Choline derivatives and compositions and uses thereof for cryopreservation

Abstract

This application provides compounds of formula I and cryopreservation compositions comprising one or more compounds of formula I. Compounds of formula I can be used to replace or significantly reduce the amount of commonly used cryopreservatives DMSO and / or glycerol in cryopreservation compositions. Compounds of formula I also exhibit ice recrystallization inhibitor activity.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202380105165.2 (22) Application Date 2023.11.10 (85) PCT International Application Entering National Phase Date 2026.07.09 (86) PCT International Application Application Data PCT / CA2023 / 051510 2023.11.10 (87) PCT International Application Publication Data WO2025 / 097235 EN 2025.05.15 (71) Applicant University of Ottawa Address Ontario, Canada (72) Inventor Robert N. Ben Elisa M. Walsh (74) Patent Agency Shanghai Shengkang Law Firm 31263 Patent Attorneys Li Xianzhong and Zhang Hua (51) Int.Cl. C07C 215 / 40 (2006.01) A01N 1 / 125 (2025.01) C12N 1 / 04 (2006.01) C12N 5 / 00 (2006.01) (54) Invention Title: Choline Derivatives and Compositions Thereof and Their Use in Cryopreservation (57) Abstract: This application provides compounds of Formula I and cryopreservation compositions comprising one or more compounds of Formula I. Compounds of Formula I can be used to replace or significantly reduce the amount of commonly used cryopreservatives DMSO and / or glycerol in cryopreservation compositions. Compounds of Formula I also exhibit ice recrystallization inhibitor activity.Claims 5 pages, Description 21 pages, Drawings 6 pages, CN 122555691 A 2026.08.11 CN 1 22 55 56 91 A 1. Compound of Formula I: wherein each of R1, R2 and R3 is independently H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl substituted at the terminal with an alcohol, aryl or heteroaryl, or one of R1, R2 and R3 is absent, is H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl substituted at the terminal with an alcohol, aryl or heteroaryl. R4 is a 4- to 8-membered heterocyclic ring consisting of a C1- to C9 alkyl group, a C2- to C9 alkenyl group, a C2- to C9 alkenyl group, or a C2- to C9 alkyl group, a C2- to C9 alkenyl group, or a C2- to C9 alkenyl group substituted with an alcohol, C(O)OH, C(O)OR5, or C(O)NR6R7. R5 is a C1- to C8 alkyl group, a C2- to C8 alkenyl group, or a C2- to C8 alkenyl group. The alkynyl group, wherein the terminal of each is optionally substituted with an aryl group, an alcohol group, or a carboxylic acid group, and wherein each of R6 and R7 is independently H, C1 to C8 alkoxy, aryl, substituted aryl, 4-8 membered heterocyclic group, or wherein the terminal of each is optionally substituted with an aryl group and is a C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl; and X- is a counter ion, such as a halide ion, phosphate ion, sulfate ion, acetate ion, or hexafluorophosphate ion, provided that when R4 is -CH2CH2OH: at least one of R1, R2, and R3 is not methyl; when R1 is methyl, then R2 and R3 cannot both be H or ethyl; and when both R1 and R2 are methyl, then R3 is not H or ethyl. 2. The compound according to claim 1, wherein: each of R1, R2, and R3 is a C1-C8 alkyl group, or a C1-C8 alkyl group terminally substituted with an aryl group; or one of R1, R2, and R3 is absent, is H, a C1-C8 alkyl group, or a C1-C8 alkyl group terminally substituted with an aryl group, and both of R1, R2, and R3, together with the nitrogen to which they are attached, form a 4- to 8-membered heterocyclic ring. 3. The compound according to claim 1 or 2, wherein each of R1, R2, and R3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, or a C1-C4 alkyl group terminally substituted with an aryl group. 4. The compound according to any one of claims 1 to 3, wherein at least one of R1, R2, and R3 is ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or isobutyl.5. The compound according to any one of claims 1 to 4, wherein at least one of R1, R2, and R3 is a C1-C4 alkyl group terminally substituted with an aryl group, such as a phenyl group. 6. The compound according to claim 1 or 2, having the following structure: (Claims 1 / 5 page 2 CN 122555691 A) where n is an integer from 0 to 4. 7. The compound according to any one of claims 1 to 6, wherein X- is F-, Cl-, Br-, I-, phosphate, sulfate, acetate, or hexafluorophosphate ion. 8. The compound according to claim 1, which is: (Claims 2 / 5 page 3 CN 122555691 A) 9. A composition comprising: (a) a compound of formula I: wherein each of R1, R2 and R3 is independently H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl substituted at the end with an alcohol, aryl or heteroaryl, or one of R1, R2 and R3 is absent and is H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl substituted at the end with an alcohol, aryl or heteroaryl, and the other two of R1, R2 and R3 together with the nitrogen to which they are attached form a 4 to 8-membered heterocyclic ring; R4 is H, C1 to C9 alkyl, C2 to C9 alkenyl, C2 to C9 alkynyl, or C2 to C9 alkyl, C2 to C9 alkenyl, or C2 to C9 alkynyl substituted at the end with an alcohol, C(O)OH, C(O)OR5, or C(O)NR6R7, wherein R5 is C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl, wherein each is optionally substituted at the end with an aryl group, an alcohol group, or a carboxylic acid group, and wherein each of R6 and R7 is independently H, C1 to C8 alkoxy, aryl, substituted aryl, 4-8 membered heterocyclic group, or C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl, wherein each is optionally substituted at the end with an aryl group; and X- is a counterion, such as a halide ion, phosphate, sulfate, acetate, or hexafluorophosphate ion; and (b) a buffer, cell culture medium, or a combination thereof. The composition thereof is free of dimethyl sulfoxide (DMSO) or glycerol, or contains <10% by weight of DMSO and / or <40% by weight of glycerol. 10. The composition of claim 9, wherein the compound is as defined in any one of claims 1 to 8. 11. The composition of claim 9 or 10, wherein the composition further comprises an ice crystal recrystallization inhibitor that does not have the structure of formula I.12. The composition of any one of claims 9 to 11, wherein the composition further comprises one or more of lactobionic acid, glycerol, polyvinyl alcohol, hydroxyethyl starch (HES), ethylene glycol (EG), propylene glycol (PG), mannitol, amino acids, urea, reducing sugars, and biocompatible disaccharides or polysaccharides. Claims 3 / 5 pages 4 CN 122555691 A 13. The composition of claim 12, wherein the reducing sugar is glucose, xylose, or trehalose. 14. The composition of any one of claims 9 to 13, wherein the buffer or cell culture medium is phosphate-buffered saline, optionally comprising albumin, Eagle's minimum essential medium, Durbeco modified Eagle's medium, RPMI, fetal bovine serum, fetal calf serum, Ham's F-10, Ham's F-12, medium 199, Hank's buffered saline, Hank's buffered saline, and glucose, or combinations thereof. 15. The cryopreservation composition of any one of claims 9 to 14, further comprising biological material from humans, animals, plants, fungi, or microorganisms. 16. The cryopreservation composition of claim 15, wherein the biological material is an organ, tissue, cell, or platelet. 17. The cryopreservation composition of claim 16, wherein the cells include stem cells, T cells, peripheral blood mononuclear cells (PMBCs), neurons, progenitor cells, hepatocytes, erythrocytes, immune cells, endothelial cells, or cells derived from a cell line; preferably, the cells include stem cells, T cells, erythrocytes, progenitor cells, or hepatocytes.18. A method for cryopreserving biological material, the method comprising: (a) suspending the biological material in a cryopreservation composition, wherein the cryopreservation composition comprises: (i) one or more compounds of formula I: wherein each of R1, R2 and R3 is independently H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl substituted at the end with an alcohol, aryl or heteroaryl, or one of R1, R2 and R3 is absent and is H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl substituted at the end with an alcohol, aryl or heteroaryl, and the remaining two of R1, R2 and R3, together with the nitrogen to which they are attached, form a 4 to 8-membered heterocyclic ring; R4 is H, C1 to C9 alkyl, C2 to C9 alkenyl, C2 to C9 alkynyl, or C2 to C9 alkyl, C2 to C9 alkenyl, or C2 to C9 alkynyl substituted at the end with an alcohol, C(O)OH, C(O)OR5, or C(O)NR6R7, wherein R5 is C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl, wherein each is optionally substituted at the end with an aryl group, an alcohol group, or a carboxylic acid group, and wherein each of R6 and R7 is independently H, C1 to C8 alkoxy, aryl, substituted aryl, 4-8 membered heterocyclic group, or C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl, wherein each is optionally substituted at the end with an aryl group; and X- is a counterion, such as a halide ion, phosphate, sulfate, acetate, or hexafluorophosphate ion; (ii) buffer, cell culture medium, or a combination thereof. The composition is free of dimethyl sulfoxide (DMSO) or glycerol, or contains <10% by weight of DMSO and / or <40% by weight of glycerol (<40%); (b) cooling the suspension from step (a) to a storage temperature equal to or less than the freezing point of the cryopreservation composition; and (c) storing the cooled suspension at the storage temperature. 19. The method of claim 18, wherein the compound of formula I is as defined in any one of claims 1 to 8. 20. The method of claim 18 or 19, wherein the biological material is derived from humans, animals, plants, fungi, or microorganisms. 21. The method of claim 20, wherein the biological material is an organ, tissue, cell, or platelet. 22. The method of claim 21, wherein the cells include stem cells, T cells, peripheral blood mononuclear cells (PMBC), neurons, progenitor cells, hepatocytes, erythrocytes, immune cells, endothelial cells, or cells derived from cell lines, preferably, the cells include stem cells, T cells, erythrocytes, progenitor cells, or hepatocytes.23. The method of any one of claims 18 to 22, wherein cooling the suspension comprises cooling at a controlled rate of about 1°C per minute over about 16 hours. Claims 5 / 5 pages 6 CN 122555691 A Choline derivatives and compositions thereof and their use in cryopreservation Technical Field

[0001] This application relates to the field of cryopreservation and sub-zero storage of biological materials. More specifically, this application relates to choline derivatives used as cryoprotectants, methods of their manufacture and their use in cryopreservation and sub-zero storage. Background Art

[0002] Cryopreservation remains the most commonly used method for long-term cell preservation and can also be used for long-term tissue preservation.

[0003] However, during cryopreservation, a large number of cells often suffer irreparable damage due to the growth of ice, ultimately leading to reduced recovery rates or impaired function after thawing. As cell-based therapies continue to define new modalities of care such as stem cell therapy, regenerative medicine and blood transfusions, ensuring that the viability and function of cells after thawing are at their highest levels becomes increasingly important.

[0004] The successful development and distribution of biomaterials and drug therapies depend on efficient cryopreservation methods. 1-3 Cryopreservation employs extremely low sub-zero storage temperatures (<-80°C) to significantly extend the shelf life of products by slowing down biological processes. However, despite the routine use of cryopreservation in clinical and industrial applications, its effectiveness (including post-thawing viability, recovery rate, and functionality) remains unsatisfactory. Ice formation during cryopreservation subjects cells to enormous physical and chemical stress, which, if not mitigated, can cause irreversible cell damage, thereby reducing the recovery rate and viability of cryopreserved biological materials.

[0005] Dimethyl sulfoxide (DMSO) and glycerol are the preferred cryoprotectants (CPAs) for cryopreservation protocols in research and clinical settings, and have been key CPAs driving the development of cryobiology over the past 50 years. 4-6 DMSO possesses a range of physical properties that enable its successful use as a CPA; for example, DMSO exhibits strong molecular stability, a high dielectric constant, a high affinity for water, and a strong hydrogen bonding ability. The ability to form strong hydrogen bonds with water imparts powerful antifreeze properties. Furthermore, DMSO can easily penetrate biological membranes with minimal damage to cells, thus enabling cryopreserved biological samples to produce beneficial antifreeze and permeation effects both intracellularly and extracellularly.

[0006] The role of DMSO and glycerol as permeable CPAs is to enhance viability after thawing; however, both DMSO and glycerol exhibit cytotoxicity and adverse biological effects, which poses challenges to their routine use in cryopreservation, particularly for highly complex or cryosensitive cell types. 5,7,8 Cells derived from T cells and B cells have been shown to be more sensitive to DMSO, with increased apoptosis, abnormal differentiation, and growth arrest observed.10 Furthermore, it has been reported that exposure to low concentrations of DMSO also reduces function; some examples include: increased erythrocyte hemolysis 10, inhibited platelet disaggregation and reduced thrombin 10, and reduced morphology, adhesion and differentiation capacity of mesenchymal stem cells (MSCs). 11 Due to the adverse biological effects of DMSO and glycerol on complex cell types commonly used in cell-based therapies (e.g., human induced pluripotent stem cells (hiPSCs), MSCs, hematopoietic stem cells and progenitor cells (HSPCs)), these CPAs are not suitable for routine cryopreservation in emerging cell-based therapies.

[0007] In addition to the harmful biological effects observed with cell-based products, DMSO and glycerol also have inherent limitations as CPAs in the clinical setting. Infusion of these CPAs into patients can lead to harmful toxic effects, therefore, a time-consuming but necessary CPA removal step is required after thawing biological samples. For example, glycerol is typically removed from erythrocyte (RBC) samples after thawing to avoid intravascular hemolysis after infusion. 5 In summary, these limitations make it necessary to design improved, non-toxic, and biocompatible CPA formulations. Specification 1 / 21 pages 7 CN 122555691 A

[0008] To mitigate the toxicity issues associated with the use of DMSO and glycerol, attempts have been made to reduce the concentration of CPA used or to combine CPA with other non-toxic cryopreservation additives (such as sugars or polymers). 12,13 However, although these approaches are improvements over DMSO / glycerol alone, they are still unsatisfactory. Therefore, despite the success of cryopreservation of various cell types using DMSO and / or glycerol, there is still a need to improve cryopreservation additives and cryopreservation protocols to completely eliminate the need for DMSO and glycerol.

[0009] During the freezing and thawing process of cryopreservation, ice recrystallization is a significant factor leading to cell damage and death. Therefore, there is considerable interest in designing CPA as an effective ice recrystallization inhibitor (IRI) to mitigate the damage caused by ice crystal growth and improve overall cryopreservation effectiveness. 14 Unfortunately, DMSO and glycerol, as industry-standard CPAs, cannot resolve the ice recrystallization process, leading to uncontrolled ice growth and reduced viability after thawing. Existing research indicates that various small molecules possess IRI activity, and this IRI activity can be optimized through simple chemical modifications of the molecules. 4,5,15 Adding these IRI-active small molecules to the cryopreservation medium has successfully improved cryopreservation outcomes (i.e., viability, recovery rate, and function after thawing) for various cell types, including complex cells such as MSCs, HSPCs, and induced pluripotent stem cells (iPSCs). 15-17 However, alternatives to DMSO and glycerol are still needed in the cryopreservation medium.

[0010] The purpose of providing the above information is to disclose information that the applicant believes may be relevant to the present invention.This is not necessarily intended to acknowledge, nor should it be construed as, any of the foregoing information constituting prior art to this invention. Summary of the Invention

[0011] The object of this application is to provide choline derivatives and compositions thereof, and their use in cryopreservation. According to an aspect of this application, a compound of formula I is provided: wherein each of R1, R2, and R3 is independently H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl substituted at the terminal with an alcohol, aryl, or heteroaryl group; or one of R1, R2, and R3 is absent and is H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl substituted at the terminal with an alcohol, aryl, or heteroaryl group; the remaining two of R1, R2, and R3, together with the nitrogen atom to which they are attached, form a 4 to 8-membered heterocyclic ring; R4 is H, C1 to C9 alkyl, C2 to C9 alkenyl, C2 to C9 alkynyl, or terminated with an alcohol, C(O)OH, C(O)OR5, or C(O). NR6R7 is a C2 to C9 alkyl, C2 to C9 alkenyl, or C2 to C9 alkynyl group, wherein R5 is a C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl group, wherein the terminal of each is optionally substituted with an aryl group, an alcohol group, or a carboxylic acid group, and wherein each of R6 and R7 is independently H, a C1 to C8 alkoxy, an aryl, a substituted aryl, a 4-8 membered heterocyclic group, or a C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl group, wherein the terminal of each is optionally substituted with an aryl group; and X- is a counter ion, such as a halide ion, a phosphate ion, a sulfate ion, an acetate ion, or a hexafluorophosphate ion. (See specification 2 / 21 page 8 CN 122555691 A) Provided that when R4 is -CH2CH2OH, then: at least one of R1, R2, and R3 is not methyl; When R1 is methyl, then R2 and R3 cannot both be H or ethyl; and when both R1 and R2 are methyl, then R3 cannot both be H or ethyl.

[0012] According to some embodiments, each of R1, R2 and R3 is a C1-C8 alkyl (e.g., ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl or isobutyl), or a C1-C8 alkyl terminally substituted with an aryl group (e.g., ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl or isobutyl); or one of R1, R2 and R3 is absent, is H, or is a C1-C8 alkyl (e.g., ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl or isobutyl), or a C1-C8 alkyl terminally substituted with an aryl group (e.g., ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl or isobutyl), and two of R1, R2 and R3 together with the nitrogen to which they are attached form a 4 to 8-membered heterocyclic ring.In some embodiments, at least one of R1, R2, and R3 is a C1-C4 alkyl group terminally substituted with an aryl group, such as a phenyl group.

[0013] According to some embodiments, the compound of formula I has the following structure: where n is an integer from 0 to 4.

[0014] According to some embodiments, the counter ion X- is F-, Cl-, Br-, I-, phosphate, sulfate, acetate, or hexafluorophosphate ion.

[0015] According to another aspect of this application, a composition comprising: (a) a compound of formula I wherein each of R1, R2 and R3 is independently H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl substituted at the end with an alcohol, aryl or heteroaryl, or one of R1, R2 and R3 is absent and is H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl substituted at the end with an alcohol, aryl or heteroaryl, and the other two of R1, R2 and R3 together with the nitrogen to which they are attached form a 4 to 8 membered heterocyclic ring; R4 is H, C1 to C9 alkyl, C2 to C9 alkenyl, C2 to C9 alkynyl, or C2 to C9 alkyl, C2 to C9 alkenyl, or C2 to C9 alkynyl substituted with an alcohol, C(O)OH, C(O)OR5, or C(O)NR6R7 at the end, wherein R5 is C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkyl. (Specification 3 / 21, page 9, CN 122555691 A) The alkynyl group, wherein the terminus of each is optionally substituted with an aryl group, an alcohol group or a carboxylic acid group, and wherein each of R6 and R7 is independently H, C1 to C8 alkoxy, aryl, substituted aryl, 4-8 membered heterocyclic group, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl, wherein the terminus of each is optionally substituted with an aryl group; and X- is a counterion, such as a halide ion, phosphate, sulfate, acetate or hexafluorophosphate ion; (b) a buffer, cell culture medium or a combination thereof, wherein the composition is free of dimethyl sulfoxide (DMSO) or glycerol, or contains <10% by weight of DMSO and / or <40% by weight of glycerol.

[0016] In some embodiments, the composition further includes an ice crystal recrystallization inhibitor that does not have the structure of Formula I, and / or the composition further includes one or more of the following: lactobionic acid, glycerol, polyvinyl alcohol, hydroxyethyl starch (HES), ethylene glycol (EG), propylene glycol (PG), mannitol, amino acids, urea, reducing sugars (e.g., glucose, xylose, or trehalose), and biocompatible disaccharides or polysaccharides. The combination of Formula I compounds with one or more additional compounds optionally forms a deep eutectic system.

[0017] In some embodiments, the composition includes a buffer or cell culture medium, which is a phosphate-buffered saline, optionally including albumin, Eagle's minimum essential medium, Durbecox modified Eagle's medium, RPMI, fetal bovine serum, fetal calf serum, Ham's F-10, Ham's F-12, medium 199, Hank's buffer solution, Hank's buffer solution, and glucose, or combinations thereof.

[0018] In some embodiments, the cryopreservation composition further includes biological material from humans, animals, plants, fungi, or microorganisms. For example, the biological material is an organ, tissue, cell, or platelet, wherein the cells may be or include stem cells, T cells, peripheral blood mononuclear cells (PMBC), neurons, progenitor cells, hepatocytes, erythrocytes, immune cells, endothelial cells, or cells derived from cell lines, preferably, the cells include stem cells, T cells, erythrocytes, progenitor cells, or hepatocytes.

[0019] According to another aspect of this application, a method for cryopreserving biological material is provided, the method comprising: (a) suspending the biological material in a cryopreservation composition, wherein the cryopreservation composition comprises: (i) one or more compounds of formula (i), wherein each of R1, R2 and R3 is independently H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl substituted at the end with an alcohol, aryl or heteroaryl, or one of R1, R2 and R3 is absent and is H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl substituted at the end with an alcohol, aryl or heteroaryl, and the other two of R1, R2 and R3 together with the nitrogen to which they are attached form a 4 to 8 membered heterocyclic ring; R4 is H, C1 to C9 alkyl, C2 to C9 alkenyl, C2 to C9 alkynyl, or C2 to C9 alkyl, C2 to C9 alkenyl, or C2 to C9 alkynyl substituted at the terminal with an alcohol, C(O)OH, C(O)OR5, or C(O)NR6R7, wherein R5 is C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl, wherein the terminal of each is optionally substituted with an aryl group, an alcohol group, or a carboxylic acid group, and wherein each of R6 and R7 is independently H, C1 to C8 alkoxy, aryl, substituted aryl, 4-8 membered heterocyclic group, or C1 to C8 alkyl, C2 to C8 alkenyl. (See page 4 / 21 of the specification, 10 CN 122555691 A) Or C2 to C8 alkynyl groups, wherein the terminal of each is optionally substituted with an aryl group; and X- is an anti-ion, such as a halide ion, phosphate ion, sulfate ion, acetate ion, or hexafluorophosphate ion; and (ii) a buffer, cell culture medium, or a combination thereof, wherein the composition is free of dimethyl sulfoxide (DMSO) or glycerol, or contains <10% by weight of DMSO and / or <40% by weight of glycerol (<40%); (b) cooling the suspension from step (a) to a storage temperature equal to or less than the freezing point of the cryopreservation composition; and (c) storing the cooled suspension at the storage temperature.

[0020] In some embodiments, the method is used for cryopreserving biological material from humans, animals, plants, fungi, or microorganisms. For example, the biological material is an organ, tissue, cell, or platelet, wherein the cells may be or include stem cells, T cells, peripheral blood mononuclear cells (PMBC), neurons, progenitor cells, hepatocytes, erythrocytes, immune cells, endothelial cells, or cells derived from cell lines, preferably, the cells include stem cells, T cells, erythrocytes, progenitor cells, or hepatocytes.

[0021] In some embodiments, the step of cooling the suspension includes cooling at a controlled rate of about 1°C per minute over about 16 hours.

[0022] To better understand the application described herein and its other aspects and features, reference is made to the following description used in conjunction with the accompanying drawings, in which: Figure 1 depicts (A) an ammonium-based compound, previously found to be usable as an IRI, but also found to be cytotoxic; and (B) choline chloride, a compatible alternative to the previously studied ammonium-based salt. Figure 2 shows the ice recrystallization inhibitory activity of choline-based compounds 1–4 as measured via a modified splash cooling assay. Images of ice crystals at each concentration were acquired three times after a 5-minute annealing period. Using the cross-sectional area of ​​all ice crystals in the images, the ice crystal growth rate was calculated and normalized (vnorm) to a phosphate-buffered saline (PBS) control (n=3, mean ± SD), and then plotted to generate dose-response curves. From the dose-response curves, the half-maximal inhibitory concentration (IC50) of each IRI was deduced, representing a quantitative measurement of IRI activity that can be directly compared between the compounds (see Table 1).

[0023] Figure 3 shows the cell viability of HepG2 cells in the presence of ChCl dissolved in supplemented minimum essential medium (MEM) and modified choline derivatives 1-4. Cells were incubated with different concentrations of IRI for 24 hours, and cell viability was then analyzed by rezinz viability dye. Data are reported as mean viability (N=2, n=18). (A) Dose-response curves were generated by plotting mean viability % versus IRI concentration on a semi-logarithmic plot. Error bars indicate the standard deviation of mean viability % at each concentration. The 24-hour median lethal dose (LD50) can be inferred from the dose-response curves. (B) The mean viability % of compounds 1 and 2 at each IRI concentration is shown. Error bars indicate % SEM. An asterisk indicates statistical significance compared to the control condition (0 mM IRI) as determined by two-way ANOVA using a multiple comparison test. (p < 0.05, p < 0.01, p < 0.001, p < 0.0001).

[0024] Figure 4 shows the cell viability of HepG2 cells as part of a glucose deep eutectic system (DES) in the presence of ChCl (1) and modified choline derivatives 2, 3, and 8. A dose-response curve was generated by plotting the mean viability % within the DES versus the IRI concentration on a semi-logarithmic plot.

[0025] Figure 5 schematically depicts the protocol used in the Tf-1a cryopreservation study.

[0026] Figure 6 shows the viability of Tf-1a cells after thawing, cryopreserved with ChCl and different concentrations of DMSO.Data are expressed as mean ± SEM, n=4, with the number of events >75,000 under all conditions. An asterisk indicates a significant difference determined by two-way ANOVA and multiple comparisons as defined in Tukey's specification, page 5 / 21, CN 122555691 A, with a confidence level ≥95% (p < 0.05, p < 0.01). Detailed Description

[0027] Definitions Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] As used in the specification and claims, the singular forms “an,” “a,” and “the” include plural references unless the context clearly specifies otherwise.

[0029] The term “comprising” as used herein should be understood to mean that the following list is not exhaustive and may or may not include any other suitable additional items, such as one or more other features, components, and / or ingredients, as appropriate.

[0030] The terms “one embodiment,” “an embodiment,” “another embodiment,” “a specific embodiment,” “a related embodiment,” “a particular embodiment,” “an additional embodiment,” or “a further embodiment,” or combinations thereof, as used in this specification, refer to a specific feature, structure, or characteristic described in relation to this embodiment being included in at least one embodiment. Therefore, the appearance of these phrases throughout this specification does not necessarily refer to the same embodiment. Furthermore, in one or more embodiments, specific features, structures, or characteristics may be combined in any suitable manner.

[0031] The term “and / or” as used herein, such as in phrases like “A and / or B,” is intended to include “A and B,” “A or B,” and “A” and “B.”

[0032] The term “alkoxy” as used herein refers to a straight-chain or branched alkyl group bonded to oxygen. In some embodiments, the alkoxy group comprises an alkyl group having 1 to about 10 carbons, 1 to about 8 carbons, or 1 to 4 carbons. Examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxy, and octoxy. For example, “C1-C4-alkoxy” refers to an alkoxy group having 1 to 4 carbon atoms, such as, but not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy, n-butoxy, 1-methylpropoxy, 2-methylpropoxy, and 1,1-dimethylethoxy. “Alkoxy” is intended to encompass all structural isomers of the alkoxy group. For example, propoxy as used herein encompasses both n-propoxy and isopropoxy, etc.

[0033] The term “alkyl” as used herein refers to a monovalent saturated hydrocarbon chain having a length of 1 to about 12, or 1 to about 8, or 1 to 4 carbon atoms, such as, but not limited to, methyl, ethyl, propyl, and butyl. Alkyl groups can be straight-chain, branched, or cyclic.For example, as used herein, the term “C1-C4-alkyl” refers to a saturated straight-chain or branched hydrocarbon having 1 to 4 carbon atoms. “alkyl” is intended to encompass all structural isomers of the alkyl group. For example, as used herein, propyl encompasses both n-propyl and isopropyl; butyl encompasses n-butyl, sec-butyl, isobutyl, and tert-butyl.

[0034] As used herein, the term “alkenyl” refers to a monovalent hydrocarbon chain of 2 to about 12, 2 to about 8, or 2 to 4 carbon atoms containing at least one carbon-carbon double bond. Alkenyl groups can be straight-chain, branched, or cyclic. For example, as used herein, the term “C2-C4-alkenyl” refers to a straight-chain or branched hydrocarbon having 2 to 4 carbon atoms and containing at least one carbon-carbon double bond. “Alkenyl” is intended to encompass all structural isomers of the alkenyl group.

[0035] As used herein, the term “alkynyl” refers to a monovalent hydrocarbon chain of 2 to 12, 2 to 8, or 2 to 4 carbon atoms containing at least one carbon-carbon triple bond. The alkynyl group can be straight-chain, branched, or cyclic. For example, as used herein, the term “C2-C4-alkynyl” refers to a straight-chain or branched hydrocarbon having 2 to 4 carbon atoms and containing at least one carbon-carbon triple bond. “Alynyl” is intended to encompass all structural isomers of the alkynyl group.

[0036] As used herein, the term “aryl” refers to a monocyclic or polycyclic aromatic group, such as, but not limited to, phenyl, naphthyl, thiophene, and indole.

[0037] As used herein, the term “biomaterial” refers to any substance suitable for cryopreservation that can be or has been removed from plants, fungi, bacterial colonies, humans, or non-human animals, such as, but not limited to, organs, tissues, cells, platelets, eggs, and embryos. Alternatively, the biomaterial may be cultured cells, tissues, or organs. Examples of “cells” include, but are not limited to, stem cells, T cells, oocytes, sperm, peripheral blood mononuclear cells (PMBCs), MSCs, iPSCs, hiPSCs, HSPCs, neurons, progenitor cells, hepatocytes, erythrocytes, immune cells, endothelial cells, fungal cells, or cells derived from cell lines. Another alternative is that “cells” may be single-celled microorganisms, including, for example, bacterial or single-celled fungal (e.g., yeast) cells. Cells may be in isolated or purified form, or they may be mixed with one or more other cell types.

[0038] In some embodiments, the biomaterial is derived from non-human animals or human sources. In alternative embodiments, the biomaterial is derived from plant, fungal, or bacterial sources. For example, plant-derived biomaterial may be plant cells, tissues, or organs, or may be plant seeds or fruits, or a portion thereof.

[0039] In this document, the terms “cell matrix,” “cell culture medium,” and “growth medium” are used interchangeably to refer to liquid or gel compositions used to support cell growth of cells, tissues, or organs in an artificial environment. Examples of cell culture media include, but are not limited to, Eagle's Minimum Essential Medium (MEM), Durbeco Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Fetal Bovine Serum (FBS), Fetal Bovine Serum (FCS), Ham's F-10, Ham's F-12, Hank's Buffered Salt Solution (HBSS), HBSS and glucose, and Medium 199.

[0040] In this document, the terms “crystaltant,” “cryoprotectant,” and “cryoprotectant” are used interchangeably to refer to compounds that facilitate the cryopreservation of biological materials. Examples of cryoprotectants include, but are not limited to, dimethyl sulfoxide (DMSO), lactobionic acid, glycerol, trehalose, mannitol, hydroxyethyl starch (HES), ethylene glycol (EG), propylene glycol (PG), and other biopolymers that can be used for cryopreservation. Examples of biopolymers that can be used as cryoprotectants include, but are not limited to, polyvinyl alcohol. The term “crystaltant” as used herein is not intended to include water, RPMI, DMEM, or MEM, HBSS, or glucose (i.e., when not used as part of a deep eutectic system).

[0041] In this document, the terms “cryopreservation composition,” “cryopreservation formulation,” and “cryoprotectant” are used interchangeably to refer to compositions containing one or more cryoprotectants (CPAs) and a carrier medium (e.g., buffer and / or growth medium) selected to support the storage of cells at low temperatures (which may be below zero degrees Celsius).

[0042] As used herein, the term "heterocyclic ring" refers to a four- to eight-membered ring containing one to four heteroatoms, such as oxygen, sulfur, and / or nitrogen. These four- to eight-membered rings can be saturated, fully unsaturated, or partially unsaturated. As used herein, "heteroaryl" refers to a cyclic aromatic radical having five to ten ring atoms, one of which is selected from S, O, and N; zero, one, or two ring atoms are additional heteroatoms independently selected from S, O, and N; the remaining ring atoms are carbon, and the radical is connected to the rest of the molecule via any ring atom, such as pyridyl, pyrazinyl, pyrimidinyl, pyrroleyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isoxazolyl, thiadiazolyl, oxadiazolyl, thiophene, furanyl, quinolinyl, isoquinolinyl, etc. Non-limiting examples of nitrogen-containing heterocyclic rings include piperidinyl, pyrrolidinyl, pyrroleyl, pyrazolyl, pyrazolylalkyl, pyridinyl, pyrimidinyl, piperazinyl, indolinel, etc.

[0043] The term “ice recrystallization inhibitor” as used herein refers to a cryoprotectant that inhibits ice recrystallization during cryopreservation.As used herein, a compound is considered to have ice recrystallization inhibitory activity if it exhibits inhibition of ice recrystallization and has a half-maximal inhibitory concentration (IC50) of 100 mM or less. As used herein, "good" ice recrystallization inhibition refers to IC50.

Claims

1. Compound of Formula I: Where R 1 R 2 and R 3 Each of these is independently H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or a C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl substituted at the terminal with an alcohol, aryl, or heteroaryl group, or R. 1 R 2 and R 3 One of them is absent, is H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl whose terminal is substituted with alcohol, aryl or heteroaryl, and R 1 R 2 and R 3 The other two, together with the nitrogen atoms they are attached to, form 4- to 8-membered heterocyclic rings; R 4 It is H, C1 to C9 alkyl, C2 to C9 alkenyl, C2 to C9 alkynyl, or C2 to C9 alkyl, C2 to C9 alkenyl, or C2 to C9 alkynyl substituted with an alcohol, C(O)OH, C(O)OR5, or C(O)NR6R7 at the end, wherein R 5 It is a C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl, wherein the terminal of each is optionally substituted with an aryl group, an alcohol group, or a carboxylic acid group, and wherein R 6 and R 7 Each of these is independently H, a C1 to C8 alkoxy group, an aryl group, a substituted aryl group, a 4-8 membered heterocyclic group, or a C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl group whose terminal is optionally substituted with an aryl group; and X - These are counter ions, such as halide ions, phosphate ions, sulfate ions, acetate ions, or hexafluorophosphate ions. The premise is that when R 4 When it is -CH2CH2OH: R 1 R 2 and R 3 At least one of them is not methyl. When R 1 When it is methyl, then R 2 and R 3 It cannot be both H or ethyl; and When R 1 and R 2 When all are methyl groups, then R 3 It is not H or ethyl.

2. The compound according to claim 1, wherein: R 1 R 2 and R 3 Each of them is a C1-C8 alkyl group, or a C1-C8 alkyl group with an aryl-substituted terminal group; or R 1 R 2 and R 3 One of them is absent, is H, a C1-C8 alkyl group, or a C1-C8 alkyl group with a terminal aryl substituted group, and R 1 R 2 and R 3 The two components, together with the nitrogen they are attached to, form 4 to 8-membered heterocyclic rings.

3. The compound according to claim 1 or 2, wherein R 1 R 2 and R 3 Each of them is independently methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, or a C1-C4 alkyl group whose terminal is substituted with an aryl group.

4. The compound according to any one of claims 1 to 3, wherein R 1 R 2 and R 3 At least one of them is ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or isobutyl.

5. The compound according to any one of claims 1 to 4, wherein R 1 R 2 and R 3 At least one of them is a C1-C4 alkyl group whose terminal is substituted with an aryl group, such as a phenyl group.

6. The compound according to claim 1 or 2, having the following structure: Where n is an integer from 0 to 4.

7. The compound according to any one of claims 1 to 6, wherein X - For F - Cl - ,Br - I - Phosphate, sulfate, acetate or hexafluorophosphate ions.

8. The compound according to claim 1, wherein the compound is: 。 9. A composition comprising: (a) Compound of Formula I: in R 1 R 2 and R 3 Each of these is independently H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or a C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl substituted at the terminal with an alcohol, aryl, or heteroaryl group. R 1 R 2 and R 3 One of them is absent, is H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl whose terminal is substituted with alcohol, aryl or heteroaryl, and R 1 R 2 and R 3 The other two, together with the nitrogen they are attached to, form 4 to 8-membered heterocyclic rings; R 4 It is H, C1 to C9 alkyl, C2 to C9 alkenyl, C2 to C9 alkynyl, or C2 to C9 alkyl, C2 to C9 alkenyl, or C2 to C9 alkynyl substituted with an alcohol, C(O)OH, C(O)OR5, or C(O)NR6R7 at the end, wherein R 5 It is a C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl, wherein the terminal of each is optionally substituted with an aryl group, an alcohol group, or a carboxylic acid group, and wherein R 6 and R 7 Each of these groups is independently H, C1 to C8 alkoxy, aryl, substituted aryl, 4-8 membered heterocyclic group, or C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl, wherein the terminal of each group is optionally substituted with an aryl group; and X - These are counter ions, such as halide ions, phosphate ions, sulfate ions, acetate ions, or hexafluorophosphate ions; and (b) Buffer solution, cell culture medium, or a combination thereof. The composition thereof is free of dimethyl sulfoxide (DMSO) or glycerol, or contains <10% by weight of DMSO and / or <40% by weight of glycerol.

10. The composition of claim 9, wherein the compound is defined as in any one of claims 1 to 8.

11. The composition of claim 9 or 10, wherein the composition further comprises an ice crystal recrystallization inhibitor that does not have the structure of formula I.

12. The composition of any one of claims 9 to 11, wherein the composition further comprises one or more of lactobionic acid, glycerol, polyvinyl alcohol, hydroxyethyl starch (HES), ethylene glycol (EG), propylene glycol (PG), mannitol, amino acids, urea, reducing sugars, and biocompatible disaccharides or polysaccharides.

13. The composition of claim 12, wherein the reducing sugar is glucose, xylose, or trehalose.

14. The composition of any one of claims 9 to 13, wherein the buffer or cell culture medium is a phosphate-buffered saline solution, optionally comprising albumin, Eagle's minimum essential medium, Durbeco modified Eagle's medium, RPMI, fetal bovine serum, fetal calf serum, Ham's F-10, Ham's F-12, medium 199, Hank's buffered saline solution, Hank's buffered saline solution and glucose, or a combination thereof.

15. The cryopreservation composition according to any one of claims 9 to 14, further comprising biological material derived from humans, animals, plants, fungi, or microorganisms.

16. The cryopreservation composition of claim 15, wherein the biomaterial is an organ, tissue, cell or platelet.

17. The cryopreservation composition of claim 16, wherein the cells comprise stem cells, T cells, peripheral blood mononuclear cells (PMBCs), neurons, progenitor cells, hepatocytes, erythrocytes, immune cells, endothelial cells, or cells derived from a cell line, preferably, the cells comprise stem cells, T cells, erythrocytes, progenitor cells, or hepatocytes.

18. A method for cryopreserving biological material, the method comprising: (a) Suspending the biological material in a cryopreservation composition, wherein the cryopreservation composition comprises: (i) One or more compounds of formula I: in R 1 R 2 and R 3 Each of these is independently H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or a C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl substituted at the terminal with an alcohol, aryl, or heteroaryl group. R 1 R 2 and R 3 One of them is absent, is H, C1 to C8 alkyl, C2 to C8 alkenyl, C2 to C8 alkynyl, or C1 to C8 alkyl, C2 to C8 alkenyl or C2 to C8 alkynyl whose terminal is substituted with alcohol, aryl or heteroaryl, and R 1 R 2 and R 3 The other two, together with the nitrogen they are attached to, form 4 to 8-membered heterocyclic rings; R 4 It is H, C1 to C9 alkyl, C2 to C9 alkenyl, C2 to C9 alkynyl, or C2 to C9 alkyl, C2 to C9 alkenyl, or C2 to C9 alkynyl substituted with an alcohol, C(O)OH, C(O)OR5, or C(O)NR6R7 at the end, wherein R 5 It is a C1 to C8 alkyl, C2 to C8 alkenyl, or C2 to C8 alkynyl, wherein the terminal of each is optionally substituted with an aryl group, an alcohol group, or a carboxylic acid group, and wherein R 6 and R 7 Each of these groups is independently H, a C1 to C8 alkoxy group, an aryl group, a substituted aryl group, a 4-8 membered heterocyclic group, or a C1 to C8 alkyl group, a C2 to C8 alkenyl group, or a C2 to C8 alkynyl group, wherein the terminal of each group is optionally substituted with an aryl group; and X - These are counter ions, such as halide ions, phosphate ions, sulfate ions, acetate ions, or hexafluorophosphate ions; (ii) Buffer solution, cell culture medium, or a combination thereof. The composition thereof is free of dimethyl sulfoxide (DMSO) or glycerol, or contains <10% by weight of DMSO and / or <40% by weight of glycerol (<40%). (b) Cooling the suspension from step (a) to a storage temperature equal to or less than the freezing point of the cryopreservation composition; and (c) The cooled suspension is stored at the storage temperature.

19. The method of claim 18, wherein the compound of formula I is defined as in any one of claims 1 to 8.

20. The method according to claim 18 or 19, wherein the biological material is derived from humans, animals, plants, fungi, or microorganisms.

21. The method of claim 20, wherein the biological material is an organ, tissue, cell, or platelet.

22. The method of claim 21, wherein the cells include stem cells, T cells, peripheral blood mononuclear cells (PMBCs), neurons, progenitor cells, hepatocytes, erythrocytes, immune cells, endothelial cells, or cells derived from cell lines, preferably, the cells include stem cells, T cells, erythrocytes, progenitor cells, or hepatocytes.

23. The method of any one of claims 18 to 22, wherein cooling the suspension comprises cooling at a controlled rate of about 1°C per minute over about 16 hours.

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