was fitted to the top of the cartridge, the cartridge was sealed, and stored at room temperature (dry solid phase agent) or refrigerated (wet solid phase agent). The cartridges can be incorporated into closed-loop processing systems as shown in Figures 2, 3, 5-8.

[0258] Example 24

[0259] Maintenance of cell culture support properties of animal serum treated with compound VI

[0260] Thermoinactivated fetal bovine serum (FBS, catalog number 89510-188, VWR) and thermoinactivated equine serum (HS, catalog number H1138, Sigma) were incubated with 100 μM compound VI at 40 ± 1°C for 60 minutes in a 50 mL sterile conical tube. The control serum was incubated with compound VI diluent only at 40 ± 1°C for 60 minutes. After incubation, compound VI was removed from the treated serum using a cartridge filled with the solid phase agent prepared as described in Examples 22 and 23. After cartridge filtration, the serum was filtered and sterilized using a 0.2 μm syringe filter. The control serum was not incubated at 40°C or exposed to the solid phase agent, but was filtered and sterilized.

[0261] These serums were used at three different concentrations, 5%, 10%, and 20%, to supplement cell growth media. The ability of these media to support the growth of bovine turbinate cells (BTT, fibroblast morphology), porcine testicular cells (PT, epithelial), and two human cell lines, A172 (glioblastoma, astrocyte-like cells) and MCF7 (epithelial mammary cancer cells), was evaluated.

[0262] Cell growth curves: BTT, PT, A172, and MCF7 cells in the early stages of confluence were trypsin-treated and seeded in 48-well plates in DMEM supplemented with the treated or control serum described above. The culture medium was changed daily. Live cells were counted every 24 hours using a standard hemocytometer with trypan blue exclusion method. Results are shown as the average cell count per well. At least three wells were used for each dilution.

[0263] Clonal Growth: BTT, PT, A172, and MCF7 cells in the early stages of confluence were trypsin-treated, serially diluted (1:2), and seeded in 6 replicates in 96-well plates in DMEM supplemented with the treated or control serum. The medium was changed every 2 days for 16 days. The presence of clonal growth was determined by visual inspection of each well. Results for the last four dilutions are shown, but cell growth was observed as the number of wells with growth from a total of 6 replicates per dilution.

[0264] Long-term culture: BTT, PT, A172, and MCF7 cell lines were grown in medium supplemented with control or treated FBS or HS (BTT cells only) for 10 passages at 3-4 day intervals, in the same manner as described above. Cell and monolayer morphology was tracked and evaluated daily using phase-contrast microscopy.

[0265] Cell Growth Results: Typical growth curves are shown in Figure 22. All growth curves exhibited similar patterns: a typical stagnation phase was observed first in all cell lines and culture media, followed by a gradual logarithmic growth phase. As expected, the highest growth rates were observed in all cell lines cultured in media containing 20% ​​serum. Growth in media supplemented with 10% serum was intermediate, while cell proliferation was significantly reduced in media containing 5% serum. No statistically significant differences in cell growth rates were observed in any cell line or at any serum concentration in the presence of control serum, pseudo-treated serum, or compound VI treated serum.

[0266] Figure 22 shows the effect of dummy-treated serum or compound VI-treated serum on the growth of four different cell lines in a 48-well plate, measured over a period of 6–7 days. A, porcine PT cells; B, human A172 cells; C, human MCF-7 cells; D, bovine BTT cells grown in FBS-containing medium; E, bovine BTT cells grown in HS-containing medium. The T0 column represents the cell count at seeding; the first of three columns (days 1–7) – the number of cells in the wells containing medium supplemented with untreated control serum; the second of three columns (days 1–7) – the number of cells in the wells containing medium supplemented with dummy-treated serum; the third of three columns (days 1–7) – the number of cells in the wells containing medium supplemented with compound VI-treated serum. Each time point represents the mean of three wells. Error bars represent the standard deviation.

[0267] Clone Growth Results: Another important characteristic of serum is its ability to support cell growth at extremely low seeding densities (clonal growth). Table 9 shows the presence of serially diluted cell growth in four final dilutions. These results indicate that clonal growth in all four cell lines was unaffected by serum treatment.

[0268] [Table 9]

[0269] Long-term culture results: No visual differences in cell growth / appearance or morphology were observed between cells maintained in a medium containing compound VI-treated serum and cells in a control medium over 10 consecutive passages, resulting in a moderate or confluent monolayer.

[0270] Example 25

[0271] Tests to determine the ability of compound VI-treated fetal bovine serum to maintain its ability to support viral development and infectivity.

[0272] Serially diluted stock solutions of porcine parvovirus (PPV, ATCC#VR-742) and bovine viral diarrhea virus (BVDV, ATCC#VR-534) were added to porcine testicular cells (PT, PT; ATCC#CRL-1746) and bovine turbinate cells (BTT, ATCC;#CRL-1390), respectively. After adsorption, media supplemented with control or compound VI-treated FBS prepared as described in Example 24 was added. Aliquots from all virus-contaminated samples (treated serum, pseudo-treated serum, or untreated serum) were serially diluted (1:5 or 1:10) with serum-free DMEM, and 25 μL from each dilution was seeded in 3 replicates into their respective indicator cells in 96-well plates. The plates were incubated in a 5% CO2 incubator at 37°C for 60 minutes to adsorb the virus. Undiluted samples were used to infect host cells in 24-well plates or 10 cm Petri dishes to increase the detection limit. After adsorption, DMEM / 5% FBS was packed into all wells without aspirating 25 μL of dilution, and the plates were further incubated in a CO2 incubator at 37°C for 6-7 days. The development of the cytopathic effect caused by the virus in each well was detected by visual inspection, and the log was read. 10 TCID 50 This was used to calculate each viral titer, expressed as / mL. The detection limit was 0.2 infectious particles per mL. In some cases, to corroborate the assay results, the supernatant from the inoculated wells was collected after 6–7 days and used to infect fresh cells in a 24-well plate.

[0273] The results of the viral titer measurements shown in Table 10 indicate that the control medium supplemented with untreated FBS and the medium supplemented with compound VI-treated serum have essentially the same viral infection support properties in the test cells.

[0274] [Table 10]

[0275] Example 26

[0276] Quality of whole blood and red blood cells (RBCs) treated with a compound of structure I

[0277] 10 mL of whole blood sample or 25 mL of concentrated red blood cell (RBCC) was treated with 500 μM compound VI for 6 hours at room temperature. The remaining compound VI was neutralized with the same volume of 10 mM sodium thiosulfate for 2 hours at room temperature. For controls, the same whole blood or RBCC sample was treated with physiological saline and sodium thiosulfate without compound VI, or with physiological saline alone without compound VI and / or thiosulfate. Aliquots of whole blood and RBCC from each sample were subjected to whole blood count and biochemical analysis using an IDEXX Procyte Dx blood analyzer and an IDEXX Catalyst Dx chemical analyzer according to the manufacturer's recommendations. Samples were analyzed immediately after treatment, and re-analyzed for whole blood one week later, and for RBCC weekly over 5 weeks while stored at 4–6°C. The following parameters were measured: RBC count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, red blood cell distribution, reticulocyte count, platelets, mean platelet volume, leukocytes, neutrophils, lymphocytes, monocytes, eosinophils, basophils, chlorine, potassium, sodium, glucose, and lactate concentrations. After weekly testing, within the precision and accuracy limits of the analyzer, no differences in cellular or biochemical characteristics were observed between the treated samples and the control for all measured parameters (RBC count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, red blood cell distribution, reticulocyte count, platelets, mean platelet volume, leukocytes, neutrophils, lymphocytes, monocytes, eosinophils, basophils, chlorine, potassium, sodium, glucose, and lactate concentrations).

[0278] Embodiments of the present invention The present invention provides the following non-limiting embodiments:

[0279] Embodiment 1. A method for inactivating or reducing pathogens or undesirable organisms from a sample, (i) Compounds having structure I:

[0280] [ka]

[0281] [During the ceremony, Each R1 is independently selected from H, CH3, CH2CH3, CH(CH3)2, Cl, F, alkyl group, alkenyl group, phenyl group, alkyloxy group, acyloxy group, or substituted alkyl group. Each R2 independently represents H, CH3, CH2CH3, CH(CH3)2, alkyl group, alkenyl group, phenyl group, cycloalkyl group, alkyloxy group, or substituted alkyl, substituted alkenyl, substituted cycloalkyl or substituted phenyl group, or part of structure II:

[0282] [ka]

[0283] Selected from, Each R3 is independently selected from H, CH3, CH2CH3, CH(CH3)2, Cl, F, alkyl group, alkenyl group, phenyl group, alkyloxy group, acyloxy group, or other substituted alkyl groups. Each instance of n is independently 3, 4, or 5. Each m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 in each occurrence. or treatment of the sample with a chemically acceptable salt, hydrate, or solvate thereof, (ii) Incubation for a period of time sufficient to inactivate or reduce pathogens or undesirable organisms from the sample. (iii) Treatment of the sample with one or more neutralizing agents that eliminate or reduce the toxicity or other undesirable properties of the compound of structure I. The method, including the method described above.

[0284] Embodiment 2. The compound having structure I has structure IA:

[0285] [ka]

[0286] [During the ceremony, Each R2 independently represents H, alkyl group, CH3, CH2CH3, CH(CH3)2, alkenyl group, phenyl group, cycloalkyl group, alkyloxy group, or substituted alkyl, alkenyl, cycloalkyl, phenyl group, or part of structure IIA:

[0287] [ka]

[0288] Selected from, Each R3 is independently selected from H, Cl, F, alkyl group, CH3, CH2CH3, CH(CH3)2, alkenyl group, phenyl group, alkyloxy group, acyloxy group, or substituted alkyl group. Each instance of 'a' is independently selected from 1, 2, or 3. Each instance of b is independently selected from 0, 1, 2, 3, 4, 5, or 6. The method according to embodiment 1, wherein the method is characterized by having the following features.

[0289] Embodiment 3. The compound having structure I has structure IB:

[0290] [ka]

[0291] [During the ceremony, Each R2 is independently selected from H, CH3, CH2CH3, or CH(CH3)2 each time it appears. Each R3 is independently selected from H, CH3, CH2CH3, or CH(CH3)2 each time it appears. Each instance of 'a' is independently selected from 1, 2, or 3. [b is selected from 0, 1, 2, 3, 4, 5, or 6] The method according to embodiment 1, wherein the method is characterized by having the following features.

[0292] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the one or more neutralizing agents are nucleophilic compounds that react with the aziridine ring of the compound of structure I, IA, or IB to open its ring, thereby eliminating the alkylation properties of the compound of structure I, IA, or IB.

[0293] Embodiment 5. The method according to Embodiment 4, wherein the one or more neutralizing agents are organic polymers that are soluble in an aqueous medium and contain thiosulfate, preferably sodium thiosulfate, thiophosphate, preferably sodium thiophosphate, thiourea or substituted thiourea, thiocarboxylic acid and its salts, dithiocarboxylic acid and its salts, thiocarbonate, dithiocarbonate, salt of thiocarbonate O-ester, salt of dithiocarbonate O-ester, mercaptan i.e. thiol or its salts, or substituted mercaptan i.e. substituted thiol, or polymercaptan i.e. polythiol and its salts, or any combination thereof, or covalently bonded mercapto i.e. thiol groups, thiosulfate, thiophosphate, thiourea, thiocarboxylic acid, dithiocarboxylic acid, thiocarbonate O-ester, dithiocarbonate O-ester group, or a combination thereof.

[0294] Embodiment 6. The one or more neutralizing agents include sodium thiosulfate, 2-mercaptoethanol, 2-(methylamino)ethanethiol, 2-aminoethanethiol, 2-(dimethylamino)ethanethiol, 2-mercapto-N,N,N-trimethylethaneaminium and its salts, thiocarboxylic acid and its salts, thioacetic acid and its salts, thiopropionic acid and its salts, thiooxalic acid and its salts, thiomalonic acid and its salts, thiosuccinic acid and its salts, thioglycolic acid and its salts, thiolactic acid and its salts, dithiocarboxylic acid and its salts, dithioacetic acid and its salts, 2-mercaptoacetic acid and its salts, 2-mercaptopropionic acid and its salts, 2-mercaptoethyl acetate, 2-mercaptosuccinic acid and its salts and esters, 2-(methylsulfonyl)methanethiol The method according to embodiment 5, wherein the substances are (ethylsulfonyl)methanethiol, sulfonyl dimethanethiol, 2,2,2-trifluoroethanethiol, 1H-imidazole-5-thiol, imidazolidine-2-thiol, 1,3-dimethylimidazolidine-2-thiol, pyridine-2-thiol, 4-thioxo-3,4-dihydropyrimidine-2(1H)-one, 2-thioxodihydropyrimidine-4,6(1H,5H)-dione, 2-mercaptobenzoic acid and its salts, 4-mercaptobenzoic acid and its salts, thiophenol, 2-,3- or 4-mercaptonisole, 2-mercaptopropane-1,2-diol, 2,3-dimercaptopropanol, or 1,3-dimercapto-2-propanol, and combinations thereof.

[0295] Embodiment 7. The pK of the dissociation of the -SH group of the mercaptan, i.e., thiol, of the neutralizing agent. a The method according to embodiment 5, wherein the pH is 4 to 10, preferably 5 to 9, more preferably 6 to 8, or near the pH of the medium to be treated.

[0296] Embodiment 8. The method according to Embodiment 5, wherein the mercaptan, i.e., the thiol, of the neutralizing agent has an -SH group that is directly bonded to a double bond, or to an aromatic ring structure, or to a fully or partially sp2 hybridized carbon atom.

[0297] Embodiment 9. The method according to Embodiment 5, wherein the neutralizing agent comprises at least one electron-accepting group, for example, a sulfone group (-S(O2)-R), a sulfoxide group (-S(O)-R), an ester group (-C(O)OR), or an amide group (-C(O)NH2, -C(O)NHR, -C(O)NR2), where R is any alkyl or substituted alkyl group, and the electron-accepting group is bonded to the carbon atom to which the SH group is bonded.

[0298] Embodiment 10. The method according to any one of Embodiments 1 to 9, wherein the neutralizing agent is optionally covalently bonded to a solid support via a linking group.

[0299] Embodiment 11. The method according to any one of Embodiments 1 to 10, wherein the sample containing the remaining amount of the compound of structure I is contacted with one or more neutralizing agents over a period of 1 minute to 48 hours, preferably 20 minutes to 24 hours, more preferably 60 minutes to 8 hours, at a temperature of 0 to 100°C, preferably 10 to 60°C, more preferably 20 to 40°C, at a pH of 1 to 14, preferably 4 to 9, more preferably 6 to 8, at a concentration of 1 M or less, preferably 0.1 M or less, more preferably 10 mM or less.

[0300] Embodiment 12. The method according to any one of Embodiments 1 to 11, wherein the concentration of the remaining compound having structure I decreases by at least 2 logarithms, preferably at least 3 logarithms, more preferably at least 4 logarithms, even more preferably at least 5 logarithms, even more preferably at least 6 logarithms, even more preferably at least 7 logarithms, even more preferably at least 8 logarithms, even more preferably at least 9 logarithms, and even more preferably at least 10 logarithms after treatment with the neutralizing agent.

[0301] Embodiment 13. After contacting the remaining compound of structure I with the neutralizing agent, the product of neutralization or decomposition of the compound of structure I, and / or the excess neutralizing agent, is a solid-phase agent insoluble in the medium being treated, and is porous, microporous, The method according to any one of embodiments 1 to 12, wherein the solid phase agent may be macroporous or gel-like or nonporous, highly dispersible, high-surface-area solid, and may have the shape of beads or particles of various diameters from 1 μm to 1 cm, and is partially or completely removed from the sample to be treated by treatment with the solid phase agent which chemically reacts with and covalently bonds, absorbs, or otherwise sequestrates the product of the neutralization or decomposition of the compound(s) of structure I, and subsequently removes the solid phase agent, preferably by filtration, sedimentation, or centrifugation, or the treatment is carried out by filtration of the medium or composition with a cartridge containing the solid phase agent, or by contact of the medium or composition with the solid phase agent via a permeable or semipermeable membrane, and the treatment may be carried out once, twice, or more times, or until a desired reduction of the compound of structure I is achieved, and the treatment may be carried out by a single solid phase agent or by two or more different solid phase agents, either used sequentially or as a mixture.

[0302] Embodiment 14. The method according to Embodiment 13, wherein the solid phase agent absorbs the products of the neutralization or decomposition of the compound of structure I, and / or any excess neutralizing agent.

[0303] Embodiment 15. The solid phase agent is activated carbon, or reversed phase resin, or porous or microporous hydrophobic organic polymer, for example, polystyrene resin, or divinylbenzene crosslinked polystyrene resin, or C4-C 18 The method according to embodiment 14, wherein the polyacrylate or polymethacrylate resin is modified with a hydrophobic organic group such as an alkyl group.

[0304] Embodiment 16. The method according to Embodiment 15, wherein the solid phase agent is a cation exchange resin or anion exchange resin, and when the neutralizing agent is anionic or cationic at the pH of the treatment, it forms ion pairs with the product of neutralization or decomposition of the compound of structure I, and / or excess neutralizing agent.

[0305] Embodiment 17. The method according to Embodiment 16, wherein the cation exchange resin is an organic polymer, preferably crosslinked, and possesses anionic groups, such as sulfo, sulfone, or carboxyl groups, which are in an ion-pairing form with cations, such as sodium, potassium, or ammonium or substituted ammonium cations or hydrogen cations.

[0306] Embodiment 18. The method according to Embodiment 16, wherein the anion exchange resin is an organic polymer, preferably crosslinked, and possesses a cationic group, for example, a protonated amino or alkyl-substituted amino group, for example, a mono-, di-, or trimethylamine group, or a quaternary ammonium group, for example, a tetramethylammonium group, wherein the group is in an ion-pairing form with an anion, for example, a chloride, sulfate, citrate, or hydroxyl anion.

[0307] Embodiment 19. The solid phase agent is a polymer, preferably crosslinked, and bonded to a thiosulfate group that forms an ion pair with an acceptable cation such as sodium, and having the formula PRS-SO3 - Na + The formula has a component where P is a polymer, R is a covalent bond or any divalent linker, and the group is replaced by an exchange reaction with the excess R of the formula. 1 SH or R 1 S - Cat + It reacts with mercapto, i.e., thiol-type neutralizing agents, in the formula, Cat + Since sodium is an acceptable cation, the following formula PRSSR 1 The covalent bond between the inactivator and the polymer is represented by a disulfide bond, and the thiosulfate anion S2O3 2-This results in the liberation of; or the polymer is bonded to an epoxy or substituted epoxy directly or via a linker, and the epoxy group is an excess of formula R 1 SH or R 1 S - Cat + The mercapto, or thiol-type, neutralizing agent reacts with the epoxy group to open the ring and covalently bond the neutralizing agent to the polymer, in which Cat + The method according to embodiment 13, wherein is an acceptable cation such as sodium.

[0308] Embodiment 20. The method according to any one of Embodiments 1 to 19, wherein the sample is a composition, a utility product, a surface, an apparatus, or a living organism.

[0309] Embodiment 21. The method according to any one of Embodiments 1 to 19, wherein the sample is blood or blood products, body fluids, culture media of eukaryotic or prokaryotic origin, vaccine formulations, biologics or biological preparations, clinical samples, biopsy materials, research samples, cosmetics, pharmaceutical compositions, consumables, equipment, underwater fluid conduits, pipes, hoses, heat exchangers or surface vessels, and their surfaces.

[0310] Embodiment 22. The method according to any one of Embodiments 1 to 19, wherein the sample is blood or a blood product.

[0311] Embodiment 23. A method for inactivating, reducing, or removing pathogens or undesirable organisms from a sample, Compounds having structure I:

[0312] [ka]

[0313] [During the ceremony, Each R1 is independently selected from H, Cl, F, alkyl groups, CH3, CH2CH3, CH(CH3)2, alkenyl groups, phenyl groups, alkyloxy groups, acyloxy groups, or other substituted alkyl groups. Each R2 independently represents H, CH3, CH2CH3, CH(CH3)2, alkyl group, alkenyl group, phenyl group, cycloalkyl group, alkyloxy group, or substituted alkyl, alkenyl, cycloalkyl or phenyl group, or part of structure II:

[0314] [ka]

[0315] Selected from, n is independently 3, 4, or 5 in each occurrence. m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 in each occurrence. or treatment of the sample with a chemically acceptable salt, hydrate, or solvate thereof, and Incubation for a sufficient period of time to enable the desired effect of the compound or the compound having structure I on the subsequent appearance of the pathogen or undesirable organism; (ii) Treatment of the sample with a solid phase agent insoluble in the medium to be treated, which may be porous, microporous, macroporous, or gel-like, or may be a nonporous, highly dispersible, high-surface-area solid, which may have the shape of beads or particles of various diameters such as 1 μm to 1 cm, and which causes chemical reactions and covalent bonding, absorption, or sequestration of the remaining compound of structure I or its decomposition products (may be multiple); (iii) Removal of the solid phase agent, preferably by filtration, sedimentation, or centrifugation. The method comprising; or the treatment being carried out by filtering the sample with a cartridge containing the solid phase agent, or by contact of the sample with the solid phase agent via a permeable or semipermeable membrane; the treatment being carried out once, twice or more times, or until a desired reduction of the compound of structure I or the products of its decomposition is achieved, and the treatment being carried out by a single solid phase agent or by two or more different solid phase agents, either used sequentially or as a mixture.

[0316] Embodiment 24. The compound having structure I has structure IA:

[0317] [ka]

[0318] [During the ceremony, Each R2 independently represents H, alkyl group, CH3, CH2CH3, CH(CH3)2, alkenyl group, phenyl group, cycloalkyl group, alkyloxy group, or substituted alkyl, alkenyl, cycloalkyl, phenyl group, or part of structure IIA:

[0319] [ka]

[0320] Selected from, Each R3 is independently selected from H, Cl, F, alkyl group, CH3, CH2CH3, CH(CH3)2, alkenyl group, phenyl group, alkyloxy group, acyloxy group, or substituted alkyl group. Each instance of 'a' is independently selected from 1, 2, or 3. Each instance of b is independently selected from 0, 1, 2, 3, 4, 5, or 6. The method according to embodiment 23, having the following characteristics.

[0321] Embodiment 25. The compound of structure I has structure IB:

[0322] [ka]

[0323] [During the ceremony, Each R2 is independently selected from H, CH3, CH2CH3, or CH(CH3)2 each time it appears. Each R3 is independently selected from H, CH3, CH2CH3, or CH(CH3)2 each time it appears. Each instance of 'a' is independently selected from 1, 2, or 3. [b is selected from 0, 1, 2, 3, 4, 5, or 6] The method according to embodiment 23, having the following characteristics.

[0324] Embodiment 26. The method according to any one of Embodiments 23 to 25, wherein the solid phase agent contains a reactive group that undergoes a chemical reaction and covalent bonding with the compound of structure I.

[0325] Embodiment 27. The reactive group is capable of reacting with the aziridine ring of the compound of structure I to open its ring, and is a nucleophilic group, for example, thiosulfate-OS(O)(O - )S - , or thiosulfonate -S(O)(O - )S - , or mercapto, i.e., thiol groups -SH, -CH2SH, -CH2CH2SH, -CF2CH2SH, -OCH2CH2SH, -NH2CH2CH2SH, -NH(Me)CH2CH2SH, -N(Me2)CH2CH2SH, -COCH2SH, -S(O2)CH2SH, -thiourea -NHC(S)NH2, or substituted thiourea groups, thiocarboxylic acid -C(O)S - , dithiocarboxylic acid -C(S)S - , thiocarbonate O-ester -OC(O)S - Dithiocarbonate O-ester or xanthaute -OC(S)S - The method according to embodiment 26, wherein the salts are thiophosphonate -PO(OH)SH, thiophosphate -OPO(OH)SH, o-, m- or p-thiophenyl group -C6H4SH, thiosalilate group, m- or p-thiobenzoate group -O2CC6H4SH, or salts thereof.

[0326] Embodiment 28. The mercapto, i.e., thiol, i.e., -SH group, is attached to a double bond, or to an aromatic ring structure, or is attached to a complete or partial sp 2 The method according to embodiment 27, wherein the hybrid carbon atoms are directly linked.

[0327] Embodiment 29. The -S of the -SH group - and H+ pK of dissociation to a The method according to embodiment 27 or embodiment 28, wherein is less than 10, preferably less than 9, and most preferably less than 8.

[0328] Embodiment 30. The method according to any one of Embodiments 23 to 29, wherein the solid phase agent is a porous, microporous, or gel-like organic polymer.

[0329] Embodiment 31. The method according to Embodiment 30, wherein the organic polymer is a hydrophilic organic polymer or a polymer capable of wetting, expanding, or swelling in an aqueous medium.

[0330] Embodiment 32. The method according to Embodiment 30 or Embodiment 31, wherein the organic polymer is preferably crosslinked and is a polystyrene polymer, or a polyacrylate polymer, or a polymethacrylate polymer, or a polyurethane polymer, or a polyamide polymer, or a dextran polymer, for example, but not limited to Sephadex®, or an agarose polymer, for example, but not limited to Sepharose®, or a cellulose polymer, or a modified cellulose polymer, for example, but not limited to carboxymethylcellulose or diethylaminoethylcellulose or methylcellulose, or another polysaccharide polymer, or any other linear, branched or crosslinked, homo or heteropolymer or iso-type or atactic-type configuration or other stereoregularity block copolymer, or any other suitable macromolecule insoluble in the medium to be treated.

[0331] Embodiment 33. The method according to any one of embodiments 27 to 32, wherein the nucleophilic group may be one of various types and may be directly bonded to the main chain of the polymer, or may be a divalent group, for example, but not limited to an oxygen atom, a sulfur atom, an -NH- group, a methylene group, a monosubstituted or disubstituted methylene group, an ethylene or substituted ethylene group, a propylene or substituted propylene group, an oxymethylene or oxyethylene group, or a divalent, trivalent or polyvalent linker, for example, but not limited to an oligo or polyoxyethylene, an oligo or polyester, or a polyamide-type linker, wherein the linker may be linear, branched or dendrimer-type and may contain one or more or many nucleophilic groups bonded thereto.

[0332] Embodiment 34. The method according to any one of embodiments 30 to 33, wherein the polymer also contains, but is not limited to, a group that supports the reaction between the nucleophilic group and the nucleophilic group without reacting with the compound of structure I, by means of so-called neighboring effects or neighboring electron pair effects, by enhancing the deprotonation of the nucleophilic group, by forming hydrogen bonds with the nucleophilic group, by interacting with the transition state formed between the compound of structure I and the nucleophilic group and lowering its energy, by forming non-covalent bonds or ion pairs with the compound of structure I and consequently increasing its local concentration, or by protonating the aziridine nitrogen of the compound(s) of structure I and consequently increasing their reactivity, thereby enhancing the nucleophilicity of the nucleophilic group.

[0333] Embodiment 35. The method according to any one of Embodiments 30 to 34, wherein a sufficient number of hydrophilic groups are bonded to the organic polymer to increase the polymer hydrophilicity or wettability, or to improve polymer properties, such as, but not limited to, inertness to the sample or the composition or components of a biological or biological fluid.

[0334] Embodiment 36. The organic polymer is divinylbenzene crosslinked polystyrene, and the polar group is an ethylene glycol oligomer or polyethylene glycol, or a sulfo group (sulfonic acid group, -SO3) having a molecular weight of 150 to 100,000 Da, preferably 2,000 to 40,000 Da, more preferably 4,000 to 20,000 Da, and a density of one or fewer groups per monomer unit. - The method according to embodiment 35, wherein the polymer is an acrylate or methacrylate polymer and the polar group is a polyol, for example, but not limited to, 2-hydroxyethyl, 2,3-dihydroxypropyl, di-, tri-, tetra-, penta- or oligo- or polyethylene glycol, and the polar group is bonded to the C1 i.e., carbonyl group of the acrylate or methacrylate polymer at a density sufficient to acquire desired hydrophilicity or other beneficial properties, and the other beneficial properties may be, but not limited to, non-immunogenicity, non-thrombogenicity, or non-binding or affinity to proteins or receptors, or to other components of the sample or composition or body fluid to be treated.

[0335] Embodiment 37. The method according to any one of Embodiments 23 to 36, wherein the solid phase agent forms multiple ion pairs with the positively charged nitrogen atoms of the remaining structure I compound.

[0336] Embodiment 38. The method according to Embodiment 37, wherein the solid phase is an organic polymer, a microporous or macroporous or gel-like organic polymer, preferably crosslinked and possessing anionic groups, such as sulfo, sulfone, or carboxyl groups, which are in an ion-pairing form with cations, such as sodium, potassium, or ammonium or substituted ammonium cations or hydrogen cations.

[0337] Embodiment 39. The method according to Embodiment 38, wherein the polymer is a divinyl crosslinked polystyrene polymer containing sodium-type sulfone groups at a density of 1.5 milliequivalents or less per gram of polymer.

[0338] Embodiment 40. The method according to Embodiment 38, wherein the polymer is a diacrylate-crosslinked polyacrylate or methacrylate, and the anionic group is a sodium-type sulfone or carboxyl group with a density of 4 milliequivalents or less per gram of polymer.

[0339] Embodiment 41. The method according to any one of Embodiments 1 to 40, wherein the pathogen or undesirable organism is an infectious disease-causing organism, for example, an infectious disease-causing organism, including, but not limited to, enveloped viruses and non-enveloped viruses, DNA or RNA viruses and viruses including bacteriophages, prions, prokaryotes, bacteria including Gram-positive or Gram-negative bacteria, spore-forming bacteria or bacterial spores, mycoplasmas, archaea, and bacterial films; eukaryotes, unicellular or multicellular eukaryotes, including but not limited to fungi, protozoa, unicellular or multicellular parasites, parasitic helminths, schistosomiasis or nematodes or their eggs, unicellular or multicellular algae, and crustaceans, or any combination thereof, including exudates, biofilms or biofouling systems.

[0340] Embodiment 42. The sample to be treated is selected from human or animal blood, leukocyte-depleted blood, and blood products, such as plasma, red blood cells, platelets, serum or plasma components, factors or enzymes, blood and blood components intended for intravenous administration, apheresis blood components, body fluids, animal serum, such as serum used as a cell culture additive, culture media of eukaryotic or prokaryotic origin, vaccine formulations, cosmetics and pharmaceutical compositions; the practical item may be any industrial or household equipment, electrical appliance, apparatus, mechanism, machine or material, or may contain pathogens, microorganisms or other organisms that are undesirable or need to be controlled. The method according to any one of embodiments 1 to 41, wherein the surface may be any other article that may be; the surface may be an electrical appliance, apparatus or device, for example, a pipe, duct, hose, pipeline, vent, heat exchanger, sewer pipe, flow path or any other conduit for fluids or gases where the presence of pathogens, microorganisms or other organisms, including biological contamination, is undesirable or needs to be controlled, or the surface of any object in contact with a fluid, for example, a ship, screen or filter; and the organism may be an animal, mammal or human, or a part thereof, for example, a biological sample, a biological preparation and a biopsy material.

[0341] Embodiment 43. The method according to any one of Embodiments 1 to 42, wherein the pathogen(s) or microorganism(s)(s) are treated with a composition containing one or more compounds of the structure I, the composition may be formulated as a liquid, solution, gel, solid, powder, or particles, or may be encapsulated, dissolved, dispersed, pulverized, micronized or converted into nanoparticles, or may be in other formulation forms or a combination thereof.

[0342] Embodiment 44. The method according to any one of Embodiments 1 to 43, wherein the sample or composition is treated with the compound of Structure I for a period of 1 minute to 48 hours, preferably 20 minutes to 24 hours, more preferably 60 minutes to 8 hours, at a temperature of 0 to 100°C, preferably 10 to 60°C, more preferably 20 to 40°C, at a pH of 1 to 14, preferably 4 to 9, more preferably 6 to 8, at a concentration of 10 nM to 10 mM, preferably 1 μM to 1 mM, and even more preferably 100 to 500 μM.

[0343] Embodiment 45. The method according to any one of Embodiments 1 to 44, wherein the titer of at least one pathogen or undesirable organism present in the sample to be treated is reduced by at least 50%, preferably at least 1 logarithmically, more preferably at least 2 logarithms, even more preferably at least 3 logarithms, even more preferably at least 4 logarithms, even more preferably at least 5 logarithms, even more preferably at least 6 logarithms, even more preferably at least 7 logarithms, even more preferably at least 8 logarithms, even more preferably at least 9 logarithms, even more preferably at least 10 logarithms or more.

[0344] Embodiment 46. The method according to any one of Embodiments 1 to 45, wherein the pathogen(s) or microorganism(s)(s) are present in a living organism, the organism may be a mammal or a human, and treatment with the compound of structure I, or a formulation of the compound of structure I, is carried out in vivo by intravenous, oral, topical, rectal, subcutaneous, intramuscular, inhalation, or a combination thereof, and the treatment can be carried out by single, multiple, or sequential administration in a dose(s) sufficient to achieve the desired reduction of the pathogen(s).

[0345] Embodiment 47. The method according to Embodiment 46, wherein the removal, neutralization, or inactivation of the compound of structure I, and the optional removal of the neutralization product and / or excess neutralizing agent of the compound of structure I, are performed by extracellular treatment of the body fluids of the organism, and the body fluids are returned to the original organism or administered intravenously.

[0346] Embodiment 48. The method according to any one of Embodiments 1 to 47, wherein the pathogen(s) or microorganism(s) are present in an animal or human, and the treatment with the compound of structure I, and the removal or neutralization of the compound of structure I and optionally the products of their neutralization or degradation and / or excess neutralizing agent are carried out in vitro by treatment of the bodily fluids of the animal or human, for example, blood or plasma which may be collected by apheresis, and after the treatment the fluid is returned to the original animal or human or administered intravenously.

[0347] Embodiment 49. The method according to any one of Embodiments 1 and 48, wherein at least one of the pathogen or undesirable organism may be resistant to one or more antipathogen treatments or may not be susceptible to any treatment other than treatment with the compound of structure I.

[0348] Embodiment 50. The method according to any one of Embodiments 1 to 49, wherein the compound of structure I is in the form of a salt with an organic or inorganic anion, preferably a less nucleophilic anion, for example, a sulfate, perchlorate, methanesulfonate or tetrafluoroborate, or in the form of a solid solution with a solid having good water solubility and a melting point of less than 40°C, greater than 40°C and less than 120°C, for example, a polyethylene glycol of various molecular weights, but not limited to these.

Claims

1. A method for inactivating or reducing pathogens or undesirable organisms from a sample, (i) Compounds having structure I: 【Chemistry 1】 [During the ceremony, Each R 1 Each instance of appearance is independent of H and CH. 3 ,CH 2 CH 3 , CH (CH 3 ) 2 Selected from Cl, F, alkyl groups, alkenyl groups, phenyl groups, alkyloxy groups, acyloxy groups, or substituted alkyl groups, Each R 2 is independently, for each occurrence, H, CH 3 , CH 2 CH 3 , CH(CH 3 ) 2 , an alkyl group, an alkenyl group, a phenyl group, a cycloalkyl group, an alkyloxy group, or a substituted alkyl, substituted alkenyl, substituted cycloalkyl or substituted phenyl group, or a moiety of Structure II: 【Chemistry 2】 Selected from, Each R 3 Each instance of appearance is independent of H and CH. 3 ,CH 2 CH 3 , CH (CH 3 ) 2 Selected from Cl, F, alkyl groups, alkenyl groups, phenyl groups, alkyloxy groups, acyloxy groups, or other substituted alkyl groups, Each instance of n is independently 3, 4, or 5. Each instance of m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. or treatment of the sample with a chemically acceptable salt, hydrate, or solvate thereof, (ii) Incubation for a period of time sufficient to inactivate or reduce pathogens or undesirable organisms from the sample. (iii) (a) by one or more neutralizing agents that eliminate or reduce the toxicity or other undesirable properties of the compound having the structure I, or (b) Absorption of the compound having structure I, or covalent bonding with it, or sequestering therewith by one or more solid phase agents, Processing of the aforementioned sample The method, including the method described above.

2. The compound with structure I has structure IA: 【Transformation 3】 [During the ceremony, Each R 2 Each instance is independently determined as H, alkyl, CH 3 ,CH 2 CH 3 , CH (CH 3 ) 2 , alkenyl group, phenyl group, cycloalkyl group, alkyloxy group, or substituted alkyl, alkenyl, cycloalkyl, phenyl group, or part of structure IIA: 【Chemistry 4】 Selected from, Each R 3 Each instance is independently determined as H, Cl, F, alkyl, CH 3 ,CH 2 CH 3 , CH (CH 3 ) 2 Selected from alkenyl groups, phenyl groups, alkyloxy groups, acyloxy groups, or substituted alkyl groups, Each instance of 'a' is independently selected from 1, 2, or 3. Each instance of b is independently selected from 0, 1, 2, 3, 4, 5, or 6. The method according to claim 1, comprising:

3. The compound with structure I has structure IB: 【Transformation 5】 [During the ceremony, Each R 2 Each instance of appearance is independent of H and CH. 3 ,CH 2 CH 3 , or CH(CH 3 ) 2 Selected from, Each R 3 Each instance of appearance is independent of H and CH. 3 ,CH 2 CH 3 , or CH(CH 3 ) 2 Selected from, Each instance of 'a' is independently selected from 1, 2, or 3. b is selected from 0, 1, 2, 3, 4, 5, or 6. The method according to claim 1, comprising:

4. The method according to any one of claims 1 to 3, wherein the one or more neutralizing agents are nucleophilic compounds that react with the aziridine ring of the compound of structure I and open its ring, thereby eliminating the alkylation properties of the compound of structure I.

5. The one or more neutralizing agents mentioned above Thiosulfates, preferably sodium thiosulfate; thiophosphates, preferably sodium thiophosphate; thiourea or substituted thiourea; thiocarboxylic acids and their salts; dithiocarboxylic acids and their salts; thiocarbonates; dithiocarbonates; salts of thiocarbonate O-esters; salts of dithiocarbonate O-esters; mercaptans, i.e., thiols or their salts; or substituted mercaptans, i.e., substituted thiols; or polymercaptans, i.e., polythiols and their salts; or any combination thereof; Organic polymers containing mercaptos, i.e., thiol groups, thiosulfates, thiophosphates, thioureas, thiocarboxylic acids, dithiocarboxylic acids, thiocarbonate O-esters, dithiocarbonate O-esters, or combinations thereof, which are covalently bonded, and which are soluble in aqueous media. The method according to claim 4.

6. The one or more neutralizing agents mentioned above include sodium thiosulfate, 2-mercaptoethanol, 2-(methylamino)ethanethiol, 2-aminoethanethiol, 2-(dimethylamino)ethanethiol, 2-mercapto-N,N,N-trimethylethaneaminium and its salts, thiocarboxylic acid and its salts, thioacetic acid and its salts, thiopropionic acid and its salts, thiooxalic acid and its salts, thiomalonic acid and its salts, thiosuccinic acid and its salts, thioglycolic acid and its salts, thiolactic acid and its salts, dithiocarboxylic acid and its salts, dithioacetic acid and its salts, 2-mercaptoacetic acid and its salts, 2-mercaptopropionic acid and its salts, 2-mercaptoethyl acetate, 2-mercaptosuccinic acid and its salts and esters, and 2-(methylsulfonyl)methanethiol. The method according to claim 5, wherein the member is (ethylsulfonyl)methanethiol, sulfonyl dimethanethiol, 2,2,2-trifluoroethanethiol, 1H-imidazole-5-thiol, imidazolidine-2-thiol, 1,3-dimethylimidazolidine-2-thiol, pyridine-2-thiol, 4-thioxo-3,4-dihydropyrimidine-2(1H)-one, 2-thioxodihydropyrimidine-4,6(1H,5H)-dione, 2-mercaptobenzoic acid and its salts, 4-mercaptobenzoic acid and its salts, thiophenol, 2-,3- or 4-mercaptonisole, 2-mercaptopropane-1,2-diol, 2,3-dimercaptopropanol, or 1,3-dimercapto-2-propanol, and combinations thereof.

7. The method according to any one of claims 1 to 5, wherein the neutralizing agent is covalently bonded to the solid support.

8. The method according to claim 7, wherein the solid phase support is a porous, microporous, or gel-like organic polymer.

9. The method according to claim 8, wherein the organic polymer is a hydrophilic organic polymer or a polymer capable of wetting, expanding, or swelling in an aqueous medium.

10. The aforementioned organic polymer is preferably crosslinked, Polystyrene polymers, or polyacrylate polymers, or polymethacrylate polymers, or polyurethane polymers, or polyamide polymers, or dextran polymers, for example, but not limited to Sephadex®, or agarose polymers, for example, but not limited to Sepharose®, or cellulose polymers, or modified cellulose polymers, for example, but not limited to carboxymethylcellulose, diethylaminoethylcellulose, or methylcellulose, or other polysaccharide polymers, or any other linear, branched, or crosslinked, homo or heteropolymer, or iso-type or atactic-type configuration or other stereoregularity-having block copolymers, or The method according to claim 8 or 9, which may be any other suitable macromolecule insoluble in the medium to be treated.

11. The nucleophilic group of the neutralizing agent is The polymer is directly bonded to the main chain, or Divalent groups, such as oxygen atoms, sulfur atoms, -NH- groups, methylene groups, monosubstituted or disubstituted methylene groups, ethylene or substituted ethylene groups, propylene or substituted propylene groups, oxymethylene or oxyethylene groups, or divalent, trivalent or polyvalent linkers, which may be linked by oligo or polyoxyethylene, oligo or polyester, or polyamide-type linkers, for example, but are not limited to these. The method according to any one of claims 1 to 10, wherein the linker may be linear, branched, or dendrimer, and may contain one, more than one, or many nucleophilic groups bonded thereto.

12. After contacting the remaining compound of structure I with the neutralizing agent, the products of neutralization or decomposition of the compound of structure I, and / or the excess neutralizing agent, Treatment of the sample to be treated with a solid phase agent that is insoluble in the medium to be treated and that neutralizes or decomposes the compound of structure I and / or reacts with the neutralizing agent, covalently, absorbs, or otherwise severs the product of the reaction and covalent bonding with the neutralizing agent, and The subsequent removal of the sample to be treated from the solid phase agent. The method according to any one of claims 1 to 6, wherein the sample to be treated is reduced or removed by the method thereof.

13. The method according to claim 12, wherein the solid phase agent absorbs the products of the neutralization or decomposition of the compound of structure I, and / or any excess neutralizing agent.

14. The method according to claim 13, wherein the solid phase agent is activated carbon, or a reversed phase resin, or a porous or microporous hydrophobic organic polymer, such as polystyrene resin, or divinylbenzene crosslinked polystyrene resin, or a polyacrylate or polymethacrylate resin modified with a hydrophobic organic group such as a C4-C18 alkyl group.

15. The method according to any one of claims 1 to 14, wherein the sample containing the remaining amount of the compound of structure I is contacted with one or more neutralizing agents for a period of 1 minute to 48 hours, preferably 20 minutes to 24 hours, more preferably 60 minutes to 8 hours, at a temperature of 0 to 100°C, preferably 10 to 60°C, more preferably 20 to 40°C, at a pH of 1 to 14, preferably 4 to 9, more preferably 6 to 8, at a concentration of 1 M or less, preferably 0.1 M or less, more preferably 10 mM or less.

16. The method according to any one of claims 1 to 15, wherein the concentration of the remaining compound having structure I decreases by at least two logarithms, preferably at least three logarithms, more preferably at least four logarithms, even more preferably at least five logarithms, even more preferably at least six logarithms, even more preferably at least seven logarithms, even more preferably at least eight logarithms, even more preferably at least nine logarithms, and even more preferably at least ten logarithms after treatment with the neutralizing agent.

17. The aforementioned pathogen or undesirable organism Enveloped viruses and non-enveloped viruses, DNA or RNA viruses, and viruses including bacteriophages, Prions, prokaryotes, bacteria including Gram-positive or Gram-negative bacteria, spore-forming bacteria or bacterial spores, mycoplasmas, archaea, and bacterial membranes; Eukaryotes, including but not limited to fungi, protozoa, unicellular or multicellular parasites, parasitic helminths, schistosomiasis or nematodes, or their eggs, unicellular or multicellular algae, and crustaceans, or Biofilm or biofouling system, Any combination of them The method according to any one of claims 1 to 16, wherein the organism is one or more of the infectious disease-causing organisms.

18. The method according to any one of claims 1 to 17, wherein the sample is a composition, a practical item, a surface, an apparatus, or a living organism.

19. The method according to any one of claims 1 to 17, wherein the sample is blood or blood products, body fluids, culture media of eukaryotic or prokaryotic origin, vaccine formulations, biologics or biological preparations, clinical samples, biopsy materials, research samples, cosmetics, pharmaceutical compositions, consumables, equipment, underwater fluid conduits, pipes, hoses, heat exchangers or surface vessels, and their surfaces.

20. The method according to any one of claims 1 to 17, wherein the sample is blood or a blood product.

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