# US 11,299,453 B2 · Compounds for use in the elimination of pluripotent stem cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,299,453 B2 - Document ID: US11299453B2 - Status: Granted - Kind: B2 - Application number: 16/761,172 - Filed: 2018-11-06 - Published: 2020-10-29 - Granted: 2022-04-12 - Earliest claimed: 2017-11-06 - Assignees: Universität zu Köln - Applicants: Universität zu Köln - Inventors: Tomo Saric; Albrecht Berkessel; Karsten Burkert; Jürgen Hescheler - Web page: https://patentgrep.com/patent/US11299453B2 ## CPC - C07C 215/64: with rings other than six-membered aromatic rings being part of the carbon skeleton - C07C 215/50: with amino groups and the six-membered aromatic ring, or the condensed ring system containing that ring, bound to the same carbon atom of the carbon chain - C07D 209/08: with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring - C07D 209/14: Radicals substituted by nitrogen atoms, not forming part of a nitro radical - C07D 333/20: by nitrogen atoms (nitro, nitroso radicals C07D333/12) - C12N 5/0081: {Purging biological preparations of unwanted cells} - C12N 5/0657: {Cardiomyocytes; Heart cells} - C07C 2601/14: The ring being saturated - C12N 2501/999: Small molecules not provided for elsewhere - C12N 2506/02: from embryonic cells - C12N 2506/45: from artificially induced pluripotent stem cells - C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS - C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to a method of reducing the number or percentage of pluripotent stem cells or of enriching differentiating or differentiated cells in a cell population comprising pluripotent stem cells and differentiating cells or differentiated cells derived from the pluripotent stem cells, the method comprising the step of contacting the cell population with a compound according to the general formula (1). ## Claims ### Claim 1 A method of reducing the number or percentage of pluripotent stem cells or of enriching differentiating or differentiated cells in a cell population comprising pluripotent stem cells and differentiating cells or differentiated cells derived from the pluripotent stem cells, the method comprising the step of contacting the cell population with a compound according to the following general formula (1) and/or racemates, enantiomers, stereoisomers, solvates, hydrates or salts thereof: wherein: R1, R2 the same or independent from each other are hydrogen or represent a double bond from the nitrogen atom to the adjacent non-cyclic carbon atom; R3, R4 are the same or independently from each other selected from the group comprising hydrogen, linear or branched C1-C8-alkyl, C2-C8-alkenyl, C3-C8-cycloalkyl, C6-C10-aryl, C1-C8-alkoxy and halogen; E1, E2 are the same or independent from each other a 5- or 6-membered aromatic or heteroaromatic ring selected from the group comprising thiophenyl, pyrrolyl, pyridyl and phenyl; A is selected from the group of structural elements of formulas (2) and (3): —(Z)n—B—(Z)m— (2) —CHR′—(Z)p—, (3) wherein: B is selected from the group comprising C3-C8-cycloalkyl, C3-C8-heterocycloalkyl, C6-C10-aryl and C5-C10-heteroaryl; Z is the same or independently from each other selected from the group comprising —CH2—, —CHR′—, O, S, NH, NR′; R′ is the same or independently from each other selected from the group comprising hydrogen, C1-C8-alkyl, C2-C8-alkenyl, C3-C8-cycloalkyl and C6-C10-aryl; n, m are the same or independent from each other 0, 1 or 2; p is 1, 2, 3, 4, or 5; or contacting the cell population with a compound of formula (6) ### Claim 2 (depends on 1) The method according to claim 1, wherein the compound is a compound according to the following general formula (4) and/or racemates, enantiomers, stereoisomers, solvates, hydrates or salts thereof: wherein: R1, R2 the same or independent from each other are hydrogen or represent a double bond from the nitrogen atom to the adjacent non-cyclic carbon atom; and R3, R4 are the same or independently from each other selected from the group comprising hydrogen, linear or branched C1-C8-alkyl, C2-C8-alkenyl, C3-C8-cycloalkyl and C6-C10-aryl. ### Claim 3 (depends on 1) The method according to claim 1, wherein the compound is selected from the group of compounds according to formulas (SM2), (SM6) and (SM8) as given as follows: ### Claim 4 (depends on 1) The method of claim 1, wherein the step of contacting the cell population with a compound comprises incubating the cell population in a cell culture medium containing a compound according to general formula (1). ### Claim 5 (depends on 1) The method of claim 1, wherein the differentiating or differentiated cells are cardiomyocytes. ### Claim 6 (depends on 1) The method of claim 1, wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells. ### Claim 7 (depends on 1) The method of claim 1, wherein the pluripotent stem cells are human cells. ### Claim 8 (depends on 1) The method of claim 1, wherein the method is for enriching cardiomyocyte subtypes selected from the group consisting of atrial, ventricular and pacemaker cells. ### Claim 9 (depends on 1) The method of claim 1, wherein the method is a non-transgenic and/or resistance or selection marker-free method of enriching cardiomyocytes. ### Claim 10 A composition comprising pluripotent stem cells and differentiating cells or differentiated cells derived from the pluripotent stem cells, and a compound according to the general formula (1) and/or racemates, enantiomers, stereoisomers, solvates, hydrates or salts thereof: wherein: R1, R2 the same or independent from each other are hydrogen or represent a double bond from the nitrogen atom to the adjacent non-cyclic carbon atom; R3, R4 are the same or independently from each other selected from the group comprising hydrogen, linear or branched C1-C8-alkyl, C2-C8-alkenyl, C3-C8-cycloalkyl, C6-C10-aryl, C1-C8-alkoxy and halogen; E1, E2 are the same or independent from each other a 5- or 6-membered aromatic or heteroaromatic ring selected from the group comprising thiophenyl, pyrrolyl, pyridyl and phenyl; A is selected from the group of structural elements of formulas (2) and (3): —(Z)n—B—(Z)m— (2) —CHR′—(Z)p—, (3) wherein: B is selected from the group comprising C3-C8-cycloalkyl, C3-C8-heterocycloalkyl, C6-C10-aryl and C5-C10-heteroaryl; Z is the same or independently from each other selected from the group comprising —CH2—, —CHR′—, O, S, NH, NR′; R′ is the same or independently from each other selected from the group comprising hydrogen, C1-C8-alkyl, C2-C8-alkenyl, C3-C8-cycloalkyl and C6-C10-aryl; n, m are the same or independent from each other 0, 1 or 2; p is 1, 2, 3, 4, or 5; or a compound according to formula (6) ### Claim 11 (depends on 10) The composition according to claim 10, wherein the compound is a compound according to general formula (4) or formula (6). ### Claim 12 (depends on 10) The composition of claim 10, wherein the compound is a compound according to the following general formula (4) and/or racemates, enantiomers, stereoisomers, solvates, hydrates or salts thereof: wherein: R1, R2 the same or independent from each other are hydrogen or represent a double bond from the nitrogen atom to the adjacent non-cyclic carbon atom; and R3, R4 are the same or independently from each other selected from the group comprising hydrogen, linear or branched C1-C8-alkyl, C2-C8-alkenyl, C3-C8-cycloalkyl and C6-C10-aryl. ### Claim 13 (depends on 11) The composition of claim 11, wherein the compound is selected from the group consisting of compounds according to formulas (SM2), (SM6) and (SM8) as given as follows: ### Claim 14 (depends on 10) The composition of claim 10, wherein the pluripotent stem cells are induced pluripotent stem cells or embryonic stem cells. ### Claim 15 (depends on 10) The composition of claim 10, wherein the pluripotent stem cells are human cells. ### Claim 16 (depends on 4) The method of claim 4, wherein the compound is a compound of formulas (4) to (7) ### Claim 17 (depends on 4) The method of claim 4, wherein the compound is a compound of formulas (SM6), (SM2) and (SM8) in a range from ≥0.01 μM to ≤100 μM, wherein the formulas have the structure ### Claim 18 (depends on 4) The method of claim 4, wherein the compound is in a range from ≥0.05 μM to ≤10 μM. ### Claim 19 (depends on 4) The method of claim 4, wherein the compound is in a range from ≥0.05 μM to ≤2 μM.