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WO2025022003A1 - Beta-peptides with cytotoxic activity on cancer cells - Google Patents

Beta-peptides with cytotoxic activity on cancer cells Download PDF

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Publication number
WO2025022003A1
WO2025022003A1 PCT/EP2024/071356 EP2024071356W WO2025022003A1 WO 2025022003 A1 WO2025022003 A1 WO 2025022003A1 EP 2024071356 W EP2024071356 W EP 2024071356W WO 2025022003 A1 WO2025022003 A1 WO 2025022003A1
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alkyl
compound
alkylene
formula
moiety
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French (fr)
Inventor
Cornelia ROSCHGER
Chiara Cabrele
Ilaria MESSINA
Andreas F. ZIERER
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Universitaet Linz
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Universitaet Linz
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C279/00—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups
    • C07C279/04—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C279/14—Derivatives of guanidine, i.e. compounds containing the group, the singly-bound nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of guanidine groups bound to acyclic carbon atoms of a carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel beta-peptide (p-peptide) compounds, or pharmaceutically acceptable salts thereof, comprising a moiety of formula (I), in particular to compounds of formula (I I) or pharmaceutically acceptable salts thereof, and to pharmaceutical compositions comprising said compounds and at least one pharmaceutically acceptable carrier.
  • the compounds and the pharmaceutical compositions of the present invention are useful as a medicament, in particular for the treatment or prevention of cancer.
  • p-Peptides are an underexplored class of peptides based on unnatural p-amino acids (p-AAs) that are considered promising for drug development because of their proteolytic stability and often well-defined secondary structures. However, up to date no compounds of this class have been approved for clinical use.
  • p-Peptides are artificial oligoamides comprising p-amino acid units. Compared to the proteinogenic o-amino acids, the p-amino acids include an additional carbon atom between the amino and carboxyl groups (H2N- pC*-o C*- COOH) and are rarely found in nature.
  • p-Peptides were developed simultaneously by two researchers in the late 1990s: Prof. Seebach (ETH Zurich) used acyclic p-amino acids (D. Seebach and J. L. Matthews, Chem Commun, 1997, 2015-2022), while Prof.
  • p-Peptides are a very promising class of molecules for drug discovery because they are more stable towards proteases and peptidases and structurally diverse from a- peptides.
  • the present inventors have developed p-heterooligomers starting from cyclic and acyclic building blocks which differ in their secondary-structure propensity.
  • acyclic p 3 -amino acids p 3 -AA
  • cyclic p-amino acids offer efficient control over the geometry of the p-peptide backbone.
  • the cyclic building block is expected to take precedence over the acyclic building block for determining the geometry of the p-peptide backbone, while the acyclic building block is expected to exert a disruptive effect, thereby weakening the stiffness of the resulting structure.
  • p-peptides with bioactive properties that are promising for cancer therapy.
  • These p-peptides constitute the combination of the cyclic subunit, for example p- amino acid trans-2-aminocyclopentanecarboxylic acid (trans-ACPC) with the acyclic subunit, for example p 3 -amino acids (p 3 -AA), with the register [acyclic-cyclic-acyclic].
  • the peptides of the present invention include a repeating unit of chain consisting of an acyclic residue attached through an amide bond to a cyclic residue, which is in turn attached through an amide bond to another acyclic residue.
  • the peptides of the present invention differ in their secondary structures from the previously known homo-oligomers [trans-ACPC] n and [p 3 -AA] n .
  • the peptides provided herewith seem to have greater flexibility, and thus can better adapt to a given cell component (membrane and/or biomolecule).
  • the p-peptides provided herewith show high lytic activity against cancer cells and are therefore promising for cancer therapy.
  • the peptides of the present invention show no or low activity towards non-cancer cells, for example MRC-5 human lung fibroblasts and BEAS-2B human bronchial epithelial cells, as demonstrated in the appended examples.
  • the present invention relates to a compound comprising a moiety of formula (I):
  • each A is independently a group according to the following formula: each R 1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-OH, -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci-6 alkyl), -S(Ci-6 alkylene)-SH, -S(Ci-6 alky
  • each Z is independently a group according to the following formula: each m is independently 0 or 1 ; and n is 2, 3 or 4; or a pharmaceutically acceptable salt thereof.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of the first embodiment of the present invention and a pharmaceutically acceptable carrier
  • the present invention relates to the compound of the first embodiment of the present invention or the pharmaceutical composition of the second embodiment of the present invention for use as a medicament.
  • the present invention relates to the compound of the first embodiment of the present invention or the pharmaceutical composition of the second embodiment of the present invention for use in the treatment or prevention of cancer.
  • the present invention relates to use of the compound of the first embodiment of the present invention or the pharmaceutical composition of the second embodiment of the present invention in the manufacture of a medicament against cancer.
  • the present invention relates to a method for treating cancer, comprising the step of administering the compound of the first embodiment of the present invention or the pharmaceutical composition of the second embodiment of the present invention to a subject in need thereof. It is to be understood that a therapeutically effective amount of the compound or the composition is to be administered.
  • Figure 1 presents CD-spectra of p-peptides with cyclic-acyclic register (1b and 1_ALA) or acyclic-cyclic-acyclic register (1a) in methanol and water, comparing the effect of alternating the monomeric cyclic subunit with a monomeric (as in 1b and 1. _ALA) or dimeric (as in 1a) acyclic subunit.
  • Figure 2 demonstrates (A) cell death induction in human cell lines after 24 h treatment with 20 pM 1aR, and (B) cell death induction in A549 cells after 1 h treatment with 20 pM 1aR. Data represent at least three independent experiments, values are expressed in mean ⁇ SEM.
  • Figure 3 shows colocalization of the peptide FAM-1aR and the mitochondria in A549 cells. Fluorescence microscopy images were taken after 1 h incubation at 37 °C with FAM-1aR at a concentration of 10 pM. Nuclei were stained with Hoechst 33342 and mitochondria with the mitochondrial probe MitoViewTM720. Scale bar: 20 pm.
  • Figure 4 depicts temperature independent uptake of the fluorescence-labeled peptide FAM-1aR into A549 cells. Fluorescence microscopy images were taken after 1 h incubation at 37°C and 4°C with FAM-1 aR at a concentration of 5 pM. Nuclei were stained with Hoechst 33342. Scale bar: 20 pm.
  • Figure 5 shows uptake of the fluorescence-labeled peptide FAM-1aR into MRC-5 cells. Fluorescence microscopy images were taken after 1 h incubation at 37 °C with FAM-1aR at a concentration of 5 pM. Nuclei were stained with Hoechst 33342. Scale bar: 20 pm.
  • Figure 6 presents brightfield images of the cancer cell line A549 and the healthy lung fibroblast cell line MRC-5 after treatment with 10 pM 1aR for 24 h.
  • Black arrows indicate membrane blebbing (formation of protrusions and blister-like bodies characteristic of pyroptosis) in A549 cells but not in healthy MRC-5 fibroblasts.
  • Scale bar 20 pM for A549, 50 pM for MRC-5.
  • Figure 7 presents LDH release from A549 and MRC-5 cells treated with 10 pM 1aR for the indicated incubation times.
  • Figure 8 shows propidium iodide (PI) uptake into A549 and MRC-5 cells treated with 10 pM 1aR for 30 min. Nuclei were stained with Hoechst 33342. Scale bar: 20 pm.
  • Figure 9 demonstrates disruption of the cellular membrane upon treatment with 1aR in cancer cells but not in healthy cells using scanning electron microscopy.
  • Figure 10 shows (A) flow cytometry analyses of caspase-1 induction, and (B) IL-1 p release after treatment of A549 and MRC-5 cells with 10 pM 1aR for the indicated incubation times. Data represent at least three independent experiments, values are expressed in mean ⁇ SEM.
  • Figure 11 displays mitochondrial membrane depolarization in A549 cells treated with 10 pM 1aR. Nuclei were stained with Hoechst 33342 and the depolarization of the mitochondrial membrane was visualized by the loss of the mitochondrial stain MitoViewTM720nm. Scale bar: 50 pm.
  • Figure 12 shows ROS induction after treatment of A549 cells with 10 pM 1aR. Data represent at least three independent experiments and values are expressed in mean ⁇ SEM.
  • Figure 13 shows (A) cell death induction of senescent A549 cells after treatment with 20 pM 1aR and (B) SA-beta- gal staining identifies senescent cells (left) in untreated A549 cells. No senescent cells were detectable in A549 cells treated with 1aR (right). Data represent at least three independent experiments and values are expressed in mean ⁇ SEM.
  • Figure 14 shows uptake of FAM-1aR into A549 cells, and pyroptosis-induced morphological characteristics.
  • White arrows indicate the formation of membrane bubbles.
  • the membrane was stained with MemBriteTM Fix 660/680 and nuclei were stained with Hoechst 33342. Scale bar: 20 pM
  • Figure 15 shows FAM-1aR uptake into A549 cells and the resulting morphological changes. Pictures on the left and on the right were taken three minutes apart. Uptake of the peptide FAM-1 aR is shown. The membrane was stained with MemBriteTM Fix 660/680, nuclei were stained with Hoechst 33342. Scale bar: 20 pM.
  • Figure 16 shows the viability of selected human cancer cells after 24 h treatment with the peptides 1b, 1_ALA, and 1a (100 pM). Data represent at least three independent experiments and values are expressed in mean ⁇ SEM.
  • the present invention relates to a compound comprising a moiety of formula (I):
  • each A is independently a group according to the following formula:
  • Each R 1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)- heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-OH, -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci.
  • one -CH 2 - unit in the Co-6 alkylene moiety in said -(Co-6 alkylene)-carbocyclyl or in said -(Co-6 alkylene)-heterocyclyl is optionally replaced with -O-, -S-, -NH- or -N(CI-6 alkyl)-. If said one -CH 2 - unit is replaced, as explained above, it is preferably replaced with -0- or -S-, more preferably it is replaced with -0-.
  • each R 1 is independently C1-6 alkyl (e.g. methyl, isopropyl, isobutyl, sec-butyl, or n-butyl), C2-6 alkenyl (e.g., allyl), C2-6 alkynyl (e.g., propargyl), -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)- heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -0(Ci-6 alkyl), -0(Ci-6 alkylene)-OH, -0(Ci-6 alkylene)-0(Ci-6 alkyl)-
  • R 1 include, in particular, methyl, isopropyl, isobutyl, sec-butyl, n-butyl, allyl, propargyl, phenethyl, benzyloxymethyl and 4-(trifluoromethyl)phenylmethyl.
  • each R 1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -0(Ci-6 alkyl), -0(Ci-6 alkylene)-OH, -0(Ci-6 alkylene)-0(Ci-6 alkyl), -SH, -S(Ci.
  • each R 1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -0(Ci-6 alkyl), -0(Ci-6 alkylene)-OH, -0(Ci-6 alkylene)-0(Ci-6 alkyl), -SH, -S(Ci.
  • each R 1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci-6 alkyl), -S(Ci-6 alkylene)-S(Ci-6 alkyl), -NH-O(CI-6 alkyl), -N(CI-6 alkyl)-O(Ci-6 alkyl),
  • each R 1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl.
  • each R 1 is independently C1-6 alkyl, C2-6 alkenyl, or C2-6 alkynyl.
  • each R 1 is independently C1-6 alkyl.
  • each R 1 is independently C2-5 alkyl.
  • each R 1 is independently C3-4 alkyl.
  • each R 1 is independently isopropyl or isobutyl.
  • each Z is independently a group according to the following formula: wherein each m is independently 0 or 1 , preferably wherein each m is 1 .
  • n 2, 3 or 4.
  • n 2 or 3.
  • n is 2. It is to be understood that with respect to formula (I) as shown hereinabove, bivalent groups A and Z are so connected to other groups that said A or Z is connected to the group on its left through its -NH- moiety and that said A or Z is connected to the group on its right through its -CO- moiety.
  • each A has the following configuration:
  • each Z has the following configuration:
  • the compound of formula (I) can be a compound of formula (II):
  • p is O or 1.
  • p is 1.
  • X is selected from a hydrophobic moiety, a PEG consisting of 2 to 10 ethylene glycol repeats, and an amino acid sequence of 1 to 5 hydrophobic amino acid residues, wherein said amino acid sequence is attached via its C-terminus to the rest of the compound of formula (II), optionally wherein said amino acid sequence has an alkanoyl group (e.g., an acetyl group) at its N-terminus.
  • X is selected from a hydrophobic moiety, a PEG consisting of 2 to 10 ethylene glycol repeats, and an amino acid sequence of 1 to 5 hydrophobic amino acid residues, wherein said amino acid sequence is attached via its C-terminus to the rest of the compound of formula (II), optionally wherein said amino acid sequence has an alkanoyl group (e.g., an acetyl group) at its N-terminus.
  • alkanoyl group e.g., an acetyl group
  • X is an amino acid sequence of 1 to 5 hydrophobic amino acid residues, wherein X is attached via its C-terminus to the rest of the compound of formula (II), optionally wherein X has an alkanoyl group at its N-terminus, preferably selected from butanoyl, propionyl and acetyl. It is preferred that X has an alkanoyl group (e.g., a butanoyl, propionyl or acetyl group; particularly an acetyl group) at its N-terminus.
  • alkanoyl group e.g., a butanoyl, propionyl or acetyl group; particularly an acetyl group
  • the amino acid sequence of 1 to 5 hydrophobic amino acid residues is preferably an amino acid sequence of 1 to 3 hydrophobic amino acid residue, more preferably an amino acid sequence of 1 to 2 hydrophobic amino acid residue, even more preferably is a single amino acid residue.
  • hydrophobic amino acid residues in X are each independently selected from Gly, Ala, Vai, Leu, lie, Pro, Phe, Tyr, Met and Trp, more preferably from Vai, Leu, lie, Phe, Tyr and Trp, even more preferably from Phe, Tyr and Trp, even more preferably from Phe and Tyr.
  • amino acid residues as referred to herein as L-amino acid residues are each independently selected from Gly, Ala, Vai, Leu, lie, Pro, Phe, Tyr, Met and Trp, more preferably from Vai, Leu, lie, Phe, Tyr and Trp, even more preferably from Phe, Tyr and Trp, even more preferably from Phe and Tyr.
  • X is alkanoyl-Tyr-, such as, e.g., butanoyl-Tyr-, propionyl-Tyr- or acetyl-Tyr- (Ac-Tyr-), particularly Ac-Tyr-.
  • X is alkanoy l-(L-Tyr)-, such as, e.g., butanoy l-(L-Tyr)-, propiony l-(L-Tyr)- or Ac-(L-Tyr)-.
  • Y is a polar moiety.
  • Y is neutral or positively charged, preferably Y displays a positive net charge.
  • the amino acid sequence of 1 to 5 polar amino acid residues is preferably an amino acid sequence of 1 to 3 polar amino acid residues, more preferably an amino acid sequence of 2 to 3 polar amino acid residues.
  • the polar amino acid residues are basic amino acid residues (e.g., selected from Arg, Lys and His) or that they are a combination of basic amino acid residues and polar neutral amino acid residues (e.g., selected from Ser, Thr, Asn and Gin).
  • the polar amino acid residues in Y are all basic amino acid residues. More preferably, the polar amino acid residues in Y are each independently selected from Arg, Lys and His, preferably from Arg and Lys, more preferably they are each Arg. Accordingly, it is particularly preferred that Y consists of basic amino acids that are each independently selected from Arg, Lys and His, preferably from Arg and Lys, more preferably that are each Arg.
  • each A is selected from
  • R 1 is independently -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-OH, -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci-6 alkyl), -S(Ci- 6 alkylene)-SH, -S(Ci.
  • n is 3 or 4.
  • n is 3.
  • p 0.
  • moiety X is a hydrophobic moiety.
  • X is (C1-20 alkyl)-CO-
  • X is a (C1-20 alkyl)-CO-.
  • Particularly suitable C1-20 alkyl is C10-20 alkyl, more preferably selected from C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl and C alkyl.
  • X is CH3-CO-.
  • X is a PEG consisting of 2 to 10 ethylene glycol repeats. In an eleventh specific embodiment, X is Ac-Tyr-, preferably wherein X is Ac-(L-Tyr)-.
  • Y is -Arg-Arg-NH2 or -Arg-Arg-Arg-NH2. Even more preferably, Y is -(D-Arg)-(D- Arg)-NH 2 or -(D-Arg)-(D-Arg)-(D-Arg)-NH 2 .
  • R 1 is selected from methyl, isopropyl, isobutyl, sec-butyl, and n-butyl.
  • R 1 is selected from allyl and propargyl. In a fifteenth specific embodiment, R 1 is selected from phenylethyl, benzyloxymethyl and 4-(trifluoromethyl)phenyl methyl.
  • the compound comprising a moiety of formula (I) or the compound of formula (II) is preferably amphipathic.
  • Particularly preferred compounds comprising the moiety of formula (I) or the compounds of formula (II) are selected from the following compounds or their pharmaceutically acceptable salts:
  • the present invention specifically relates to each and every combination of features and embodiments described herein, including any combination of general and/or preferred features/embodiments.
  • the invention specifically relates to each combination of meanings (including general and/or preferred meanings) for the various groups and variables comprised in formula (I) or (II).
  • the following definitions apply, unless specifically indicated to the contrary.
  • hydrocarbon group refers to a group consisting of carbon atoms and hydrogen atoms.
  • alkyl refers to a monovalent saturated acyclic (i.e. , non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an “alkyl” group does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond.
  • a “C1-5 alkyl” denotes an alkyl group having 1 to 5 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, secbutyl, or tert-butyl). Unless defined otherwise, the term “alkyl” preferably refers to C1-4 alkyl.
  • alkenyl refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond.
  • C2-5 alkenyl denotes an alkenyl group having 2 to 5 carbon atoms.
  • Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-1 -en-1-yl, prop-1 -en-2-yl, or prop-2- en-1-yl), butenyl, butadienyl (e.g., buta-1 ,3-dien-1-yl or buta-1,3-dien-2-yl), pentenyl, or pentadienyl (e.g., isoprenyl).
  • alkenyl preferably refers to C2-4 alkenyl.
  • alkynyl refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds.
  • C2-5 alkynyl denotes an alkynyl group having 2 to 5 carbon atoms.
  • Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl.
  • alkynyl preferably refers to C2-4 alkynyl.
  • alkylene refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched.
  • a “C1-5 alkylene” denotes an alkylene group having 1 to 5 carbon atoms, and the term “C0-3 alkylene” indicates that a covalent bond (corresponding to the option "Co alkylene”) or a C1-3 alkylene is present.
  • Preferred exemplary alkylene groups are methylene (-CH2-), ethylene (e.g., -CH2-CH2- or CH(CH3)-), propylene (e.g., -CH2-CH2-CH2-, -CH(-CH 2 -CH 3 )-, -CH 2 -CH(-CH 3 )-, or -CH(-CH 3 )-CH 2 -), or butylene (e.g., -CH 2 - CH2-CH2-CH2-).
  • alkylene preferably refers to C1-4 alkylene (including, in particular, linear C1-4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.
  • carbocyclyl refers to a hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic.
  • “carbocyclyl” preferably refers to aryl, cycloalkyl or cycloalkenyl.
  • heterocyclyl refers to a ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic.
  • each heteroatom-containing ring comprised in said ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • heterocyclyl preferably refers to heteroaryl, heterocycloalkyl or heterocycloalkenyl.
  • aryl refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic).
  • Aryl may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1,2-dihydronaphthyl), tetralinyl (i.e., 1 ,2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., 1 H-indenyl), anthracenyl, phenanthrenyl, 9H-fluorenyl, or azulenyl.
  • an "aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.
  • heteroaryl refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group).
  • aromatic ring group comprises one or more (such as, e.g., one, two, three
  • each heteroatom-containing ring comprised in said aromatic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • Heteroaryl may, e.g., refer to thienyl (i.e., thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-1 -benzopyranyl or 4H-1 -benzopyranyl), isochromenyl (e.g., 1 H-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 1 H-pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyr
  • heteroaryl preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a “heteroaryl” refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized,
  • cycloalkyl refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings, such as, e.g., a fused ring system composed of two or three fused rings).
  • Cycloalkyl may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl.
  • cycloalkyl preferably refers to a C3-11 cycloalkyl, and more preferably refers to a C3-7 cycloalkyl.
  • a particularly preferred "cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members (e.g., cyclopropyl or cyclohexyl).
  • heterocycloalkyl refers to a saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group).
  • each heteroatom-containing ring comprised in said saturated ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • Heterocycloalkyl may, e.g., refer to aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, azepanyl, diazepanyl (e.g.,
  • oxazolidinyl isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl (e.g., morpholin-4-yl), thiomorpholinyl (e.g., thiomorpholin-4-yl), oxazepanyl, oxiranyl, oxetanyl, tetrahydrofuranyl, 1 ,3-dioxolanyl, tetrahydropyranyl, 1 ,4-dioxanyl, oxepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl (i.e., thiolanyl), 1,3-dithiolanyl, thianyl, 1 ,1-dioxothianyl, thiepanyl, decahydroquinolinyl, decahydrois
  • heterocycloalkyl preferably refers to a 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, "heterocycloalkyl” refers to a 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms
  • cycloalkenyl refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond.
  • Cycloalkenyl may, e.g., refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl.
  • cycloalkenyl preferably refers to a C3-11 cycloalkenyl, and more preferably refers to a C3-7 cycloalkenyl.
  • a particularly preferred "cycloalkenyl” is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds.
  • heterocycloalkenyl refers to an unsaturated alicyclic (non-aromatic) ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group comprises at least one double bond between adjacent
  • each heteroatom-containing ring comprised in said unsaturated alicyclic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • Heterocycloalkenyl may, e.g., refer to imidazolinyl (e.g., 2-imidazolinyl (i.e., 4,5- dihydro-1 H-imidazolyl), 3-imidazolinyl, or 4-imidazolinyl), tetrahydropyridinyl (e.g., 1 ,2,3,6-tetrahydropyridinyl), dihydropyridinyl (e.g., 1,2-dihydropyridinyl or 2,3-dihydropyridinyl), pyranyl (e.g., 2H-pyranyl or 4H-pyranyl), thiopyranyl (e.g., 2H-thiopyranyl or 4H-thiopyranyl), dihydropyranyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrazinyl, dihydroisoindolyl, oc
  • heterocycloalkenyl preferably refers to a 3 to 11 membered unsaturated alicyclic ring group, which is a monocyclic ring or afused ring system (e.g., afused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms; more preferably, "heterocycloalkenyl” refers to a 5 to 7 membered monocyclic unsaturated non-aromatic ring group containing one or more (e.g
  • halogen refers to fluoro (-F), chloro (-CI), bromo (-Br), or iodo (-1).
  • haloalky I refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) halogen atoms which are selected independently from fluoro, chloro, bromo and iodo, and are preferably all fluoro atoms. It will be understood that the maximum number of halogen atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the haloalkyl group.
  • Haloalkyl may, e.g., refer to -CF3, -CHF2, -CH2F, -CF2-CH3, -CH2-CF3, -CH2-CHF2, -CH2-CF2-CH3, -CH2-CF2-CF3, or -CH(CF 3 ) 2 .
  • a particularly preferred "haloalkyl” group is -CF3.
  • alkanoyl refers to a moiety of the formula -CO-alkyl, wherein alkyl is as defined herein.
  • Preferred examples of alkanoyl are acetyl (-CO-CH3), propionyl (-CO-CH2CH3), or butanoyl (e.g., n-butanoyl, i.e. -CO-CH2CH2CH3, or isobutanoyl, i.e. -CO-CH(-CH3)2; preferably n-butanoyl).
  • a particularly preferred alkanoyl is acetyl (-CO-CH3).
  • hydrophobic moiety is defined herein as a group that does not include any hydrogen bond donors or acceptors, optionally except for its point of attachment to the rest of the compound. Accordingly, said hydrophobic moiety can be attached to the rest of the compound, for example, through -0-, -S-, -NH-, -N(CI-6 alkyl), -CO-, -CONH-, -C0N(CI-6 alkyl)-, -NHCO-, or -N(Ci-6 alkyl)CO-.
  • Typical and exemplary hydrophobic groups include (C1-20 alkyl)-O- , (C1.20 alkyl)-S-, (C1.20 alkyl)-NH-, (C1.20 alkyl)-N(Ci. 6 alkyl), (C1.20 alkyl)-CO-, (C1.20 alkyl)-CONH-, (C1.20 alkyl)- C0N(CI-6 alkyl)-, (C1-20 alkylj-NHCO-, and (C1-20 alkyl)-N(Ci-6 alkyl)CO-.
  • a hydrophobic moiety is to be attached to a Z or A moiety through its -NH- group, a particularly preferred hydrophobic moiety is (C1-20 alkyl)- C0-.
  • amphiphilic compound is a compound comprising both a polar moiety and a hydrophobic moiety. Accordingly, an amphiphilic compound is soluble in both polar (for example in water, ionic liquid or ethanol) and non-polar liquids (for example, in hexane or benzene). If an amphiphilic compound is exposed to a biphasic system made of a polar phase (e.g. of water, ionic liquid or ethanol) and non-polar liquid phase (e.g. benzene, hexane), said amphiphilic compound will be enriched at the phase boundary between both phases.
  • a polar phase e.g. of water, ionic liquid or ethanol
  • non-polar liquid phase e.g. benzene, hexane
  • a "polar moiety” is preferably a moiety characterized by affinity for water and characterized by the presence of at least 2 hydrogen bond donors or acceptors.
  • a polar moiety comprises a charged group, or a polar moiety comprises a group selected from -OH, -SH, -COOH (in particular in its charged form -COO ), -NH2, -NH(CI-5 alkyl), -N(0I-5 alkyl)(Ci-5 alkyl) (which can be present in a charged form) and -N(0I-5 alkyl)(Ci-5 alkyl)(Ci-5 alkyl) + .
  • Another example of a polar moiety is a PEG moiety, as defined herein.
  • PEG or a “PEG moiety” is a moiety comprising at least two repeating units according to formula -(OCH2CH2)-.
  • PEG is a moiety according to formula (C1-5 alky l)-(OCH2CH2) m - wherein m is a number of PEG repeats, preferably being between 2 and 20, more preferably between 2 and 10.
  • PEG is a moiety according to formula (C1-5 alkyl)-(OCH2CH2) m -V-, wherein V is selected from -O-, -S-, -NH-, -N(CI-6 alkyl), -CO-, -CONH-, -CON(CI-6 alkyl)-, -NHCO-, and -N(CI-6 alkyl)CO-, preferably wherein the left side of V is connected to the PEG repeating unit, and wherein m is a number of PEG repeats, preferably being between 2 and 20, more preferably between 2 and 10.
  • the terms “optional”, “optionally” and “may” denote that the indicated feature may be present but can also be absent.
  • the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent.
  • the expression “X is optionally substituted with Y” (or “X may be substituted with Y”) means that X is either substituted with Y or is unsubstituted.
  • a component of a composition is indicated to be “optional”, the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.
  • substituents such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety.
  • the "optionally substituted” groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent.
  • the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.
  • substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via several different positions of the corresponding specific substituent group. Unless defined otherwise, the preferred attachment positions for the various specific substituent groups are as illustrated in the examples.
  • Co-6 alkylene moiety may be a covalent bond (corresponding to a Co alkylene) or it may be a C1-6 alkylene in which one -CH2- unit (comprised in the C1-6 alkylene) is optionally replaced by one of the aforementioned groups.
  • Examples of a Co-6 alkylene moiety wherein one -CH2- unit is replaced with -0- include, inter alia, -0-, -O-CH2-, -CH2-O-, -O-CH2-CH2-, -CH2-O-CH2-, -CH2-CH2-O-, -O-CH2-CH2-, -O-CH(-CH 3 )-, -CH(- CH 3 )-O-, -O-CH2-CH2-, -CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-, or -CH2-CH2-CH2-O-, particularly -CH2-O-CH2-.
  • compositions comprising “a” compound of formula (I) can be interpreted as referring to a composition comprising "one or more” compounds of formula (I).
  • the term “comprising” (or “comprise”, “comprises”, “contain”, “contains”, or “containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of “containing, inter alia”, i.e. , “containing, among further optional elements, In addition thereto, this term also includes the narrower meanings of “consisting essentially of' and “consisting of'.
  • a comprising B and C has the meaning of "A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., "A containing B, C and D” would also be encompassed), but this term also includes the meaning of "A consisting essentially of B and C” and the meaning of "A consisting of B and C” (i.e., no other components than B and C are comprised in A).
  • the scope of the invention embraces all pharmaceutically acceptable salt forms of the compounds comprising the moiety of formula (I) or the compounds of formula (II) which may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation.
  • Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as N,N-dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylam
  • Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nic
  • Preferred pharmaceutically acceptable salts of the compounds of formula (I) include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, and a phosphate salt.
  • a particularly preferred pharmaceutically acceptable salt of the compound comprising the moiety of formula (I) or the compound of formula (II) is a hydrochloride salt.
  • the compound comprising the moiety of formula (I) or the compound of formula (II), including any one of the specific compounds comprising the moiety of formula (I) or the compounds of formula (II) described herein is in the form of a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, or a phosphate salt, and it is particularly preferred that the compound comprising the moiety of formula (I) or the compound of formula (II) is in the form of a hydrochloride salt.
  • the present invention also specifically relates to the compound comprising the moiety of formula (I) or the compound of formula (II), including any one of the specific compounds comprising the moiety of formula (I) or the compounds of formula (II) described herein, in non-salt form.
  • the scope of the invention embraces the compounds comprising the moiety of formula (I) or the compounds of formula (II) in any solvated form, including, e.g., solvates with water (i.e., as a hydrate) or solvates with organic solvents such as, e.g., methanol, ethanol, isopropanol, acetic acid, ethyl acetate, ethanolamine, DMSO, or acetonitrile. All physical forms, including any amorphous or crystalline forms (i.e., polymorphs), of the compounds comprising the moiety of formula (I) or the compounds of formula (II) are also encompassed within the scope of the invention. It is to be understood that such solvates and physical forms of pharmaceutically acceptable salts of the compounds comprising the moiety of formula (I) or the compounds of formula (II) are likewise embraced by the invention.
  • the compounds comprising the moiety of formula (I) or the compounds of formula (II) may exist in the form of different isomers, in particular stereoisomers (including, e.g., geometric isomers (or cis/trans isomers), enantiomers and diastereomers) or tautomers (including, in particular, prototropic tautomers, such as keto/enol tautomers or thione/thiol tautomers). All such isomers of the compounds comprising the moiety of formula (I) or the compounds of formula (II) are contemplated as being part of the present invention, either in admixture or in pure or substantially pure form.
  • the invention embraces the isolated optical isomers of the compounds according to the invention as well as any mixtures thereof (including, in particular, racemic mixtures/racemates).
  • the racemates can be resolved by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography.
  • the individual optical isomers can also be obtained from the racemates via salt formation with an optically active acid followed by crystallization.
  • the present invention further encompasses any tautomers of the compounds comprising the moiety of formula (I) or the compounds of formula (II). It will be understood that some compounds may exhibit tautomerism.
  • the scope of the invention also embraces compounds comprising the moiety of formula (I) or compounds of formula (II), in which one or more atoms are replaced by a specific isotope of the corresponding atom.
  • the invention encompasses compounds of formula (I), in which one or more hydrogen atoms (or, e.g., all hydrogen atoms) are replaced by deuterium atoms (i.e. , 2 H; also referred to as “D”).
  • the invention also embraces compounds comprising the moiety of formula (I) or compounds of formula (II) which are enriched in deuterium.
  • Naturally occurring hydrogen is an isotopic mixture comprising about 99.98 mol-% hydrogen-1 ( 1 H) and about 0.0156 mol-% deuterium ( 2 H or D).
  • the content of deuterium in one or more hydrogen positions in the compounds comprising the moiety of formula (I) or the compounds of formula (II) can be increased using deuteration techniques known in the art.
  • a compound comprising the moiety of formula (I) or the compound of formula (I I) or a reactant or precursor to be used in the synthesis of the compound comprising the moiety of formula (I) or the compound of formula (II) can be subjected to an H/D exchange reaction using, e.g., heavy water (D2O).
  • deuteration techniques are described in: Atzrodt J et al., Bioorg Med Chem, 20(18), 5658-5667, 2012; William JS et al., Journal of Labelled Compounds and Radiopharmaceuticals, 53(11-12), 635-644, 2010; Modvig A et al., J Org Chem, 79, 5861-5868, 2014.
  • the content of deuterium can be determined, e.g., using mass spectrometry or NMR spectroscopy.
  • it is preferred that the compound comprising the moiety of formula (I) or the compound of formula (II) is not enriched in deuterium. Accordingly, the presence of naturally occurring hydrogen atoms or 1 H hydrogen atoms in the compounds comprising the moiety of formula (I) or the compounds of formula (II) is preferred.
  • the present invention also embraces compounds comprising the moiety of formula (I) and the compound of formula (II), in which one or more atoms are replaced by a positron-emitting isotope of the corresponding atom, such as, e.g., 18 F, 11 C, 13 N, 15 O, 76 Br, 77 Br, 120 l and/or 124 l.
  • a positron-emitting isotope of the corresponding atom such as, e.g., 18 F, 11 C, 13 N, 15 O, 76 Br, 77 Br, 120 l and/or 124 l.
  • Such compounds can be used as tracers, trackers or imaging probes in positron emission tomography (PET).
  • the invention thus includes (I) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more fluorine atoms (or, e.g., all fluorine atoms) are replaced by 18 F atoms, (II) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more carbon atoms (or, e.g., all carbon atoms) are replaced by 11 C atoms, (ill) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more nitrogen atoms (or, e.g., all nitrogen atoms) are replaced by 13 N atoms, (iv) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more oxygen atoms (or, e.g., all oxygen atoms) are replaced by 15 O atoms, (v) compounds comprising the moiety of formula (I) and the compounds of formula (
  • the compounds provided herein may be administered as compounds perse or may be formulated as medicaments.
  • the medicaments/pharmaceutical compositions may optionally comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and/or solubility enhancers.
  • the pharmaceutical compositions may comprise one or more solubility enhancers, such as, e.g., polyethylene glycol), including poly (ethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da (e.g., PEG 200, PEG 300, PEG 400, or PEG 600), ethylene glycol, propylene glycol, glycerol, a non-ionic surfactant, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate (e.g., Kolliphor® HS 15, CAS 70142-34-6), a phospholipid, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, a cyclodextrin, o-cyclodextrin, p-cyclodextrin, y-cyclodextrin, hydroxyethyl-p-cyclodextrin
  • the pharmaceutical compositions may also comprise one or more preservatives, particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2-ethoxyethanol, m-cresol, chlorocresol (e.g., 2-chloro-3-methyl-phenol or 4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic acid (or a pharmaceutically acceptable salt thereof), sorbic acid (or a pharmaceutically acceptable salt thereof), chlorhexidine, thimerosal, or any combination thereof.
  • preservatives particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2-ethoxyethanol, m-cresol, chlorocresol (e.g., 2-chloro-3-methyl-phenol or 4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic
  • compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in "Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press, 22 nd edition.
  • the pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, rectal, nasal, topical, aerosol or vaginal administration.
  • Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets.
  • Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration.
  • Dosage forms for rectal and vaginal administration include suppositories and ovula.
  • Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler.
  • Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.
  • the compounds comprising the moiety of formula (I) or the compounds of formula (II) or the above described pharmaceutical compositions comprising a compound comprising the moiety of formula (I) or a compound of formula (II) may be administered to a subject by any convenient route of administration, whether systemically/peripheral ly or at the site of desired action, including but not limited to one or more of: oral (e.g., as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e.g., using injection techniques or infusion techniques, and including, for example, by injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrastern
  • examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracardially, intracranially, intramuscularly or subcutaneously administering the compounds or pharmaceutical compositions, and/or by using infusion techniques.
  • parenteral administration the compounds are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • Said compounds or pharmaceutical compositions can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
  • the tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glyco
  • Preferred excipients in this regard include lactose, starch, a cellulose, or high molecular weight polyethylene glycols.
  • the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the compounds or pharmaceutical compositions are preferably administered by oral ingestion, particularly by swallowing. The compounds or pharmaceutical compositions can thus be administered to pass through the mouth into the gastrointestinal tract, which can also be referred to as "oral-gastrointestinal” administration.
  • said compounds or pharmaceutical compositions can be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder.
  • the compounds of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch.
  • sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules.
  • Sustained-release matrices include, e.g., polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(— )-3-hydroxybutyric acid.
  • Sustained-release pharmaceutical compositions also include liposomally entrapped compounds. The present invention thus also relates to liposomes containing a compound of the invention.
  • Said compounds or pharmaceutical compositions may also be administered by the pulmonary route, rectal routes, or the ocular route.
  • they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzalkonium chloride.
  • they may be formulated in an ointment such as petrolatum.
  • dry powder formulations of the compounds comprising the moiety of formula (I) or the compounds of formula (II) for pulmonary administration, particularly inhalation may be prepared by spray drying under conditions which result in a substantially amorphous glassy or a substantially crystalline bioactive powder. Accordingly, dry powders of the compounds of the present invention can be made according to an emulsification/spray drying process.
  • said compounds or pharmaceutical compositions can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying waxwax, and water.
  • they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzyl alcohol and water.
  • the present invention thus relates to the compounds or the pharmaceutical compositions provided herein, wherein the corresponding compound or pharmaceutical composition is to be administered by any one of: an oral route; topical route, including by transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, including by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, intrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, including by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, including by intravitreal, or intracameral route; rectal route; or vaginal route.
  • Preferred routes of administration are oral administration or parenteral administration.
  • a physician will determine the actual dosage which will be most suitable for an individual subject.
  • the specific dose level and frequency of dosage for any particular individual subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual subject undergoing therapy.
  • a proposed, yet non-limiting dose of the compounds according to the invention for oral administration to a human may be 0.05 to 2000 mg, preferably 0.1 mg to 1000 mg, of the active ingredient per unit dose.
  • the unit dose may be administered, e.g., 1 to 3 times per day.
  • the unit dose may also be administered 1 to 7 times per week, e.g., with no more than one administration per day. It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the pati ent/su bject as well as the severity of the condition to be treated. The precise dose and also the route of administration will ultimately be at the discretion of the attendant physician or veterinarian.
  • the present invention relates to the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) for the treatment or prevention of cancer.
  • Data presented in the Examples demonstrate clear selectivity of the compounds and compositions of the invention for cancer cells over healthy cells.
  • the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) have also been demonstrated to be effective on chemoresistant and senescent cancer cells.
  • the cancer to be treated or prevented in accordance with the present invention may be a solid cancer or a hematological cancer.
  • the cancer is selected from lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, large cell lung carcinoma, lung adenocarcinoma, including also lung adenocarcinoma with EGFR mutation AE746-A750, or squamous cell carcinoma of the lung), renal cancer (or kidney cancer; e.g., renal carcinoma), gastrointestinal cancer, stomach cancer, colorectal cancer (e.g., colorectal carcinoma), colon cancer, anal cancer, genitourinary cancer, bladder cancer, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer (e.g., pancreatic adenocarcinoma or pancreatic ductal adenocarcinoma), ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, ovarian cancer (e.g., ovarian
  • the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) can be administered in monotherapy (e.g., without concomitantly administering any further therapeutic agents, or without concomitantly administering any further therapeutic agents against the same disease that is to be treated with the compound of formula (I)).
  • monotherapy e.g., without concomitantly administering any further therapeutic agents, or without concomitantly administering any further therapeutic agents against the same disease that is to be treated with the compound of formula (I)
  • the present invention relates to the compound comprising the moiety of formula (I), the compound of formula (II) or a corresponding pharmaceutical composition for use in the monotherapeutic treatment or prevention of cancer.
  • the invention relates to the monotherapeutic administration of the compound comprising the moiety of formula (I), the compound of formula (II) or a corresponding pharmaceutical composition, without concomitantly administer
  • the compound comprising the moiety of formula (I), the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) can also be administered in combination with one or more further therapeutic agents. If the compound comprising the moiety of formula (I) or the compound of formula (II) is used in combination with a second therapeutic agent active against the same disease or condition, the dose of each compound may differ from that when the corresponding compound is used alone, in particular, a lower dose of each compound may be used.
  • the combination of the compound comprising the moiety of formula (I) or the compound of formula (II) with one or more further therapeutic agents may comprise the simultaneous/concomitant administration of the compound comprising the moiety of formula (I) or the compound of formula (II) and the further therapeutic agent(s) (either in a single pharmaceutical formulation or in separate pharmaceutical formulations), or the sequential/separate administration of the compound comprising the moiety of formula (I) or the compound of formula (II) and the further therapeutic agent(s). If administration is sequential, either the compound comprising the moiety of formula (I) or the compound of formula (II) according to the invention or the one or more further therapeutic agents may be administered first. If administration is simultaneous, the one or more further therapeutic agents may be included in the same pharmaceutical formulation as the compound comprising the moiety of formula (I) or the compound of formula (II), or they may be administered in two or more different (separate) pharmaceutical formulations.
  • An alkylating agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a nitrogen mustard (such as cyclophosphamide, mechlorethamine (chlormethine), uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, or trofosfamide), a nitrosourea (such as carmustine, streptozocin, fotemustine, lomustine, nimustine, prednimustine, ranimustine, or semustine), an alkyl sulfonate (such as busulfan, mannosulfan, or treosulfan), an aziridine (such as hexamethylmelamine (altretamine), triethylenemelamine, ThioTEPA (N, N'N'-triethylenethiophosphoramide), carboquone, or triaziquone), a hydrazine (such as procarbazine),
  • a platinum coordination complex which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, or triplatin tetranitrate.
  • An antimitotic agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a taxane (such as docetaxel, larotaxel, ortataxel, paclitaxel/taxol, tesetaxel, or nab- paclitaxel (e.g., Abraxane®)), a Vinca alkaloid (such as vinblastine, vincristine, vinflunine, vindesine, or vinorelbine), an epothilone (such as epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, or epothilone F) or an epothilone B analogue (such as ixabepilone/azaepothilone B).
  • a taxane such as docetaxel, larotaxel, ortataxel, paclitaxel/taxo
  • An anti-tumor antibiotic which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an anthracycline (such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin), an anthracenedione (such as mitoxantrone, or pixantrone) or an anti-tumor antibiotic isolated from Streptomyces (such as actinomycin (including actinomycin D), bleomycin, mitomycin (including mitomycin C), or plicamycin).
  • an anthracycline such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin
  • a tyrosine kinase inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sorafenib, sunitinib, axitinib, nintedanib, ponatinib, vandetanib, or vemurafenib.
  • a topoisomerase inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a topoisomerase I inhibitor (such as irinotecan, topotecan, camptothecin, belotecan, rubitecan, or lamellarin D) or a topoisomerase II inhibitor (such as amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin).
  • a topoisomerase I inhibitor such as irinotecan, topotecan, camptothecin, belotecan, rubitecan, or lamellarin D
  • a topoisomerase II inhibitor such as amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin.
  • a PARP inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, niraparib, olaparib, rucaparib, talazoparib, veliparib, pamiparib (BGB-290), BMN-673, CEP 9722, MK 4827, E7016, or 3-aminobenzamide.
  • An EGFR inhibitor/antagonist which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, gefitinib, erlotinib, lapatinib, afatinib, neratinib, osimertinib, brigatinib, dacomitinib, vandetanib, pelitinib, canertinib, icotinib, poziotinib, ABT-414, AV-412, PD 153035, PKI-166, BMS- 690514, CUDC-101, AP26113, XL647, cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.
  • anticancer drugs may also be used in combination with a compound of the present invention.
  • the anticancer drugs may comprise biological or chemical molecules, like TNF-related apoptosis-inducing ligand (TRAIL), tamoxifen, amsacrine, bexarotene, estramustine, irofulven, trabectedin, cetuximab, panitumumab, tositumomab, alemtuzumab, bevacizumab, edrecolomab, gemtuzumab, alvocidib, seliciclib, aminolevulinic acid, methyl aminolevulinate, efaproxiral, porfimer sodium, talaporfin, temoporfin, verteporf in, alitretinoin, tretinoin, anagrelide, arsenic trioxide, atrasentan, bortezomib, carmofur,
  • biological drugs like antibodies, antibody fragments, antibody constructs (for example, single-chain constructs), and/or modified antibodies (like CDR-grafted antibodies, humanized antibodies, "fully human” antibodies, etc.) directed against cancer or tumor markers/factors/cytokines involved in proliferative diseases can be employed in cotherapy approaches with the compounds of the invention.
  • biological molecules are anti-HER2 antibodies (e.g. trastuzumab, Herceptin®), anti-CD20 antibodies (e.g.
  • An anticancer drug which can be used in combination with a compound of the present invention may, in particular, be an immunooncology therapeutic (such as an antibody (e.g., a monoclonal antibody or a polyclonal antibody), an antibody fragment, an antibody construct (e.g., a single-chain construct), or a modified antibody (e.g., a CDR- grafted antibody, a humanized antibody, or a "fully human” antibody) targeting any one of CTLA-4, PD-1 , PD-L1 , TIM3, LAG3, 0X40, CSF1 R, IDO, or CD40.
  • an immunooncology therapeutic such as an antibody (e.g., a monoclonal antibody or a polyclonal antibody), an antibody fragment, an antibody construct (e.g., a single-chain construct), or a modified antibody (e.g., a CDR- grafted antibody, a humanized antibody, or a "fully human” antibody) targeting any one of CTLA-4
  • the combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation.
  • the individual components of such combinations may be administered either sequentially or simultaneously/concomitantly in separate or combined pharmaceutical formulations by any convenient route.
  • administration is sequential, either the compound of the present invention (i.e., the compound comprising the moiety of formula (I), the compound of formula (II) or a pharmaceutically acceptable salt thereof) or the further therapeutic agent(s) may be administered first.
  • administration is simultaneous the combination may be administered either in the same pharmaceutical composition or in different pharmaceutical compositions.
  • the two or more compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately, they may be provided in any convenient formulation.
  • the present invention further relates to a method of treatment of cancer, as described herein, in an individual in the need thereof, the method comprising administering the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) to said individual.
  • the administration is preferably as described herein.
  • the present invention further relates to use of the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) for the manufacture of a medicament against cancer, as described herein.
  • the subject or patient to be treated in accordance with the present invention may be an animal (e.g., a non-human animal).
  • the subject/patient is a mammal.
  • the subject/patient is a human (e.g., a male human or a female human) or a non-human mammal (such as, e.g., a guinea pig, a hamster, a rat, a mouse, a rabbit, a dog, a cat, a horse, a monkey, an ape, a marmoset, a baboon, a gorilla, a chimpanzee, an orangutan, a gibbon, a sheep, cattle, or a pig).
  • the subject/patient to be treated in accordance with the invention is a human.
  • Treatment of a disorder or disease, as used herein, is well known in the art.
  • Treatment of a disorder or disease implies that a disorder or disease is suspected or has been diagnosed in a patient/su bject.
  • a pati ent/su bject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e., diagnose a disorder or disease).
  • the "treatment” of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only).
  • the "treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease.
  • the "treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease.
  • Such a partial or complete response may be followed by a relapse.
  • a subject/patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above).
  • the treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).
  • prevention of a disorder or disease is also well known in the art.
  • a patient/subject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease.
  • the subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition.
  • Such a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators.
  • a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms).
  • prevention comprises the use of a compound of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
  • treatment or prevention preferably refers to "treatment”. Accordingly, the different grammatic form of the same term, i.e. “treating or preventing”, as used herein, preferably refers to “treating”.
  • the compounds of the present invention i.e. the compound comprising the moiety of formula (I) or the compound of formula (II) further show antibacterial properties. Accordingly, in further embodiments the present invention relates to the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) for use in the treatment or prevention of a bacterial infectious disease, as well as to use of the compound comprising the moiety of formula (I) or the compound of formula (II) as an antibacterial agent, preferably wherein the use is cosmetic and/or non-therapeutic.
  • amphiphilic p-heterooligomers starting from cyclic and acyclic building blocks with different secondary-structure propensities if only hydrophobic cyclic and acyclic building blocks are used, a hydrophobic structure is inevitably formed. Conjugation of this hydrophobic p-heterooligomer with a hydrophilic element allows the formation of an amphiphilic water-soluble molecule. At the same time, the tendency towards self-association and interaction with other amphiphilic molecules/systems is increased.
  • the hydrophilic element can be of different chemical nature and can carry polar or basic groups, preferably polar neutral groups and basic groups. Examples of amphiphilic, homochiral p-heterooligomers based on cyclic and acyclic building blocks are provided in Scheme 2.
  • the p-peptides in Schemes 2 and 3 consist of commercially available cyclic and acyclic building blocks. They were assembled by solid-phase synthesis using Fmoc chemistry and standard protocols. The peptides in Scheme 2 were prepared in house. Peptide 1aR and its analogues, which are reported in Scheme 3 (1aR-iBu3, 1aR-Me3,(CF3)5, 1aR-Me3,(CF3)8, 1aR-Me3,5, 1aR-Me6,8, and 1aR-mix1-3), were respectively resynthesized and synthesized at GenicBio (China) and provided as trifluoroacetate or chloride peptide salts with a purity of at least 95%.
  • Fmoc-p-Ala-OH and N,N'-diisopropylcarbodiimide (DIG) were purchased from Novabiochem- Merck Millipore (Germany).
  • Fmoc-frans-(1s,2s)-ACPC-OH was purchased from Chem-lmpex International (USA).
  • 2-(1 H-Benzotriazol-1-yl)-1,1 ,3,3-tetramethyluronium-hexafluorphosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) were purchased from Biosolve (The Netherlands).
  • Trifluoroacetic acid (TFA) for HPLC was purchased from Alfa Aesar (Thermo Fisher Scientific, Germany).
  • Triisopropylsilane (TIS), acetic anhydride, acetonitrile for HPLC (ACN), MeOH, and N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich (Merck, Germany).
  • D2O and deuterated solvents were purchased from Armar GmbH (Germany).
  • the solid-phase peptide syntheses of 1b, 1_ALA, and 1a were carried out manually.
  • the Fmoc-Rink-amide-MBHA resin was swollen in 500 pL DMF/NMP (80:20 v/v) for 30 min, then the solvent was sucked off.
  • the Fmoc-protecting group was cleaved by adding 500 pL 40% piperidine in DMF/NMP (80:20 v/v), shaking for 3 min, sucking the solution off, then adding 500 pL 20% piperidine in DMF/NMP (80:20 v/v), shaking for 12 min, sucking the solution off.
  • the resin was washed with DMF five times.
  • each Fmoc-amino acid (3 equiv. for the p-amino acid, 5 equiv. for the a-amino acid) was dissolved in DMF/NMP (80:20 v/v) containing 3 equiv. or 5 equiv. HOBt, added to the resin, followed by 2.8 equiv. or 4.8 equiv. HBTU, and 6 equiv. or 10 equiv. DIPEA, then the suspension was shaken for 1 h. The reaction mixture was sucked off, and the resin was washed with DMF. The coupling procedure was repeated.
  • the acetylation reaction was performed at the end of the chain elongation using 10 equiv. DIPEA and 10 equiv. acetic anhydride in 600 pL DMF/NMP (80:20 v/v).
  • the N-terminus of the resin-bound peptide was acylated with 5 equiv. FAM in the presence of HOBt/DIC (5 equiv. each) for 45 min.
  • the acylation procedure was repeated, followed by treatments with 20% piperidine in DMF (2x30 min). Finally, the resin was washed with DMF, DCM, Et20 (five times each).
  • the NMR spectra were recorded on a Broker (Germany) AVANCE III HD 600 MHz spectrometer equipped with a QXI ( 1 H/ 13 C/ 15 N/ 31 P) probe.
  • the NMR peptide samples (1.1-1 .8 mM in water with 7% D2O, 500 JJ.L) were measured in standard 5-mm TA tubes (Armar, Germany) at 298 K.
  • the spectra were 1 H calibrated using the Broker standard sample of 2 mM sucrose-0.5 mM DSS (4, 4-di methyl-4-silapentane- 1 -sulfonic acid). 13 C frequencies were calibrated indirectly using the recommended scaling factor of 0.25144953.
  • CD circular dichroism
  • the latter were prepared as follows: dried POPC was suspended in phosphate buffer, incubated for 1 h at room temperature, then vortexed vigorously to obtain a milky, uniform POPC suspension. Successively, the suspension was sonicated until the suspension changed from milky to nearly clear in appearance (max. 30 min sonication time).
  • the peptide concentrations were determined spectrophotometrically by using a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Germany) and the UV absorbance of tyrosine at about 280 nm with the molar extinction coefficient 1480 M’ 1 cm 1 (H. Mach, C. R. Middaugh, R. V. Lewis, Anal Biochem 1992, 200, 74-80).
  • the peptide concentrations were as follows: 1b: 0.19 mM in water, 0.12 mM in MeOH; 1_ALA: 0.23 mM in water, 0.14 mM in MeOH; 1a: 0.39 mM in water, 0.34 mM in MeOH.
  • 3 scans were accumulated using a step resolution of 1 nm, a bandwidth of 1 nm, and a time-per-point of 1 s.
  • the CD spectrum of the solvent was subtracted, and the difference spectrum was normalized to express the ellipticity in mean-residue molar ellipticity, divided by 10 3 and represented in the graphs as [0]R X 10 3 (deg cm 2 dmol 1 ).
  • the secondary structure of the p-peptides in Scheme 2 was investigated in water and in MeOH using CD spectroscopy ( Figure 1).
  • the two p-peptides with the register cyclic-acyclic show a maximum at 223 nm, a minimum at 204 nm and a zero crossing at 215 nm.
  • the ratio min./max. is 4.6 for the shorter p-peptide 1b and 7.6 for the longer p-peptide 1_ALA.
  • the p-peptide with the register acyclic-cyclic-acyclic (1a) shows a maximum at 222 nm, a minimum at 204 nm and a zero crossing at 214 nm.
  • the ratio min. /max. is 3.5.
  • the CD spectrum of 1a differs from the CD spectra of 1b and 1_ALA (shift to the left, as already observed in MeOH). It is believed that the p-peptides are less structured in water than in MeOH. This suggests that these peptides adopt a more defined structure in a less polar environment (e.g. in the cell membrane).
  • the formation of these secondary structures is particularly favored in a less polar environment.
  • Dulbecco's modified Eagle's medium (DMEM)-high glucose and Roswell Park Memorial Institute (RPMI) 1640 medium and 1X Dulbecco's Phosphate Buffered Saline (DPBS) were purchased from Szabo-Scandic (Austria).
  • DMSO dimethyl sulfoxide
  • the primary human lung fibroblasts were a gift from Prof. Dr. Jutta Horejs-Hock, University of Salzburg (Austria).
  • the cell line MCF-7 was a gift from Prof. Dr. Barbara Krammer, University of Salzburg (Austria).
  • the cell lines HT1975, A427 and SKLU-1 were a gift from Prof. Dr.
  • the cell lines A549 (ATCC: CLL-185), H460 (ATCC: HTB-177), H520 (ATCC: HTB-182) and HCC827 (ATCC: CRL-2868) were purchased from ATCC.
  • the cisplatin resistant A549 (ddA549) cell line was purchased from Szabo Scandic.
  • MitoViewTM720 and MemBriteTM Fix 660/680 were purchased from Biotium, Dihydrorhodamine 123 (DHR 123) was from Sigma.
  • Human primary lung fibroblasts and MCF-7 cell line were cultured in DMEM-high glucose supplemented with 10% fetal bovine serum, 1 % penicillin— streptomycin and 1 % L-glutamine. All other cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin— streptomycin and 1 % L-glutamine. Cells were grown in a humidified atmosphere at 37 °C in 5% CO2. For all experiments, 80% confluent cells were used.
  • Cells (1x10 4 cells/well) in complete growth medium were seeded into 96-well culture plates. The day after, the medium was changed to a serum free medium with peptides at various concentrations. After an additional incubation period of 24 h, the cell viability was determined by adding 10 pL MTT solution (5 mg/mL in DPBS) to the treated and non-treated control cells for 2 h at 37 °C in the dark. Then the medium was aspirated, cells were lysed with 100 pl DMSO and the absorbance of the resulting product formazan in viable cells was measured at 550 nm with a GloMax® multimode microplate reader. At least three independent experiments (with each sample in triplicate) were performed and cell viability was normalized to the untreated control.
  • MTT solution 5 mg/mL in DPBS
  • Cells (1x10 4 cells/well) in complete growth medium were seeded into 96-well culture plates. The day after, the medium was changed to a serum free medium with 20 piM 1aR. After an additional incubation period of 1 h, 2 h, 4 h and 24 h, the plate was centrifuged for 5 min with 300 rpm to avoid any cellular debris in the supernatant. Afterwards, 50 piL of the supernatant was transferred to a new 96 well plate, and 50 piL LDH working solution were added. The plate was incubated at room temperature for 30 min in the dark.
  • Cytotoxicity(%) ((peptide-low control)/(high control-low control))*100 Propidium iodide (PI) staining for observation of peptide-induced cell membrane damage
  • Cells (3x10 4 /chamber) were grown overnight in 8-well chamber pi-Slides (Ibidi). The day after, the medium was changed to a serum free medium (control) or 10 piM 1aR. The cells were incubated for 1 h and 24 h and subseguently stained with propidium iodide (PI) and Hoechst 33342 for 10 min.
  • PI propidium iodide
  • Cells (1x10 5 cells/well) in complete growth medium were seeded into 12-well culture plates. The day after, the medium was changed to a serum free medium with peptides at various concentrations. After an additional incubation period of 1 h and 24 h, the supernatants were harvested and transferred into 2 mL Eppendorf tubes. Cells were detached with 500 pL Accutase® solution and added to the respective supernatant, the suspension was then centrifuged for 5 min at 1500 rpm.
  • the cell pellet was washed twice with 1 mL DPBS and resuspended in 98.5 piL of 1x annexin V buffer with 1 pL annexin V-APC and 0.5 pL of 1 mg/mL PI. After 15 min incubation at room temperature in the dark, 200 pL of 1x annexin V buffer were added and subseguently measured with the CytoFLEX flow cytometer. Analyses from at least three independent experiments were performed with the Kaluza 1 ,5a flow cytometer analysis software (Beckman Coulter).
  • Cells (5x10 5 cells/well) in complete growth medium were seeded into 35 mm cell culture dishes. The day after, the medium was changed to a serum free medium with peptides at various concentrations. After an additional incubation period of 24 h, the supernatants were harvested and transferred into 2 mL Eppendorf tubes. Cells were detached with 500 pL Accutase® solution and added to the respective supernatant, the suspension was then centrifuged for 5 min at 1500 rpm. After removal of the supernatant, the cell pellet was resuspended in 200 pL DPBS. For fixation, 2 mL of ice-cold 70% ethanol was added dropwise to the suspension under gentle agitation.
  • the cell suspension was centrifuged for 5 min at 1500 rpm and washed twice with 2 mL DPBS. The cells were then stained with a solution of 25 pL 0.4 mg/mL PI, 25 pL of 1 mg/mL RNase solution and 450 pL DPBS. After incubation for 15 min at 37 °C in the dark, the fluorescence of PI bound to DNA was measured with the CytoFLEX flow cytometer. Cell-cycle analyses from at least three independent experiments were performed with the Kaluza 1 ,5a flow cytometer analysis software (Beckman Coulter).
  • Cells (1x10 5 cells/well) in complete growth medium were seeded into 12-well culture plates. The day after, the medium was changed to a serum free medium with peptides at various concentrations. After an additional incubation period of 1 h, 4 h and 24 h, the supernatants were harvested and transferred into 2 mL Eppendorf tubes. Cells were detached with 500 pL Accutase® solution and added to the respective supernatant, the suspension was then centrifuged for 5 min at 1500 rpm. After removal of the supernatant, the cell pellet was stained with 1.6 piL FLICA working solution in 98.4 piL medium.
  • the cells were fixed with 2.5% glutaraldehyde in 0.1 M PBS for 30 min. Afterwards, the cells were washed 3x for 10 min in 0.1 M PBS. Samples were dehydrated in a graded ethanol series. Once in 100% ethanol, the cells were dried with a critical point dryer (EM-CPD300, Leica Microsystems) and subsequently coated with 5 nm platinum using a sputter coater (EM-ACE600, Leica Microsystems). The cover slips with the dried cells were finally imaged with a scanning electron microscope (FE-SEM Merlin compact VP, Carl Zeiss) at 5 kV using a secondary electron detector.
  • FE-SEM Merlin compact VP Carl Zeiss
  • 1x10 4 cells were seeded in a BIOFLOAT FLEX (faCellitate) coated, non-TC treated U-bottom 96-well plate. Subsequently, the plate was centrifuged for 5 minutes at 1500 rpm, to allow the cells to gather in the middle of the well to facilitate spheroid formation. After an incubation period of 4 days, the spheroids were treated with increasing concentrations of the compounds in medium without FCS. To measure cell viability with the Cel ITiter-G/o® 3D Cell Viability Assay (Promega), the spheroids were transferred to a white opaque 96-well plate after 48 h and as described in manufacturer's protocol.
  • the human lung adenocarcinoma cells A549 were seeded in flasks for senescence induction at a concentration of 1x10 5 /mL in fully supplemented RPM1 1640 medium containing 0.1 pig/mL doxorubicin. Medium change was done every 48 h with RPM1 1640 with 0.1 pig/mL doxorubicin until 60% confluence was reached.
  • A549 cells were seeded in 6-well plates at a concentration of 1.25x10 5 /mL and incubated for 18 h at 37 °C. Subsequently, cells were washed with PBS and fixed (2% formaldehyde and 0.25% glutaraldehyde) for 2 min at room temperature.
  • the effect of the peptide on the viability of various human cancer cell lines was analyzed with the MTT assay and annexin V/PI staining.
  • the IC50 values for various human lung cancer cell lines after 24 h of peptide treatment were 3.5- to 12-fold lower than those for the healthy lung fibroblast cell line MRC-5 and the healthy human bronchial epithelial cell line BEAS-2B.
  • the corresponding IC50 values are summarized in Table 13.
  • annexin V/ propidium iodide PI staining was performed.
  • PI staining Upon treatment with 20 piM 1aR, 94.02% (lung adenocarcinoma, A549), 91.43% (lung adenocarcinoma with EGFR mutation AE746-A750, HCC827) and 92.12% (squamous cell carcinoma (SCO) of the lung, H520) underwent cell death (% cells not double negative for annexin V and PI).
  • SCO squamous cell carcinoma
  • the healthy lung fibroblast cell line MRC-5 was largely insensitive to the treatment, showing cell-death induction of only 8.15% compared to untreated cells (Figure 2A).
  • 63.46% dead cells occurred already 1 h after treatment with the majority of dead cells double positive for annexin V and PI ( Figure 2B). This indicates that the mode of induced cell death is not apoptosis but rather necrosis or pyroptosis.
  • the peptide 1aR possesses good cell permeability, as shown by the cellular uptake of a fluorescence-labeled analog bearing 5,6-carboxyfluorescein in place of the N-terminal acetyl group (FAM-1aR) that was found well distributed along the mitochondrial network. As shown in Figure 3, the green fluorescence of FAM-1aR overlapped with the red fluorescence of the mitochondrial probe MitoViewTM 720 in A549 cells. In addition, peptide uptake is energy independent, as peptide accumulation within the cells appeared upon incubation at 37 °C as well as at 4 °C ( Figure 4). The specificity of 1aR for cancer cells does not seem to be due to inferior uptake of the peptide ( Figure 5).
  • 1aR induces significant morphological changes on cancer cell lines.
  • Cells became swollen and displayed pronounced large bubbles budding from the plasma membrane, which is characteristic of pyroptosis ( Figures 6, 14 and 15).
  • the healthy lung fibroblast cell line MRC-5 did not undergo morphological changes ( Figure 6).
  • Peptide 1aR can also induce pyroptosis.
  • Pyroptosis refers to the process of gasdermin (GSDM)-mediated programmed cell death.
  • the GSDM-family members (GSDMA-E) can be cleaved by inflammatory caspases (caspase-1, -4, -5 and -11) as well as by apoptosis-related caspases (caspase-3, -6, -7 and -8).
  • the N-terminal domain executes pyroptosis by the formation of pores within the cell membrane, resulting in characteristic morphological changes and the release of proinflammatory mediators like interleukin-18 (IL-18) and interleukin- 1 p (IL-113) into the extracellular environment, thus amplifying the systemic immune response.
  • IL-18 interleukin-18
  • IL-113 interleukin- 1 p
  • LDH lactate dehydrogenase
  • Cleaved GSDMD and GSDME are able to form pores within the mitochondrial membrane leading to their permeabilization, dissipation of mitochondrial membrane potential, fragmentation of the mitochondrial network and as a result to the production of mitochondrial reactive oxygen species (ROS) (doi: 10.1073/pnas.1414859111 , https://doi.org/10.1038/s41467-019-09397-2, doi: 10.1080/23723556.2019.1621501). Therefore, the effect of 1aR on the mitochondria was analyzed. Indeed, treatment of A549 cells led to mitochondrial membrane depolarization (Figure 11) and a ⁇ 18-fold increase in ROS production ( Figure 12).
  • ROS mitochondrial reactive oxygen species
  • TIS Therapy induced senescence
  • TIS has been associated with reduced tumor growth and is contribute to chemoresistance of cancer cells.
  • TIS has been associated with reduced tumor growth
  • TIS and spontaneously senescent cancer cells are able to reactivate their proliferative potential leading to cancer recurrence accompanied by increased aggressiveness.
  • improved treatments are needed that either avoid induction of TIS or enable targeting of senescent or otherwise chemoresistant cancer cells.
  • Methyl (alkyl group, much less hydrophobic and much less sterically demanding than isopropyl and isobutyl).
  • R 1 in 1aR isopropyl (pos. 3), isobutyl (pos. 5, 6, 8).
  • R 1 in 1aR-Me3,(CF3)8 methyl (pos. 3), isobutyl (pos. 5, 6), 4-(trifluoromethyl)-phenylmethyl (pos. 8).
  • R 1 in 1aR-mix1 allyl (pos. 3); butyl (pos. 5), benzyloxymethyl (pos. 6), methyl (pos. 8).
  • R 1 in 1aR-mix2 propargyl (pos. 3); butyl (pos. 5); benzyloxymethyl (pos. 6); methyl (pos. 8).
  • R 1 in 1aR-Me3,(CF3)5 methyl (pos. 3); 4-(trifluoromethyl)-phenylmethyl (pos. 5), isobutyl (pos. 6, 8).
  • R 1 in 1aR-mix3 methyl (pos. 3), 2-phenylethyl (pos. 5), butyl (pos. 6), sec-butyl (pos. 8).
  • R 1 in 1aR-Me3,5 methyl (pos. 3, 5), isobutyl (pos. 6, 8).
  • R 1 in 1aR-Me6,8 isopropyl (pos. 3), isobutyl (pos. 5), methyl (pos. 6, 8).
  • the peptides embodying the combinations of the selected R 1 groups have been characterized with respect to the following properties: • IC50 values at the A549 cancer cells.
  • the IC50 values of the new peptides at the A549 cancer cells can be classified as follows:
  • the IC50 values at the A549 cancer cells of all peptides fall in a narrow range (4-111 mM), which indicates a good up to excellent tolerance of the R 1 group for chemical moieties with different degree of hydrophobicity and steric demand.
  • the following considerations can be made:
  • 1aR-Cbu displays the following substitutions with respect to the lead peptide 1aR: •
  • the R 1 group iso-propyl in pos. 3 of 1aR is replaced by the moderately more hydrophobic and moderately more sterically demanding R 1 group iso-butyl.

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Abstract

The present invention relates to novel β-peptide compounds, or pharmaceutically acceptable salts thereof, comprising a moiety of formula (I): -[A-Z-A]n-, (I) in particular to compounds of formula (II) or pharmaceutically acceptable salts thereof: X-[Z]p-[A-Z-A]n-Y, (II) and to pharmaceutical compositions comprising said compounds and at least one pharmaceutically acceptable carrier. The compounds and the pharmaceutical compositions of the present invention are useful as medicaments, in particular for the treatment or prevention of cancer.

Description

Beta-peptides with cytotoxic activity on cancer cells
The present application claims the benefit of priority of European patent application EP23188230.9, filed on July 27, 2023, which is incorporated herein by reference in its entirety.
The present invention relates to novel beta-peptide (p-peptide) compounds, or pharmaceutically acceptable salts thereof, comprising a moiety of formula (I), in particular to compounds of formula (I I) or pharmaceutically acceptable salts thereof, and to pharmaceutical compositions comprising said compounds and at least one pharmaceutically acceptable carrier. The compounds and the pharmaceutical compositions of the present invention are useful as a medicament, in particular for the treatment or prevention of cancer. p-Peptides are an underexplored class of peptides based on unnatural p-amino acids (p-AAs) that are considered promising for drug development because of their proteolytic stability and often well-defined secondary structures. However, up to date no compounds of this class have been approved for clinical use. There are few examples of biologically active p-peptides that exhibit antibacterial or fungicidal activity. p-Peptides are artificial oligoamides comprising p-amino acid units. Compared to the proteinogenic o-amino acids, the p-amino acids include an additional carbon atom between the amino and carboxyl groups (H2N- pC*-o C*- COOH) and are rarely found in nature. p-Peptides were developed simultaneously by two researchers in the late 1990s: Prof. Seebach (ETH Zurich) used acyclic p-amino acids (D. Seebach and J. L. Matthews, Chem Commun, 1997, 2015-2022), while Prof. Gellman (Wisconsin University) used cyclic p-amino acids (S. H. Gellman, Accounts Chem Res, 1998, 31, 173-180). These novel oligoamides show the ability to adopt periodic secondary structures, but not overlapping with those of the o-peptides.
Homooligomers of the cyclic p-amino acids trans-2-aminocyclopentanecarboxylic acid ([trans-ACPC]n=68) (D. H. Appella, L. A. Christianson, D. A. Klein, D. R. Powell, X. L. Huang, J. J. Barchi and S. H. Gellman, Nature, 1997, 387, 381-384) and trans-2-aminocyclobutanecarboxylic acid ([trans-ACBC]n=68) (C. Fernandes, S. Faure, E. Pereira, V. Thery, V. Declerck, R. Guillot and D. J. Aitken, Org Lett, 2010, 12, 3606-3609) are known to form so- called 12(=2.5i2)-helices in solution as well as in crystals (R. P. Cheng, S. H. Gellman and W. F. DeGrado, Chem Rev, 2001, 101, 3219-3232) (Scheme 1). Homooligomers of the cyclic p-amino acids trans-2- aminocyclohexanecarboxylic acid ([trans-ACHC]n=e) (D. H. Appella, L. A. Christianson, I. L. Karie, D. R. Powell and S. H. Gellman, J Am Chem Soc, 1996, 118, 13071-13072; D. H. Appella, L. A. Christianson, I. L. Karie, D. R. Powell and S. H. Gellman, J Am Chem Soc, 1999, 121, 6206-6212) and the acyclic p3-amino acids ([p3-AA]n=e) (D. Seebach, P. E. Ciceri, M. Overhand, B. Jaun, D. Rigo, L. Oberer, U. Hommel, R. Amstutz and H. Widmer, Helv Chim Acta, 1996, 79, 2043-2066; D. Seebach, M. Overhand, F. N. M. Kuhnle, B. Martinoni, L. Oberer, U. Hommel and H. Widmer, Helv Chim Acta, 1996, 79, 913-941) as well as the corresponding heterooligomers (L. Fulop, I. M. Mandity, G. Juhasz, V. Szegedi, A. Hetenyi, E. Weber, Z. Bozso, D. Simon, R. Benko, Z. Kiraly and T. A. Martinek, Pios One, 2012, 7) tend to form 14(=3i4)-helices in solution, but shorter oligomers (n=3) form extended structures (Scheme 1). The combination of different p-amino acids can lead to different structures: for example, alternation of cis-ACPC and p3-amino acids has been shown to result in a mixed 14/16-helix (I. M. Mandity, E. Weber, T. A. Martinek, G. Olajos, G. K. Toth, E. Vass, F. Fulop, Angew Chem Int Ed 2009, 48, 2171-2175).
Scheme 1. Examples of p-homooligomers according to Gellman (Appella et al., 1996 and 1997), Aitken (Fernandes et al., 2010) and Seebach (Seebach et al., 1997), and of a p-heterooligomer according to Martinek (Fulop et al., 2012), all displaying well-defined non-natural helical structures (2.5i2- or 3i4-helices).
Figure imgf000003_0002
Aitken, 2010 left-handed 2.512-Helix (H12)
Figure imgf000003_0001
[(fl,R CBC]n
Figure imgf000003_0003
Only a few examples of biologically active p-peptides have been reported in the last three decades. These are mostly antibacterial and fungicidal p-peptides with amphipathic helical structures (C. Cabrele, T. A. Martinek, O. Reiser and L. Berlicki, J Med Chem, 2014, 57, 9718-9739). p-Peptides are a very promising class of molecules for drug discovery because they are more stable towards proteases and peptidases and structurally diverse from a- peptides.
It is an object of the present invention to provide new highly active p-peptides with anticancer activity.
The present inventors have developed p-heterooligomers starting from cyclic and acyclic building blocks which differ in their secondary-structure propensity. On the one hand, the use of acyclic p3-amino acids (p3-AA) as building blocks of p-peptides allows the insertion of naturally occurring amino acid side chains into the sequence. On the other hand, cyclic p-amino acids offer efficient control over the geometry of the p-peptide backbone. To combine the advantages of acyclic and cyclic p-amino acids, p-peptides that contain both acyclic and cyclic p-amino acids have been developed. The cyclic building block is expected to take precedence over the acyclic building block for determining the geometry of the p-peptide backbone, while the acyclic building block is expected to exert a disruptive effect, thereby weakening the stiffness of the resulting structure.
Beyond the state of the art, provided by the present invention are p-peptides with bioactive properties that are promising for cancer therapy. These p-peptides constitute the combination of the cyclic subunit, for example p- amino acid trans-2-aminocyclopentanecarboxylic acid (trans-ACPC) with the acyclic subunit, for example p3-amino acids (p3-AA), with the register [acyclic-cyclic-acyclic]. In other words, the peptides of the present invention include a repeating unit of chain consisting of an acyclic residue attached through an amide bond to a cyclic residue, which is in turn attached through an amide bond to another acyclic residue. The peptides of the present invention differ in their secondary structures from the previously known homo-oligomers [trans-ACPC]n and [p3-AA]n. In particular, the peptides provided herewith seem to have greater flexibility, and thus can better adapt to a given cell component (membrane and/or biomolecule). The p-peptides provided herewith show high lytic activity against cancer cells and are therefore promising for cancer therapy. At the same time, the peptides of the present invention show no or low activity towards non-cancer cells, for example MRC-5 human lung fibroblasts and BEAS-2B human bronchial epithelial cells, as demonstrated in the appended examples.
The invention will be summarized in the following embodiments.
In a first embodiment, the present invention relates to a compound comprising a moiety of formula (I):
-[A-Z-A]n-
(I) wherein: each A is independently a group according to the following formula:
Figure imgf000004_0001
each R1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-OH, -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci-6 alkyl), -S(Ci-6 alkylene)-SH, -S(Ci.6 alkylene)-S(Ci.6 alkyl), -NH-OH, -N(CI.6 alkyl)-OH, -NH-O(CI.6 alkyl), -N(CI.6 alkyl)-O(Ci-6 alkyl), halogen, C1-6 haloalkyl, -O-(Ci-6 haloalkyl), -CF3, -CN, -NO2, -CHO, -C0-(Ci-6 alkyl), -C0-0-(Ci-6 alkyl), -O-CO-(Ci.6 alkyl), -CO-NH2, -CO-NH(CI.6 alkyl), -CO-N(CI.6 alkyl)(Ci.6 alkyl), -NH-CO-(CI.6 alkyl), -N(CI.6 alkyl)-CO-(Ci.6 alkyl), -NH-CO-O-(CI.6 alkyl), -N(CI.6 alkyl)-CO- 0-(Ci-6 alkyl), -O-CO-NH-(CI.6 alkyl), -O-CO-N(CI.6 alkyl)-(Ci_6 alkyl), -SO2-NH2, -SO2-NH(CI.6 alkyl), -SO2-N(CI-6 alkyl)(Ci.6 alkyl), -NH-SO2-(CI.6 alkyl), -N(CI_6 alkyl)-SO2-(Ci.6 alkyl), -SO2-(Ci.6 alkyl), -S0-(Ci-6 alkyl), cycloalkyl, and heterocycloalkyl, and wherein one -CH2- unit in the Co-6 alkylene moiety in said -(Co-6 alkylene)-carbocyclyl or in said -(Co-6 alkylene)-heterocyclyl is optionally replaced with -O-, -S-, -NH- or -N(CI-6 alkyl)-; each Z is independently a group according to the following formula:
Figure imgf000005_0001
each m is independently 0 or 1 ; and n is 2, 3 or 4; or a pharmaceutically acceptable salt thereof.
In a second embodiment, the present invention relates to a pharmaceutical composition comprising the compound of the first embodiment of the present invention and a pharmaceutically acceptable carrier
In a third embodiment, the present invention relates to the compound of the first embodiment of the present invention or the pharmaceutical composition of the second embodiment of the present invention for use as a medicament.
In a fourth embodiment, the present invention relates to the compound of the first embodiment of the present invention or the pharmaceutical composition of the second embodiment of the present invention for use in the treatment or prevention of cancer.
In a fifth embodiment, the present invention relates to use of the compound of the first embodiment of the present invention or the pharmaceutical composition of the second embodiment of the present invention in the manufacture of a medicament against cancer.
In a sixth embodiment, the present invention relates to a method for treating cancer, comprising the step of administering the compound of the first embodiment of the present invention or the pharmaceutical composition of the second embodiment of the present invention to a subject in need thereof. It is to be understood that a therapeutically effective amount of the compound or the composition is to be administered.
The invention will be illustrated using the following figures. Figure 1 presents CD-spectra of p-peptides with cyclic-acyclic register (1b and 1_ALA) or acyclic-cyclic-acyclic register (1a) in methanol and water, comparing the effect of alternating the monomeric cyclic subunit with a monomeric (as in 1b and 1. _ALA) or dimeric (as in 1a) acyclic subunit.
Figure 2 demonstrates (A) cell death induction in human cell lines after 24 h treatment with 20 pM 1aR, and (B) cell death induction in A549 cells after 1 h treatment with 20 pM 1aR. Data represent at least three independent experiments, values are expressed in mean ± SEM.
Figure 3 shows colocalization of the peptide FAM-1aR and the mitochondria in A549 cells. Fluorescence microscopy images were taken after 1 h incubation at 37 °C with FAM-1aR at a concentration of 10 pM. Nuclei were stained with Hoechst 33342 and mitochondria with the mitochondrial probe MitoView™720. Scale bar: 20 pm.
Figure 4 depicts temperature independent uptake of the fluorescence-labeled peptide FAM-1aR into A549 cells. Fluorescence microscopy images were taken after 1 h incubation at 37°C and 4°C with FAM-1 aR at a concentration of 5 pM. Nuclei were stained with Hoechst 33342. Scale bar: 20 pm.
Figure 5 shows uptake of the fluorescence-labeled peptide FAM-1aR into MRC-5 cells. Fluorescence microscopy images were taken after 1 h incubation at 37 °C with FAM-1aR at a concentration of 5 pM. Nuclei were stained with Hoechst 33342. Scale bar: 20 pm.
Figure 6 presents brightfield images of the cancer cell line A549 and the healthy lung fibroblast cell line MRC-5 after treatment with 10 pM 1aR for 24 h. Black arrows indicate membrane blebbing (formation of protrusions and blister-like bodies characteristic of pyroptosis) in A549 cells but not in healthy MRC-5 fibroblasts. Scale bar: 20 pM for A549, 50 pM for MRC-5.
Figure 7 presents LDH release from A549 and MRC-5 cells treated with 10 pM 1aR for the indicated incubation times.
Figure 8 shows propidium iodide (PI) uptake into A549 and MRC-5 cells treated with 10 pM 1aR for 30 min. Nuclei were stained with Hoechst 33342. Scale bar: 20 pm.
Figure 9 demonstrates disruption of the cellular membrane upon treatment with 1aR in cancer cells but not in healthy cells using scanning electron microscopy.
Figure 10 shows (A) flow cytometry analyses of caspase-1 induction, and (B) IL-1 p release after treatment of A549 and MRC-5 cells with 10 pM 1aR for the indicated incubation times. Data represent at least three independent experiments, values are expressed in mean ± SEM. Figure 11 displays mitochondrial membrane depolarization in A549 cells treated with 10 pM 1aR. Nuclei were stained with Hoechst 33342 and the depolarization of the mitochondrial membrane was visualized by the loss of the mitochondrial stain MitoView™720nm. Scale bar: 50 pm.
Figure 12 shows ROS induction after treatment of A549 cells with 10 pM 1aR. Data represent at least three independent experiments and values are expressed in mean ± SEM.
Figure 13 shows (A) cell death induction of senescent A549 cells after treatment with 20 pM 1aR and (B) SA-beta- gal staining identifies senescent cells (left) in untreated A549 cells. No senescent cells were detectable in A549 cells treated with 1aR (right). Data represent at least three independent experiments and values are expressed in mean ± SEM.
Figure 14 shows uptake of FAM-1aR into A549 cells, and pyroptosis-induced morphological characteristics. White arrows indicate the formation of membrane bubbles. The membrane was stained with MemBrite™ Fix 660/680 and nuclei were stained with Hoechst 33342. Scale bar: 20 pM
Figure 15 shows FAM-1aR uptake into A549 cells and the resulting morphological changes. Pictures on the left and on the right were taken three minutes apart. Uptake of the peptide FAM-1 aR is shown. The membrane was stained with MemBrite™ Fix 660/680, nuclei were stained with Hoechst 33342. Scale bar: 20 pM.
Figure 16 shows the viability of selected human cancer cells after 24 h treatment with the peptides 1b, 1_ALA, and 1a (100 pM). Data represent at least three independent experiments and values are expressed in mean ± SEM.
Accordingly, and as mentioned before, in one embodiment, the present invention relates to a compound comprising a moiety of formula (I):
-[A-Z-A]n-
(I) or a pharmaceutically acceptable salt thereof.
In formula (I), each A is independently a group according to the following formula:
Figure imgf000007_0001
Each R1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)- heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-OH, -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci.6 alkyl), -S(Ci.6 alkylene)-SH, -S(Ci.6 alkylene)-S(Ci.6 alkyl), -NH-OH, -N(CI.6 alkyl)-OH, -NH- 0(Ci-6 alkyl), -N(CI-6 alkyl)-0(Ci-6 alkyl), halogen, C1-6 haloalkyl, -O-(Ci-6 haloalkyl), -CF3, -CN, -NO2, -CHO, -CO-(Ci.6 alkyl), -CO-O-(Ci.6 alkyl), -O-CO-(Ci.6 alkyl), -CO-NH2, -CO-NH(CI.6 alkyl), -CO-N(CI-6 alkyl)(Ci.6 alkyl), -NH-CO-(CI.6 alkyl), -N(CI.6 alkyl)-CO-(Ci.6 alkyl), -NH-CO-O-(CI.6 alkyl), -N(CI.6 alkyl)-CO-O-(Ci-6 alkyl), -O-CO-NH-(CI.6 alkyl), -O-CO-N(CI.6 alkyl)-(Ci_6 alkyl), -SO2-NH2, -SO2-NH(CI.6 alkyl), -SO2-N(CI-6 alkyl)(Ci_6 alkyl), -NH-SO2-(CI.6 alkyl), -N(CI.6 alkyl)-SO2-(Ci.6 alkyl), -SO2-(Ci.6 alkyl), -SO-(Ci.6 alkyl), cycloalkyl, and heterocycloalkyl, and wherein one -CH2- unit in the Co-6 alkylene moiety in said -(Co-6 alkylene)-carbocyclyl or in said -(Co-6 alkylene)-heterocyclyl is optionally replaced with -O-, -S-, -NH- or -N(CI-6 alkyl)-. If said one -CH2- unit is replaced, as explained above, it is preferably replaced with -0- or -S-, more preferably it is replaced with -0-.
Preferably, each R1 is independently C1-6 alkyl (e.g. methyl, isopropyl, isobutyl, sec-butyl, or n-butyl), C2-6 alkenyl (e.g., allyl), C2-6 alkynyl (e.g., propargyl), -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)- heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -0(Ci-6 alkyl), -0(Ci-6 alkylene)-OH, -0(Ci-6 alkylene)-0(Ci-6 alkyl), -SH, -S(Ci-6 alkyl), -S(Ci.6 alkylene)-SH, -S(Ci.6 alkylene)-S(Ci.6 alkyl), -NH-OH, -N(CI.6 alkyl)-OH, -NH-O(CI.6 alkyl), -N(CI.6 alkyl)-0(Ci-6 alkyl), halogen, C1-6 haloalkyl, -0-(Ci-6 haloalkyl), -CF3, -CN, -N02, -CHO, -C0-(Ci-6 alkyl), -C0-0-(Ci-6 alkyl), -0-C0-(0i-6 alkyl), -CO-NH2, -CO-NH(CI.6 alkyl), -CO-N(CI.6 alkyl)(Ci.6 alkyl), -NH-CO-(CI.6 alkyl), -N(CI.6 alkyl)-C0-(Ci.6 alkyl), -NH-CO-O-(CI.6 alkyl), -N(CI.6 alkyl)-CO-O-(Ci.6 alkyl), -O-CO-NH-(CI.6 alkyl), -O-CO-N(CI.6 alkyl)-(Ci.6 alkyl), -SO2-NH2, -SO2-NH(CI.6 alkyl), -SO2-N(CI.6 alkyl)(Ci.6 alkyl), -NH-SO2-(CI.6 alkyl), -N(CI.6 alkyl)-SO2-(Ci-6 alkyl), -SO2-(Ci-6 alkyl), -S0-(Ci-6 alkyl), cycloalkyl, and heterocycloalkyl. Corresponding examples of R1 include, in particular, methyl, isopropyl, isobutyl, sec-butyl, n-butyl, allyl, propargyl, phenethyl, benzyloxymethyl and 4-(trifluoromethyl)phenylmethyl.
More preferably, each R1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -0(Ci-6 alkyl), -0(Ci-6 alkylene)-OH, -0(Ci-6 alkylene)-0(Ci-6 alkyl), -SH, -S(Ci.6 alkyl), -S(Ci.6 alkylene)-SH, -S(Ci.6 alkylene)-S(Ci.6 alkyl), -NH-OH, -N(CI.6 alkyl)-OH, -NH- 0(Ci-6 alkyl), -N(CI-6 alkyl)-0(Ci-6 alkyl), halogen, C1-6 haloalkyl, -0-(Ci-6 haloalkyl), -CF3, -CN, -N02, -CHO, -C0-(Ci.6 alkyl), -C0-0-(Ci.6 alkyl), -0-C0-(Ci.6 alkyl), -CO-NH2, -CO-NH(CI.6 alkyl), -C0-N(CI-6 alkyl)(Ci.6 alkyl), -NH-CO-(CI.6 alkyl), -N(CI.6 alkyl)-CO-(Ci.6 alkyl), -NH-CO-O-(CI.6 alkyl), -N(CI.6 alkyl)-C0-0-(Ci.6 alkyl), -O-CO-NH-(CI.6 alkyl), -O-CO-N(CI.6 alkyl)-(Ci.6 alkyl), -SO2-NH2, -SO2-NH(CI.6 alkyl), -SO2-N(CI.6 alkyl)(Ci.6 alkyl), -NH-SO2-(CI.6 alkyl), -N(CI.6 alkyl)-SO2-(Ci.6 alkyl), -SO2-(Ci.6 alkyl), and -S0-(Ci-6 alkyl).
Even more preferably, each R1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -0(Ci-6 alkyl), -0(Ci-6 alkylene)-OH, -0(Ci-6 alkylene)-0(Ci-6 alkyl), -SH, -S(Ci.6 alkyl), -S(Ci_6 alkylene)-SH, -S(Ci_6 alkylene)-S(Ci.6 alkyl), -NH-OH, -N(Ci_6 alkyl)-OH, -NH- O(Ci-6 alkyl), -N(CI-6 alkyl)-O(Ci-6 alkyl), halogen, C1-6 haloalkyl, -O-(Ci-6 haloalkyl), -CF3, -CN, and -NO2.
Even more preferably, each R1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci-6 alkyl), -S(Ci-6 alkylene)-S(Ci-6 alkyl), -NH-O(CI-6 alkyl), -N(CI-6 alkyl)-O(Ci-6 alkyl), halogen, C1-6 haloalkyl, -O-(Ci-6 haloalkyl), and -CF3.
Even more preferably, each R1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, and C2-6 alkynyl.
Even more preferably, each R1 is independently C1-6 alkyl, C2-6 alkenyl, or C2-6 alkynyl.
Even more preferably, each R1 is independently C1-6 alkyl.
Even more preferably, each R1 is independently C2-5 alkyl.
Even more preferably each R1 is independently C3-4 alkyl.
Most preferably, each R1 is independently isopropyl or isobutyl.
In formula (I), each Z is independently a group according to the following formula:
Figure imgf000009_0001
wherein each m is independently 0 or 1 , preferably wherein each m is 1 .
In formula (I), n is 2, 3 or 4.
Preferably, n is 2 or 3.
More preferably, n is 2. It is to be understood that with respect to formula (I) as shown hereinabove, bivalent groups A and Z are so connected to other groups that said A or Z is connected to the group on its left through its -NH- moiety and that said A or Z is connected to the group on its right through its -CO- moiety.
Preferably, in formula (I), each A has the following configuration:
Figure imgf000010_0001
Preferably, in formula (I), each Z has the following configuration:
Figure imgf000010_0002
The compound of formula (I) can be a compound of formula (II):
X-[Z]p-[A-Z-A]n-Y
(II) or a pharmaceutically acceptable salt thereof.
In formula (II), A, Z and n are as defined for formula (I) hereinabove.
In formula (II), p is O or 1. Preferably, p is 1.
In formula (II), X is selected from a hydrophobic moiety, a PEG consisting of 2 to 10 ethylene glycol repeats, and an amino acid sequence of 1 to 5 hydrophobic amino acid residues, wherein said amino acid sequence is attached via its C-terminus to the rest of the compound of formula (II), optionally wherein said amino acid sequence has an alkanoyl group (e.g., an acetyl group) at its N-terminus.
More preferably, X is an amino acid sequence of 1 to 5 hydrophobic amino acid residues, wherein X is attached via its C-terminus to the rest of the compound of formula (II), optionally wherein X has an alkanoyl group at its N-terminus, preferably selected from butanoyl, propionyl and acetyl. It is preferred that X has an alkanoyl group (e.g., a butanoyl, propionyl or acetyl group; particularly an acetyl group) at its N-terminus.
The amino acid sequence of 1 to 5 hydrophobic amino acid residues is preferably an amino acid sequence of 1 to 3 hydrophobic amino acid residue, more preferably an amino acid sequence of 1 to 2 hydrophobic amino acid residue, even more preferably is a single amino acid residue.
Preferably, hydrophobic amino acid residues in X are each independently selected from Gly, Ala, Vai, Leu, lie, Pro, Phe, Tyr, Met and Trp, more preferably from Vai, Leu, lie, Phe, Tyr and Trp, even more preferably from Phe, Tyr and Trp, even more preferably from Phe and Tyr. Preferably, amino acid residues as referred to herein as L-amino acid residues.
Thus, even more preferably, X is alkanoyl-Tyr-, such as, e.g., butanoyl-Tyr-, propionyl-Tyr- or acetyl-Tyr- (Ac-Tyr-), particularly Ac-Tyr-. Even more preferably, X is alkanoy l-(L-Tyr)-, such as, e.g., butanoy l-(L-Tyr)-, propiony l-(L-Tyr)- or Ac-(L-Tyr)-.
In formula (II), Y is a polar moiety. Preferably, Y is neutral or positively charged, preferably Y displays a positive net charge.
Preferably, Y is an amino acid sequence of 1 to 5 polar amino acid residues (preferably basic amino acid residues, or a combination of basic amino acid residues and polar neutral amino acid residues), wherein Y is attached via its N-terminus to the rest of the compound of formula (II), optionally wherein the C-terminal COOH group of Y is replaced by -CONH2 or -CH2OH. It is preferred that the C-terminal COOH group of Y is replaced by -CONH2 or -CH2OH, preferably with -CONH2.
The amino acid sequence of 1 to 5 polar amino acid residues is preferably an amino acid sequence of 1 to 3 polar amino acid residues, more preferably an amino acid sequence of 2 to 3 polar amino acid residues. As explained above, it is preferred that the polar amino acid residues are basic amino acid residues (e.g., selected from Arg, Lys and His) or that they are a combination of basic amino acid residues and polar neutral amino acid residues (e.g., selected from Ser, Thr, Asn and Gin).
Preferably, the polar amino acid residues in Y are all basic amino acid residues. More preferably, the polar amino acid residues in Y are each independently selected from Arg, Lys and His, preferably from Arg and Lys, more preferably they are each Arg. Accordingly, it is particularly preferred that Y consists of basic amino acids that are each independently selected from Arg, Lys and His, preferably from Arg and Lys, more preferably that are each Arg.
Thus, more preferably Y is -Arg-Arg-NH2 or -Arg-Arg-Arg-NH2. Even more preferably Y is -(D-Arg)-(D-Arg)-NH2 or -(D-Arg)-(D-Arg)-(D-Arg)-NH2.
In a first specific embodiment, each A is selected from
Figure imgf000011_0001
In a second specific embodiment, R1 is independently -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-OH, -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci-6 alkyl), -S(Ci-6 alkylene)-SH, -S(Ci.6 alkylene)-S(Ci.6 alkyl), -NH-OH, -N(CI.6 alkyl)-OH, -NH-O(CI.6 alkyl), -N(CI.6 alkyl)-O(Ci-6 alkyl), halogen, C1-6 haloalkyl, -O-(Ci-6 haloalkyl), -CF3, -ON, -NO2, -CHO, -CO-(Ci-6 alkyl), -CO-O-(Ci-6 alkyl), -O-CO-(Ci-6 alkyl), -CO-NH2, -CO-NH(CI.6 alkyl), -CO-N(CI.6 alkyl)(Ci.6 alkyl), -NH-CO-(CI.6 alkyl), -N(CI.6 alkyl)-CO-(Ci.6 alkyl), -NH-CO-O-(Ci.6 alkyl), -N(Ci_6 alkyl)-CO-O-(Ci.6 alkyl), -O-CO-NH-(CI.6 alkyl), -O-CO-N(CI.6 alkyl)-(Ci-6 alkyl), -SO2-NH2, -SO2-NH(Ci.6 alkyl), -SO2-N(CI.6 alkyl)(Ci.6 alkyl), -NH-SO2-(CI.6 alkyl), -N(CI.6 alkyl)-SO2-(Ci-6 alkyl), -SO2-(Ci-6 alkyl), -SO-(Ci-6 alkyl), cycloalkyl, and heterocycloalkyl.
In this second specific embodiment, preferably the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci-6 alkyl), -S(Ci-6 alkylene)-S(Ci-6 alkyl), -NH-O(CI-6 alkyl), -N(CI-6 alkyl)-O(Ci-6 alkyl), halogen, C1-6 haloalkyl, -O-(Ci-6 haloalkyl), and -CF3, more preferably independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 haloalkyl, and -CF3, even more preferably independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, even more preferably independently being C1-6 alkyl. Particularly preferred C1-6 alkyl are C1-3 alkyl groups, in particular methyl and ethyl.
In a third specific embodiment, R1 is independently benzyl or pyrrole, wherein the phenyl in said benzyl and the pyrrole are each optionally substituted with one or more C1-6 alkyl groups. Particularly preferred C1-6 alkyl are C1-3 alkyl groups, in particular methyl and ethyl.
In a fourth specific embodiment, each Z is:
Figure imgf000012_0001
In a fifth specific embodiment, n is 3 or 4. Preferably, n is 3.
In a sixth specific embodiment, p is 0.
In a seventh specific embodiment, moiety X is a hydrophobic moiety. Preferably, X is (C1-20 alkyl)-CO-
In an eighth specific embodiment, X is a (C1-20 alkyl)-CO-. Particularly suitable C1-20 alkyl is C10-20 alkyl, more preferably selected from C10 alkyl, C12 alkyl, C14 alkyl, C16 alkyl and C alkyl.
In a ninth specific embodiment, X is CH3-CO-.
In a tenth specific embodiment, X is a PEG consisting of 2 to 10 ethylene glycol repeats. In an eleventh specific embodiment, X is Ac-Tyr-, preferably wherein X is Ac-(L-Tyr)-.
In a twelfth specific embodiment, Y is -Arg-Arg-NH2 or -Arg-Arg-Arg-NH2. Even more preferably, Y is -(D-Arg)-(D- Arg)-NH2 or -(D-Arg)-(D-Arg)-(D-Arg)-NH2.
In a thirteenth specific embodiment, R1 is selected from methyl, isopropyl, isobutyl, sec-butyl, and n-butyl.
In a fourteenth specific embodiment, R1 is selected from allyl and propargyl. In a fifteenth specific embodiment, R1 is selected from phenylethyl, benzyloxymethyl and 4-(trifluoromethyl)phenyl methyl.
It is to be understood that the compound comprising a moiety of formula (I) or the compound of formula (II) is preferably amphipathic.
Particularly preferred compounds comprising the moiety of formula (I) or the compounds of formula (II) are selected from the following compounds or their pharmaceutically acceptable salts:
Figure imgf000013_0001
It is to be understood that the present invention specifically relates to each and every combination of features and embodiments described herein, including any combination of general and/or preferred features/embodiments. In particular, the invention specifically relates to each combination of meanings (including general and/or preferred meanings) for the various groups and variables comprised in formula (I) or (II). In the present specification as well as the appended claims the following definitions apply, unless specifically indicated to the contrary.
The term "hydrocarbon group” refers to a group consisting of carbon atoms and hydrogen atoms.
As used herein, the term "alkyl” refers to a monovalent saturated acyclic (i.e. , non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an "alkyl” group does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond. A “C1-5 alkyl” denotes an alkyl group having 1 to 5 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, secbutyl, or tert-butyl). Unless defined otherwise, the term "alkyl” preferably refers to C1-4 alkyl.
As used herein, the term "alkenyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond. The term "C2-5 alkenyl” denotes an alkenyl group having 2 to 5 carbon atoms. Preferred exemplary alkenyl groups are ethenyl, propenyl (e.g., prop-1 -en-1-yl, prop-1 -en-2-yl, or prop-2- en-1-yl), butenyl, butadienyl (e.g., buta-1 ,3-dien-1-yl or buta-1,3-dien-2-yl), pentenyl, or pentadienyl (e.g., isoprenyl). Unless defined otherwise, the term "alkenyl” preferably refers to C2-4 alkenyl.
As used herein, the term “alkynyl” refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon-to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds. The term "C2-5 alkynyl” denotes an alkynyl group having 2 to 5 carbon atoms. Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propargyl), or butynyl. Unless defined otherwise, the term "alkynyl” preferably refers to C2-4 alkynyl.
As used herein, the term "alkylene” refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched. A “C1-5 alkylene” denotes an alkylene group having 1 to 5 carbon atoms, and the term "C0-3 alkylene” indicates that a covalent bond (corresponding to the option "Co alkylene”) or a C1-3 alkylene is present. Preferred exemplary alkylene groups are methylene (-CH2-), ethylene (e.g., -CH2-CH2- or CH(CH3)-), propylene (e.g., -CH2-CH2-CH2-, -CH(-CH2-CH3)-, -CH2-CH(-CH3)-, or -CH(-CH3)-CH2-), or butylene (e.g., -CH2- CH2-CH2-CH2-). Unless defined otherwise, the term "alkylene” preferably refers to C1-4 alkylene (including, in particular, linear C1-4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.
As used herein, the term "carbocyclyl” refers to a hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. Unless defined otherwise, "carbocyclyl” preferably refers to aryl, cycloalkyl or cycloalkenyl.
As used herein, the term “heterocyclyl” refers to a ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic. For example, each heteroatom-containing ring comprised in said ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. Unless defined otherwise, "heterocyclyl” preferably refers to heteroaryl, heterocycloalkyl or heterocycloalkenyl.
As used herein, the term "aryl” refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic). "Aryl” may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1,2-dihydronaphthyl), tetralinyl (i.e., 1 ,2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., 1 H-indenyl), anthracenyl, phenanthrenyl, 9H-fluorenyl, or azulenyl. Unless defined otherwise, an "aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.
As used herein, the term "heteroaryl” refers to an aromatic ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic), wherein said aromatic ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said aromatic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. "Heteroaryl” may, e.g., refer to thienyl (i.e., thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-1 -benzopyranyl or 4H-1 -benzopyranyl), isochromenyl (e.g., 1 H-2-benzopyranyl), chromonyl, xanthenyl, phenoxathiinyl, pyrrolyl (e.g., 1 H-pyrrolyl), imidazolyl, pyrazolyl, pyridyl (i.e., pyridinyl; e.g., 2-pyridyl, 3-pyridyl, or 4-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indolyl (e.g., 3H-indolyl), isoindolyl, indazolyl, indolizinyl, purinyl, quinolyl, isoquinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl, pteridinyl, carbazolyl, p-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl (e.g., [1, 10]phenanthrolinyl, [1,7]phenanthrolinyl, or [4,7]phenanthrolinyl), phenazinyl, thiazolyl, isothiazolyl, phenothiazinyl, oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1 ,2,4-oxadiazolyl, 1,2,5-oxadiazolyl (i.e., furazanyl), or 1,3,4- oxadiazolyl), thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, or 1,3,4-thiadiazolyl), phenoxazinyl, pyrazolo[1 ,5-a]pyrimidinyl (e.g., pyrazolo[1,5-a]pyrimidin-3-yl), 1,2-benzoisoxazol-3-yl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzo[b]thiophenyl (i.e., benzothienyl), triazolyl (e.g., 1 H-1,2,3-triazolyl, 2H-1 ,2,3-triazolyl, 1 H-1,2,4-triazolyl, or 4H-1,2,4-triazolyl), benzotriazolyl, 1 H-tetrazolyl, 2H-tetrazolyl, triazinyl (e.g., 1 ,2,3-triazinyl, 1 ,2,4-triazinyl, or 1 ,3,5-triazinyl), furo[2,3-c]pyridinyl, dihydrofuropyridinyl (e.g., 2,3-dihydrofuro[2,3-c]pyridinyl or 1,3-dihydrofuro[3,4-c]pyridinyl), imidazopyridinyl (e.g., imidazo[1 ,2- a]pyridinyl or imidazo[3,2-a]pyridinyl), quinazolinyl, thienopyridinyl, tetrahydrothienopyridinyl (e.g., 4, 5,6,7- tetrahydrothieno[3,2-c]pyridinyl), dibenzofuranyl, 1 ,3-benzodioxolyl, benzodioxanyl (e.g., 1,3-benzodioxanyl or
1.4-benzodioxanyl), or coumarinyl. Unless defined otherwise, the term "heteroaryl” preferably refers to a 5 to 14 membered (more preferably 5 to 10 membered) monocyclic ring or fused ring system comprising one or more (e.g., one, two, three or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; even more preferably, a "heteroaryl” refers to a 5 or 6 membered monocyclic ring comprising one or more (e.g., one, two or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized.
As used herein, the term "cycloalkyl” refers to a saturated hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings, such as, e.g., a fused ring system composed of two or three fused rings). "Cycloalkyl” may, e.g., refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl), or adamantyl. Unless defined otherwise, "cycloalkyl” preferably refers to a C3-11 cycloalkyl, and more preferably refers to a C3-7 cycloalkyl. A particularly preferred "cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members (e.g., cyclopropyl or cyclohexyl).
As used herein, the term "heterocycloalkyl” refers to a saturated ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, and further wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group). For example, each heteroatom-containing ring comprised in said saturated ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. "Heterocycloalkyl” may, e.g., refer to aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, azepanyl, diazepanyl (e.g.,
1.4-diazepanyl), oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, morpholinyl (e.g., morpholin-4-yl), thiomorpholinyl (e.g., thiomorpholin-4-yl), oxazepanyl, oxiranyl, oxetanyl, tetrahydrofuranyl, 1 ,3-dioxolanyl, tetrahydropyranyl, 1 ,4-dioxanyl, oxepanyl, thiiranyl, thietanyl, tetrahydrothiophenyl (i.e., thiolanyl), 1,3-dithiolanyl, thianyl, 1 ,1-dioxothianyl, thiepanyl, decahydroquinolinyl, decahydroisoquinolinyl, or 2-oxa-5-aza-bicyclo[2.2.1]hept- 5-yl. Unless defined otherwise, "heterocycloalkyl” preferably refers to a 3 to 11 membered saturated ring group, which is a monocyclic ring or a fused ring system (e.g., a fused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized; more preferably, "heterocycloalkyl” refers to a 5 to 7 membered saturated monocyclic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbon ring atoms are optionally oxidized.
As used herein, the term "cycloalkenyl” refers to an unsaturated alicyclic (non-aromatic) hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said hydrocarbon ring group comprises one or more (e.g., one or two) carbon-to-carbon double bonds and does not comprise any carbon-to-carbon triple bond. "Cycloalkenyl” may, e.g., refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. Unless defined otherwise, "cycloalkenyl” preferably refers to a C3-11 cycloalkenyl, and more preferably refers to a C3-7 cycloalkenyl. A particularly preferred "cycloalkenyl” is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-to-carbon double bonds.
As used herein, the term "heterocycloalkenyl” refers to an unsaturated alicyclic (non-aromatic) ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings; such as, e.g., a fused ring system composed of two or three fused rings), wherein said ring group contains one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms. For example, each heteroatom-containing ring comprised in said unsaturated alicyclic ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring. "Heterocycloalkenyl” may, e.g., refer to imidazolinyl (e.g., 2-imidazolinyl (i.e., 4,5- dihydro-1 H-imidazolyl), 3-imidazolinyl, or 4-imidazolinyl), tetrahydropyridinyl (e.g., 1 ,2,3,6-tetrahydropyridinyl), dihydropyridinyl (e.g., 1,2-dihydropyridinyl or 2,3-dihydropyridinyl), pyranyl (e.g., 2H-pyranyl or 4H-pyranyl), thiopyranyl (e.g., 2H-thiopyranyl or 4H-thiopyranyl), dihydropyranyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrazinyl, dihydroisoindolyl, octahydroquinolinyl (e.g., 1 ,2,3,4,4a,5,6,7-octahydroquinolinyl), or octahydroisoquinolinyl (e.g., 1,2,3,4,5,6,7,8-octahydroisoquinolinyl). Unless defined otherwise, "heterocycloalkenyl” preferably refers to a 3 to 11 membered unsaturated alicyclic ring group, which is a monocyclic ring or afused ring system (e.g., afused ring system composed of two fused rings), wherein said ring group contains one or more (e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms; more preferably, "heterocycloalkenyl” refers to a 5 to 7 membered monocyclic unsaturated non-aromatic ring group containing one or more (e.g., one, two, or three) ring heteroatoms independently selected from 0, S and N, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) are optionally oxidized, wherein one or more carbon ring atoms are optionally oxidized, and wherein said ring group comprises at least one double bond between adjacent ring atoms and does not comprise any triple bond between adjacent ring atoms.
As used herein, the term "halogen” refers to fluoro (-F), chloro (-CI), bromo (-Br), or iodo (-1).
As used herein, the term “haloalky I” refers to an alkyl group substituted with one or more (preferably 1 to 6, more preferably 1 to 3) halogen atoms which are selected independently from fluoro, chloro, bromo and iodo, and are preferably all fluoro atoms. It will be understood that the maximum number of halogen atoms is limited by the number of available attachment sites and, thus, depends on the number of carbon atoms comprised in the alkyl moiety of the haloalkyl group. “Haloalkyl" may, e.g., refer to -CF3, -CHF2, -CH2F, -CF2-CH3, -CH2-CF3, -CH2-CHF2, -CH2-CF2-CH3, -CH2-CF2-CF3, or -CH(CF3)2. A particularly preferred "haloalkyl” group is -CF3.
As used herein, the term "alkanoyl” refers to a moiety of the formula -CO-alkyl, wherein alkyl is as defined herein. Preferred examples of alkanoyl are acetyl (-CO-CH3), propionyl (-CO-CH2CH3), or butanoyl (e.g., n-butanoyl, i.e. -CO-CH2CH2CH3, or isobutanoyl, i.e. -CO-CH(-CH3)2; preferably n-butanoyl). A particularly preferred alkanoyl is acetyl (-CO-CH3).
The terms "bond” and "covalent bond” are used herein synonymously, unless explicitly indicated otherwise or contradicted by context.
A "hydrophobic moiety” is defined herein as a group that does not include any hydrogen bond donors or acceptors, optionally except for its point of attachment to the rest of the compound. Accordingly, said hydrophobic moiety can be attached to the rest of the compound, for example, through -0-, -S-, -NH-, -N(CI-6 alkyl), -CO-, -CONH-, -C0N(CI-6 alkyl)-, -NHCO-, or -N(Ci-6 alkyl)CO-. Typical and exemplary hydrophobic groups include (C1-20 alkyl)-O- , (C1.20 alkyl)-S-, (C1.20 alkyl)-NH-, (C1.20 alkyl)-N(Ci.6 alkyl), (C1.20 alkyl)-CO-, (C1.20 alkyl)-CONH-, (C1.20 alkyl)- C0N(CI-6 alkyl)-, (C1-20 alkylj-NHCO-, and (C1-20 alkyl)-N(Ci-6 alkyl)CO-. As in formula (II) a hydrophobic moiety is to be attached to a Z or A moiety through its -NH- group, a particularly preferred hydrophobic moiety is (C1-20 alkyl)- C0-.
An "amphiphilic compound” is a compound comprising both a polar moiety and a hydrophobic moiety. Accordingly, an amphiphilic compound is soluble in both polar (for example in water, ionic liquid or ethanol) and non-polar liquids (for example, in hexane or benzene). If an amphiphilic compound is exposed to a biphasic system made of a polar phase (e.g. of water, ionic liquid or ethanol) and non-polar liquid phase (e.g. benzene, hexane), said amphiphilic compound will be enriched at the phase boundary between both phases.
A "polar moiety” is preferably a moiety characterized by affinity for water and characterized by the presence of at least 2 hydrogen bond donors or acceptors. Preferably, a polar moiety comprises a charged group, or a polar moiety comprises a group selected from -OH, -SH, -COOH (in particular in its charged form -COO ), -NH2, -NH(CI-5 alkyl), -N(0I-5 alkyl)(Ci-5 alkyl) (which can be present in a charged form) and -N(0I-5 alkyl)(Ci-5 alkyl)(Ci-5 alkyl)+. Another example of a polar moiety is a PEG moiety, as defined herein.
"PEG” or a "PEG moiety” is a moiety comprising at least two repeating units according to formula -(OCH2CH2)-. Preferably, PEG is a moiety according to formula (C1-5 alky l)-(OCH2CH2)m- wherein m is a number of PEG repeats, preferably being between 2 and 20, more preferably between 2 and 10. More preferably, PEG is a moiety according to formula (C1-5 alkyl)-(OCH2CH2)m-V-, wherein V is selected from -O-, -S-, -NH-, -N(CI-6 alkyl), -CO-, -CONH-, -CON(CI-6 alkyl)-, -NHCO-, and -N(CI-6 alkyl)CO-, preferably wherein the left side of V is connected to the PEG repeating unit, and wherein m is a number of PEG repeats, preferably being between 2 and 20, more preferably between 2 and 10.
As used herein, the terms "optional”, "optionally” and "may” denote that the indicated feature may be present but can also be absent. Whenever the term "optional”, "optionally” or "may” is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent. For example, the expression "X is optionally substituted with Y” (or "X may be substituted with Y”) means that X is either substituted with Y or is unsubstituted. Likewise, if a component of a composition is indicated to be "optional”, the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.
Various groups are referred to as being "optionally substituted” in this specification. Generally, these groups may carry one or more substituents, such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety. Unless defined otherwise, the "optionally substituted” groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent. Moreover, unless defined otherwise, it is preferred that the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.
A skilled person will appreciate that the substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via several different positions of the corresponding specific substituent group. Unless defined otherwise, the preferred attachment positions for the various specific substituent groups are as illustrated in the examples.
Certain groups are referred to as being "optionally replaced” in this specification. It will be understood that a corresponding group can be optionally replaced only if said group is present. Accordingly, if, for example, it is specified that one -CH2- unit in a Co-6 alkylene moiety is optionally replaced with -O-, -S-, -NH- or -N(CI-6 alkyl)-, this means that the Co-6 alkylene moiety may be a covalent bond (corresponding to a Co alkylene) or it may be a C1-6 alkylene in which one -CH2- unit (comprised in the C1-6 alkylene) is optionally replaced by one of the aforementioned groups. Examples of a Co-6 alkylene moiety wherein one -CH2- unit is replaced with -0- include, inter alia, -0-, -O-CH2-, -CH2-O-, -O-CH2-CH2-, -CH2-O-CH2-, -CH2-CH2-O-, -O-CH2-CH2-, -O-CH(-CH3)-, -CH(- CH3)-O-, -O-CH2-CH2-CH2-, -CH2-O-CH2-CH2-, -CH2-CH2-O-CH2-, or -CH2-CH2-CH2-O-, particularly -CH2-O-CH2-.
As used herein, unless explicitly indicated otherwise or contradicted by context, the terms "a”, "an” and "the” are used interchangeably with "one or more” and "at least one”. Thus, for example, a composition comprising "a” compound of formula (I) can be interpreted as referring to a composition comprising "one or more” compounds of formula (I).
It is to be understood that wherever numerical ranges are provided/disclosed herein, all values and subranges encompassed by the respective numerical range are meant to be encompassed within the scope of the invention. Accordingly, the present invention specifically and individually relates to each value that falls within a numerical range disclosed herein, as well as each subrange encompassed by a numerical range disclosed herein.
As used herein, the term "comprising” (or "comprise”, "comprises”, "contain”, "contains”, or "containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of "containing, inter alia”, i.e. , "containing, among further optional elements, In addition thereto, this term also includes the narrower meanings of "consisting essentially of' and "consisting of'. For example, the term "A comprising B and C” has the meaning of "A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., "A containing B, C and D” would also be encompassed), but this term also includes the meaning of "A consisting essentially of B and C” and the meaning of "A consisting of B and C” (i.e., no other components than B and C are comprised in A).
The scope of the invention embraces all pharmaceutically acceptable salt forms of the compounds comprising the moiety of formula (I) or the compounds of formula (II) which may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation. Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as N,N-dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts or tetrabutylammonium salts; and basic amino acid salts such as arginine salts, lysine salts, or histidine salts. Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nicotinate, benzoate, salicylate, ascorbate, pamoate (embonate), camphorate, glucoheptanoate, or pivalate salts; sulfonate salts such as methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate (isethionate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), 2-naphthalenesulfonate (napsylate), 3-phenylsulfonate, or camphorsulfonate salts; glycerophosphate salts; and acidic amino acid salts such as aspartate or glutamate salts. Preferred pharmaceutically acceptable salts of the compounds of formula (I) include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, and a phosphate salt. A particularly preferred pharmaceutically acceptable salt of the compound comprising the moiety of formula (I) or the compound of formula (II) is a hydrochloride salt. Accordingly, it is preferred that the compound comprising the moiety of formula (I) or the compound of formula (II), including any one of the specific compounds comprising the moiety of formula (I) or the compounds of formula (II) described herein, is in the form of a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, or a phosphate salt, and it is particularly preferred that the compound comprising the moiety of formula (I) or the compound of formula (II) is in the form of a hydrochloride salt.
The present invention also specifically relates to the compound comprising the moiety of formula (I) or the compound of formula (II), including any one of the specific compounds comprising the moiety of formula (I) or the compounds of formula (II) described herein, in non-salt form.
Moreover, the scope of the invention embraces the compounds comprising the moiety of formula (I) or the compounds of formula (II) in any solvated form, including, e.g., solvates with water (i.e., as a hydrate) or solvates with organic solvents such as, e.g., methanol, ethanol, isopropanol, acetic acid, ethyl acetate, ethanolamine, DMSO, or acetonitrile. All physical forms, including any amorphous or crystalline forms (i.e., polymorphs), of the compounds comprising the moiety of formula (I) or the compounds of formula (II) are also encompassed within the scope of the invention. It is to be understood that such solvates and physical forms of pharmaceutically acceptable salts of the compounds comprising the moiety of formula (I) or the compounds of formula (II) are likewise embraced by the invention.
Furthermore, the compounds comprising the moiety of formula (I) or the compounds of formula (II) may exist in the form of different isomers, in particular stereoisomers (including, e.g., geometric isomers (or cis/trans isomers), enantiomers and diastereomers) or tautomers (including, in particular, prototropic tautomers, such as keto/enol tautomers or thione/thiol tautomers). All such isomers of the compounds comprising the moiety of formula (I) or the compounds of formula (II) are contemplated as being part of the present invention, either in admixture or in pure or substantially pure form. As for stereoisomers, the invention embraces the isolated optical isomers of the compounds according to the invention as well as any mixtures thereof (including, in particular, racemic mixtures/racemates). The racemates can be resolved by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography. The individual optical isomers can also be obtained from the racemates via salt formation with an optically active acid followed by crystallization. The present invention further encompasses any tautomers of the compounds comprising the moiety of formula (I) or the compounds of formula (II). It will be understood that some compounds may exhibit tautomerism. In such cases, the formulae provided herein expressly depict only one of the possible tautomeric forms. The formulae and chemical names as provided herein are intended to encompass any tautomeric form of the corresponding compound and not to be limited merely to the specific tautomeric form depicted by the drawing or identified by the name of the compound.
The scope of the invention also embraces compounds comprising the moiety of formula (I) or compounds of formula (II), in which one or more atoms are replaced by a specific isotope of the corresponding atom. For example, the invention encompasses compounds of formula (I), in which one or more hydrogen atoms (or, e.g., all hydrogen atoms) are replaced by deuterium atoms (i.e. , 2H; also referred to as “D”). Accordingly, the invention also embraces compounds comprising the moiety of formula (I) or compounds of formula (II) which are enriched in deuterium. Naturally occurring hydrogen is an isotopic mixture comprising about 99.98 mol-% hydrogen-1 (1H) and about 0.0156 mol-% deuterium (2H or D). The content of deuterium in one or more hydrogen positions in the compounds comprising the moiety of formula (I) or the compounds of formula (II) can be increased using deuteration techniques known in the art. For example, a compound comprising the moiety of formula (I) or the compound of formula (I I) or a reactant or precursor to be used in the synthesis of the compound comprising the moiety of formula (I) or the compound of formula (II) can be subjected to an H/D exchange reaction using, e.g., heavy water (D2O). Further suitable deuteration techniques are described in: Atzrodt J et al., Bioorg Med Chem, 20(18), 5658-5667, 2012; William JS et al., Journal of Labelled Compounds and Radiopharmaceuticals, 53(11-12), 635-644, 2010; Modvig A et al., J Org Chem, 79, 5861-5868, 2014. The content of deuterium can be determined, e.g., using mass spectrometry or NMR spectroscopy. Unless specifically indicated otherwise, it is preferred that the compound comprising the moiety of formula (I) or the compound of formula (II) is not enriched in deuterium. Accordingly, the presence of naturally occurring hydrogen atoms or 1H hydrogen atoms in the compounds comprising the moiety of formula (I) or the compounds of formula (II) is preferred.
The present invention also embraces compounds comprising the moiety of formula (I) and the compound of formula (II), in which one or more atoms are replaced by a positron-emitting isotope of the corresponding atom, such as, e.g., 18F, 11C, 13N, 15O, 76Br, 77Br, 120l and/or 124l. Such compounds can be used as tracers, trackers or imaging probes in positron emission tomography (PET). The invention thus includes (I) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more fluorine atoms (or, e.g., all fluorine atoms) are replaced by 18F atoms, (II) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more carbon atoms (or, e.g., all carbon atoms) are replaced by 11C atoms, (ill) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more nitrogen atoms (or, e.g., all nitrogen atoms) are replaced by 13N atoms, (iv) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more oxygen atoms (or, e.g., all oxygen atoms) are replaced by 15O atoms, (v) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by 76Br atoms, (vi) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more bromine atoms (or, e.g., all bromine atoms) are replaced by 77Br atoms, (vii) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more iodine atoms (or, e.g., all iodine atoms) are replaced by 120l atoms, and (viii) compounds comprising the moiety of formula (I) and the compounds of formula (II), in which one or more iodine atoms (or, e.g., all iodine atoms) are replaced by 124l atoms. In general, it is preferred that none of the atoms in the compounds comprising the moiety of formula (I) or the compounds of formula (II) are replaced by specific isotopes.
The compounds provided herein may be administered as compounds perse or may be formulated as medicaments. The medicaments/pharmaceutical compositions may optionally comprise one or more pharmaceutically acceptable excipients, such as carriers, diluents, fillers, disintegrants, lubricating agents, binders, colorants, pigments, stabilizers, preservatives, antioxidants, and/or solubility enhancers.
The pharmaceutical compositions may comprise one or more solubility enhancers, such as, e.g., polyethylene glycol), including poly (ethylene glycol) having a molecular weight in the range of about 200 to about 5,000 Da (e.g., PEG 200, PEG 300, PEG 400, or PEG 600), ethylene glycol, propylene glycol, glycerol, a non-ionic surfactant, tyloxapol, polysorbate 80, macrogol-15-hydroxystearate (e.g., Kolliphor® HS 15, CAS 70142-34-6), a phospholipid, lecithin, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, a cyclodextrin, o-cyclodextrin, p-cyclodextrin, y-cyclodextrin, hydroxyethyl-p-cyclodextrin, hydroxypropyl-p- cyclodextrin, hydroxyethyl-y-cyclodextrin, hydroxypropyl-y-cyclodextrin, dihydroxypropyl-p-cyclodextrin, sulfobutylether-p-cyclodextrin, sulfobutylether-y-cyclodextrin, glucosyl-o-cyclodextrin, glucosyl-p-cyclodextrin, diglucosyl-p-cyclodextrin, maltosyl-a-cyclodextrin, maltosyl-p-cyclodextrin, maltosyl-y-cyclodextrin, maltotriosyl-p- cyclodextrin, maltotriosyl-y-cyclodextrin, dimaltosyl-p-cyclodextrin, methyl-p-cyclodextrin, a carboxyalkyl thioether, hydroxypropyl methylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, a vinyl acetate copolymer, vinyl pyrrolidone, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, or any combination thereof.
The pharmaceutical compositions may also comprise one or more preservatives, particularly one or more antimicrobial preservatives, such as, e.g., benzyl alcohol, chlorobutanol, 2-ethoxyethanol, m-cresol, chlorocresol (e.g., 2-chloro-3-methyl-phenol or 4-chloro-3-methyl-phenol), benzalkonium chloride, benzethonium chloride, benzoic acid (or a pharmaceutically acceptable salt thereof), sorbic acid (or a pharmaceutically acceptable salt thereof), chlorhexidine, thimerosal, or any combination thereof.
The pharmaceutical compositions can be formulated by techniques known to the person skilled in the art, such as the techniques published in "Remington: The Science and Practice of Pharmacy”, Pharmaceutical Press, 22nd edition. The pharmaceutical compositions can be formulated as dosage forms for oral, parenteral, such as intramuscular, intravenous, subcutaneous, intradermal, intraarterial, intracardial, rectal, nasal, topical, aerosol or vaginal administration. Dosage forms for oral administration include coated and uncoated tablets, soft gelatin capsules, hard gelatin capsules, lozenges, troches, solutions, emulsions, suspensions, syrups, elixirs, powders and granules for reconstitution, dispersible powders and granules, medicated gums, chewing tablets and effervescent tablets. Dosage forms for parenteral administration include solutions, emulsions, suspensions, dispersions and powders and granules for reconstitution. Emulsions are a preferred dosage form for parenteral administration. Dosage forms for rectal and vaginal administration include suppositories and ovula. Dosage forms for nasal administration can be administered via inhalation and insufflation, for example by a metered inhaler. Dosage forms for topical administration include creams, gels, ointments, salves, patches and transdermal delivery systems.
The compounds comprising the moiety of formula (I) or the compounds of formula (II) or the above described pharmaceutical compositions comprising a compound comprising the moiety of formula (I) or a compound of formula (II) may be administered to a subject by any convenient route of administration, whether systemically/peripheral ly or at the site of desired action, including but not limited to one or more of: oral (e.g., as a tablet, capsule, or as an ingestible solution), topical (e.g., transdermal, intranasal, ocular, buccal, and sublingual), parenteral (e.g., using injection techniques or infusion techniques, and including, for example, by injection, e.g., subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, or intrasternal by, e.g., implant of a depot, for example, subcutaneously or intramuscularly), pulmonary (e.g., by inhalation or insufflation therapy using, e.g., an aerosol, e.g., through mouth or nose), gastrointestinal, intrauterine, intraocular, subcutaneous, ophthalmic (including intravitreal or intracameral), rectal, or vaginal administration.
If said compounds or pharmaceutical compositions are administered parenterally, then examples of such administration include one or more of: intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intraurethrally, intrasternally, intracardially, intracranially, intramuscularly or subcutaneously administering the compounds or pharmaceutical compositions, and/or by using infusion techniques. For parenteral administration, the compounds are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
Said compounds or pharmaceutical compositions can also be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.
The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof. For oral administration, the compounds or pharmaceutical compositions are preferably administered by oral ingestion, particularly by swallowing. The compounds or pharmaceutical compositions can thus be administered to pass through the mouth into the gastrointestinal tract, which can also be referred to as "oral-gastrointestinal” administration.
Alternatively, said compounds or pharmaceutical compositions can be administered in the form of a suppository or pessary, or may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The compounds of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch.
Said compounds or pharmaceutical compositions may also be administered by sustained release systems. Suitable examples of sustained-release compositions include semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include, e.g., polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D-(— )-3-hydroxybutyric acid. Sustained-release pharmaceutical compositions also include liposomally entrapped compounds. The present invention thus also relates to liposomes containing a compound of the invention.
Said compounds or pharmaceutical compositions may also be administered by the pulmonary route, rectal routes, or the ocular route. For ophthalmic use, they can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.
It is also envisaged to prepare dry powder formulations of the compounds comprising the moiety of formula (I) or the compounds of formula (II) for pulmonary administration, particularly inhalation. Such dry powders may be prepared by spray drying under conditions which result in a substantially amorphous glassy or a substantially crystalline bioactive powder. Accordingly, dry powders of the compounds of the present invention can be made according to an emulsification/spray drying process.
For topical application to the skin, said compounds or pharmaceutical compositions can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying waxwax, and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, 2-octyldodecanol, benzyl alcohol and water.
The present invention thus relates to the compounds or the pharmaceutical compositions provided herein, wherein the corresponding compound or pharmaceutical composition is to be administered by any one of: an oral route; topical route, including by transdermal, intranasal, ocular, buccal, or sublingual route; parenteral route using injection techniques or infusion techniques, including by subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, intrasternal, intraventricular, intraurethral, or intracranial route; pulmonary route, including by inhalation or insufflation therapy; gastrointestinal route; intrauterine route; intraocular route; subcutaneous route; ophthalmic route, including by intravitreal, or intracameral route; rectal route; or vaginal route. Preferred routes of administration are oral administration or parenteral administration. For each of the compounds or pharmaceutical compositions provided herein, it is particularly preferred that the respective compound or pharmaceutical composition is to be administered orally (particularly by oral ingestion).
Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual subject may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual subject undergoing therapy.
A proposed, yet non-limiting dose of the compounds according to the invention for oral administration to a human (of approximately 70 kg body weight) may be 0.05 to 2000 mg, preferably 0.1 mg to 1000 mg, of the active ingredient per unit dose. The unit dose may be administered, e.g., 1 to 3 times per day. The unit dose may also be administered 1 to 7 times per week, e.g., with no more than one administration per day. It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the pati ent/su bject as well as the severity of the condition to be treated. The precise dose and also the route of administration will ultimately be at the discretion of the attendant physician or veterinarian.
The present invention relates to the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) for the treatment or prevention of cancer. Data presented in the Examples demonstrate clear selectivity of the compounds and compositions of the invention for cancer cells over healthy cells. Importantly, the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) have also been demonstrated to be effective on chemoresistant and senescent cancer cells.
The cancer to be treated or prevented in accordance with the present invention may be a solid cancer or a hematological cancer. Preferably, the cancer is selected from lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, large cell lung carcinoma, lung adenocarcinoma, including also lung adenocarcinoma with EGFR mutation AE746-A750, or squamous cell carcinoma of the lung), renal cancer (or kidney cancer; e.g., renal carcinoma), gastrointestinal cancer, stomach cancer, colorectal cancer (e.g., colorectal carcinoma), colon cancer, anal cancer, genitourinary cancer, bladder cancer, liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer (e.g., pancreatic adenocarcinoma or pancreatic ductal adenocarcinoma), ovarian cancer, cervical cancer, endometrial cancer, vaginal cancer, vulvar cancer, ovarian cancer (e.g., ovarian carcinoma), uterine cancer, prostate cancer (e.g., hormone-refractory prostate cancer), testicular cancer, biliary tract cancer, hepatobiliary cancer, neuroblastoma, brain cancer (e.g., glioblastoma), breast cancer (e.g., triple-negative breast cancer, breast cancer having a BRCA1 and/or BRCA2 gene mutation, or breast adenocarcinoma), head and/or neck cancer (e.g., head and neck squamous cell carcinoma), skin cancer, melanoma, Merkel-cell cancer (e.g., Merkel-cell carcinoma), epidermoid cancer, squamous cell cancer (or squamous cell carcinoma; including, e.g., oral squamous cell carcinoma/squamous-cell mouth carcinoma, squamous-cell skin cancer, squamous-cell lung carcinoma, squamous-cell thyroid carcinoma, squamous-cell esophageal carcinoma, or squamous-cell vaginal carcinoma), bone cancer (e.g., osteosarcoma or osteogenic sarcoma), fibrosarcoma, Ewing's sarcoma, malignant mesothelioma, esophageal cancer, laryngeal cancer, mouth cancer, thymoma, neuroendocrine cancer (e.g., neuroendocrine carcinoma), goblet cell cancer (e.g., goblet cell carcinoid), hematological cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, or chronic myeloid leukemia), lymphoma (e.g., Hodgkin lymphoma or non-Hodgkin lymphoma, such as, e.g., follicular lymphoma or diffuse large B-cell lymphoma), and multiple myeloma. Moreover, the cancer to be treated (including any one of the aforementioned specific types of cancer) may also be a chemoresistant and/or a metastatic cancer.
The compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) can be administered in monotherapy (e.g., without concomitantly administering any further therapeutic agents, or without concomitantly administering any further therapeutic agents against the same disease that is to be treated with the compound of formula (I)). Thus, the present invention relates to the compound comprising the moiety of formula (I), the compound of formula (II) or a corresponding pharmaceutical composition for use in the monotherapeutic treatment or prevention of cancer. In particular, the invention relates to the monotherapeutic administration of the compound comprising the moiety of formula (I), the compound of formula (II) or a corresponding pharmaceutical composition, without concomitantly administering any further anticancer agents.
However, the compound comprising the moiety of formula (I), the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) can also be administered in combination with one or more further therapeutic agents. If the compound comprising the moiety of formula (I) or the compound of formula (II) is used in combination with a second therapeutic agent active against the same disease or condition, the dose of each compound may differ from that when the corresponding compound is used alone, in particular, a lower dose of each compound may be used. The combination of the compound comprising the moiety of formula (I) or the compound of formula (II) with one or more further therapeutic agents may comprise the simultaneous/concomitant administration of the compound comprising the moiety of formula (I) or the compound of formula (II) and the further therapeutic agent(s) (either in a single pharmaceutical formulation or in separate pharmaceutical formulations), or the sequential/separate administration of the compound comprising the moiety of formula (I) or the compound of formula (II) and the further therapeutic agent(s). If administration is sequential, either the compound comprising the moiety of formula (I) or the compound of formula (II) according to the invention or the one or more further therapeutic agents may be administered first. If administration is simultaneous, the one or more further therapeutic agents may be included in the same pharmaceutical formulation as the compound comprising the moiety of formula (I) or the compound of formula (II), or they may be administered in two or more different (separate) pharmaceutical formulations.
Preferably, in the context of the treatment (or prevention) of cancer, the one or more further therapeutic agents to be administered in combination with a compound of the present invention are anticancer drugs. The anticancer drug(s) to be administered in combination with a compound comprising the moiety of formula (I) or a compound of formula (II) according to the invention may, e.g., be selected from: a tumor angiogenesis inhibitor (e.g., a protease inhibitor, an epidermal growth factor receptor kinase inhibitor, or a vascular endothelial growth factor receptor kinase inhibitor); a cytotoxic drug (e.g., an antimetabolite, such as purine and pyrimidine analog antimetabolites); an antimitotic agent (e.g., a microtubule stabilizing drug or an antimitotic alkaloid); a platinum coordination complex; an anti-tumor antibiotic; an alkylating agent (e.g., a nitrogen mustard or a nitrosourea); an endocrine agent (e.g., an adrenocorticosteroid, an androgen, an anti-androgen, an estrogen, an anti-estrogen, an aromatase inhibitor, a gonadotropin-releasing hormone agonist, or a somatostatin analog); or a compound that targets an enzyme or receptor that is overexpressed and/or otherwise involved in a specific metabolic pathway that is deregulated (or misregulated) in the tumor cell (e.g., ATP and GTP phosphodiesterase inhibitors, histone deacetylase inhibitors, protein kinase inhibitors (such as serine, threonine and tyrosine kinase inhibitors, e.g., Abelson protein tyrosine kinase inhibitors) and the various growth factors, their receptors and corresponding kinase inhibitors (such as epidermal growth factor receptor kinase inhibitors, vascular endothelial growth factor receptor kinase inhibitors, fibroblast growth factor inhibitors, insulin-like growth factor receptor inhibitors and platelet-derived growth factor receptor kinase inhibitors)); methionine aminopeptidase inhibitors (e.g., methionine aminopeptidase 2 inhibitors), proteasome inhibitors, cyclooxygenase inhibitors (e.g., cyclooxygenase-1 or cyclooxygenase-2 inhibitors), topoisomerase inhibitors (e.g., topoisomerase I inhibitors or topoisomerase II inhibitors), poly ADP ribose polymerase inhibitors (PARP inhibitors), and epidermal growth factor receptor (EGFR) inhibitors/antagonists.
An alkylating agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a nitrogen mustard (such as cyclophosphamide, mechlorethamine (chlormethine), uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, or trofosfamide), a nitrosourea (such as carmustine, streptozocin, fotemustine, lomustine, nimustine, prednimustine, ranimustine, or semustine), an alkyl sulfonate (such as busulfan, mannosulfan, or treosulfan), an aziridine (such as hexamethylmelamine (altretamine), triethylenemelamine, ThioTEPA (N, N'N'-triethylenethiophosphoramide), carboquone, or triaziquone), a hydrazine (such as procarbazine), a triazene (such as dacarbazine), or an imidazotetrazine (such as temozolomide).
A platinum coordination complex which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, or triplatin tetranitrate.
A cytotoxic drug which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an antimetabolite, including folic acid analogue anti metabolites (such as aminopterin, methotrexate, pemetrexed, or raltitrexed), purine analogue antimetabolites (such as cladribine, clofarabine, fludarabine, 6-mercaptopurine (including its prodrug form azathioprine), pentostatin, or 6-thioguanine), and pyrimidine analogue antimetabolites (such as cytarabine, decitabine, 5-fluorouracil (including its prodrug forms capecitabine and tegafur), floxuridine, gemcitabine, enocitabine, or sapacitabine).
An antimitotic agent which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a taxane (such as docetaxel, larotaxel, ortataxel, paclitaxel/taxol, tesetaxel, or nab- paclitaxel (e.g., Abraxane®)), a Vinca alkaloid (such as vinblastine, vincristine, vinflunine, vindesine, or vinorelbine), an epothilone (such as epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, or epothilone F) or an epothilone B analogue (such as ixabepilone/azaepothilone B).
An anti-tumor antibiotic which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, an anthracycline (such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, or zorubicin), an anthracenedione (such as mitoxantrone, or pixantrone) or an anti-tumor antibiotic isolated from Streptomyces (such as actinomycin (including actinomycin D), bleomycin, mitomycin (including mitomycin C), or plicamycin).
A tyrosine kinase inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, axitinib, bosutinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sorafenib, sunitinib, axitinib, nintedanib, ponatinib, vandetanib, or vemurafenib.
A topoisomerase inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, a topoisomerase I inhibitor (such as irinotecan, topotecan, camptothecin, belotecan, rubitecan, or lamellarin D) or a topoisomerase II inhibitor (such as amsacrine, etoposide, etoposide phosphate, teniposide, or doxorubicin).
A PARP inhibitor which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, niraparib, olaparib, rucaparib, talazoparib, veliparib, pamiparib (BGB-290), BMN-673, CEP 9722, MK 4827, E7016, or 3-aminobenzamide.
An EGFR inhibitor/antagonist which can be used as an anticancer drug in combination with a compound of the present invention may be, for example, gefitinib, erlotinib, lapatinib, afatinib, neratinib, osimertinib, brigatinib, dacomitinib, vandetanib, pelitinib, canertinib, icotinib, poziotinib, ABT-414, AV-412, PD 153035, PKI-166, BMS- 690514, CUDC-101, AP26113, XL647, cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.
Further anticancer drugs may also be used in combination with a compound of the present invention. The anticancer drugs may comprise biological or chemical molecules, like TNF-related apoptosis-inducing ligand (TRAIL), tamoxifen, amsacrine, bexarotene, estramustine, irofulven, trabectedin, cetuximab, panitumumab, tositumomab, alemtuzumab, bevacizumab, edrecolomab, gemtuzumab, alvocidib, seliciclib, aminolevulinic acid, methyl aminolevulinate, efaproxiral, porfimer sodium, talaporfin, temoporfin, verteporf in, alitretinoin, tretinoin, anagrelide, arsenic trioxide, atrasentan, bortezomib, carmofur, celecoxib, demecolcine, elesclomol, elsamitrucin, etoglucid, lonidamine, lucanthone, masoprocol, mitobronitol, mitoguazone, mitotane, oblimersen, omacetaxine, sitimagene, ceradenovec, tegafur, testolactone, tiazofurine, tipifarnib, vorinostat, iniparib, or copanlisib.
Also biological drugs, like antibodies, antibody fragments, antibody constructs (for example, single-chain constructs), and/or modified antibodies (like CDR-grafted antibodies, humanized antibodies, "fully human” antibodies, etc.) directed against cancer or tumor markers/factors/cytokines involved in proliferative diseases can be employed in cotherapy approaches with the compounds of the invention. Examples of such biological molecules are anti-HER2 antibodies (e.g. trastuzumab, Herceptin®), anti-CD20 antibodies (e.g. Rituximab, Rituxan®, MabThera®, Reditux®), anti-CD19/CD3 constructs (see, e.g., EP1071752) and anti-TNF antibodies (see, e.g., Taylor PC, Curr Opin Pharmacol, 2003, 3(3):323-328). Further antibodies, antibody fragments, antibody constructs and/or modified antibodies to be used in cotherapy approaches with the compounds of the invention can be found, e.g., in: Taylor PC, Curr Opin Pharmacol, 2003, 3(3):323-328; or Roxana A, Maedica, 2006, 1 (1):63-65.
An anticancer drug which can be used in combination with a compound of the present invention may, in particular, be an immunooncology therapeutic (such as an antibody (e.g., a monoclonal antibody or a polyclonal antibody), an antibody fragment, an antibody construct (e.g., a single-chain construct), or a modified antibody (e.g., a CDR- grafted antibody, a humanized antibody, or a "fully human” antibody) targeting any one of CTLA-4, PD-1 , PD-L1 , TIM3, LAG3, 0X40, CSF1 R, IDO, or CD40. Such immunooncology therapeutics include, e.g., an anti-CTLA-4 antibody (particularly an antagonistic or pathway-blocking anti-CTLA-4 antibody; e.g., ipilimumab or tremelimumab), an anti-PD-1 antibody (particularly an antagonistic or pathway-blocking anti-PD-1 antibody; e.g., nivolumab (BMS-936558), pembrolizumab (MK-3475), pidilizumab (CT-011), AMP-224, or APE02058), an anti-PD- L1 antibody (particularly a pathway-blocking anti-PD-L1 antibody; e.g., BMS-936559, MEDI4736, MPDL3280A (RG7446), MDX-1105, or MEDI6469), an anti-TIM3 antibody (particularly a pathway-blocking anti-TIM3 antibody), an anti-LAG3 antibody (particularly an antagonistic or pathway-blocking anti-LAG3 antibody; e.g., BMS-986016, IMP701 , or IMP731), an anti-CX40 antibody (particularly an agonistic anti-CX40 antibody; e.g., MEDI0562), an anti-CSF1 R antibody (particularly a pathway-blocking anti-CSF1 R antibody; e.g., IMC-CS4 or RG7155), an anti- IDO antibody (particularly a pathway-blocking anti-IDO antibody), or an anti-CD40 antibody (particularly an agonistic anti-CD40 antibody; e.g., CP-870,893 or Chi Lob 7/4). Further immunooncology therapeutics are known in the art and are described, e.g., in: Kyi C et al., FEBS Lett, 2014, 588(2):368-76; Intlekofer AM et al., J Leukoc Biol, 2013, 94(1):25-39; Callahan MK et al., J Leukoc Biol, 2013, 94(1):41-53; Ngiow SF et al., Cancer Res, 2011, 71 (21):6567-71 ; and Blattman JN et al., Science, 2004, 305(5681 ):200-5.
The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation. The individual components of such combinations may be administered either sequentially or simultaneously/concomitantly in separate or combined pharmaceutical formulations by any convenient route. When administration is sequential, either the compound of the present invention (i.e., the compound comprising the moiety of formula (I), the compound of formula (II) or a pharmaceutically acceptable salt thereof) or the further therapeutic agent(s) may be administered first. When administration is simultaneous, the combination may be administered either in the same pharmaceutical composition or in different pharmaceutical compositions. When combined in the same formulation, it will be appreciated that the two or more compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately, they may be provided in any convenient formulation.
The present invention further relates to a method of treatment of cancer, as described herein, in an individual in the need thereof, the method comprising administering the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) to said individual. The administration is preferably as described herein.
The present invention further relates to use of the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) for the manufacture of a medicament against cancer, as described herein.
The subject or patient to be treated in accordance with the present invention may be an animal (e.g., a non-human animal). Preferably, the subject/patient is a mammal. More preferably, the subject/patient is a human (e.g., a male human or a female human) or a non-human mammal (such as, e.g., a guinea pig, a hamster, a rat, a mouse, a rabbit, a dog, a cat, a horse, a monkey, an ape, a marmoset, a baboon, a gorilla, a chimpanzee, an orangutan, a gibbon, a sheep, cattle, or a pig). Most preferably, the subject/patient to be treated in accordance with the invention is a human.
The term "treatment” of a disorder or disease, as used herein, is well known in the art. "Treatment” of a disorder or disease implies that a disorder or disease is suspected or has been diagnosed in a patient/su bject. A pati ent/su bject suspected of suffering from a disorder or disease typically shows specific clinical and/or pathological symptoms which a skilled person can easily attribute to a specific pathological condition (i.e., diagnose a disorder or disease).
The "treatment” of a disorder or disease may, for example, lead to a halt in the progression of the disorder or disease (e.g., no deterioration of symptoms) or a delay in the progression of the disorder or disease (in case the halt in progression is of a transient nature only). The "treatment” of a disorder or disease may also lead to a partial response (e.g., amelioration of symptoms) or complete response (e.g., disappearance of symptoms) of the subject/patient suffering from the disorder or disease. Accordingly, the "treatment” of a disorder or disease may also refer to an amelioration of the disorder or disease, which may, e.g., lead to a halt in the progression of the disorder or disease or a delay in the progression of the disorder or disease. Such a partial or complete response may be followed by a relapse. It is to be understood that a subject/patient may experience a broad range of responses to a treatment (such as the exemplary responses as described herein above). The treatment of a disorder or disease may, inter alia, comprise curative treatment (preferably leading to a complete response and eventually to healing of the disorder or disease) and palliative treatment (including symptomatic relief).
The term "prevention” of a disorder or disease, as used herein, is also well known in the art. For example, a patient/subject suspected of being prone to suffer from a disorder or disease may particularly benefit from a prevention of the disorder or disease. The subject/patient may have a susceptibility or predisposition for a disorder or disease, including but not limited to hereditary predisposition. Such a predisposition can be determined by standard methods or assays, using, e.g., genetic markers or phenotypic indicators. It is to be understood that a disorder or disease to be prevented in accordance with the present invention has not been diagnosed or cannot be diagnosed in the patient/subject (for example, the patient/subject does not show any clinical or pathological symptoms). Thus, the term "prevention” comprises the use of a compound of the present invention before any clinical and/or pathological symptoms are diagnosed or determined or can be diagnosed or determined by the attending physician.
The term "treatment or prevention”, as used herein, preferably refers to "treatment”. Accordingly, the different grammatic form of the same term, i.e. "treating or preventing”, as used herein, preferably refers to "treating”.
The compounds of the present invention, i.e. the compound comprising the moiety of formula (I) or the compound of formula (II) further show antibacterial properties. Accordingly, in further embodiments the present invention relates to the compound comprising the moiety of formula (I) or the compound of formula (II) or a pharmaceutical composition comprising the compound comprising the moiety of formula (I) or the compound of formula (II) for use in the treatment or prevention of a bacterial infectious disease, as well as to use of the compound comprising the moiety of formula (I) or the compound of formula (II) as an antibacterial agent, preferably wherein the use is cosmetic and/or non-therapeutic.
The invention will now be described by reference to the following examples which are merely illustrative and are not to be construed as a limitation of the scope of the present invention.
EXAMPLES
Development of amphiphilic p-heterooligomers starting from cyclic and acyclic building blocks with different secondary-structure propensities if only hydrophobic cyclic and acyclic building blocks are used, a hydrophobic structure is inevitably formed. Conjugation of this hydrophobic p-heterooligomer with a hydrophilic element allows the formation of an amphiphilic water-soluble molecule. At the same time, the tendency towards self-association and interaction with other amphiphilic molecules/systems is increased. The hydrophilic element can be of different chemical nature and can carry polar or basic groups, preferably polar neutral groups and basic groups. Examples of amphiphilic, homochiral p-heterooligomers based on cyclic and acyclic building blocks are provided in Scheme 2.
Scheme 2
Figure imgf000033_0001
Synthesis
The p-peptides in Schemes 2 and 3 consist of commercially available cyclic and acyclic building blocks. They were assembled by solid-phase synthesis using Fmoc chemistry and standard protocols. The peptides in Scheme 2 were prepared in house. Peptide 1aR and its analogues, which are reported in Scheme 3 (1aR-iBu3, 1aR-Me3,(CF3)5, 1aR-Me3,(CF3)8, 1aR-Me3,5, 1aR-Me6,8, and 1aR-mix1-3), were respectively resynthesized and synthesized at GenicBio (China) and provided as trifluoroacetate or chloride peptide salts with a purity of at least 95%. The materials and methods for the in-house synthesis are described hereafter. Fmoc-L-p3-Val-OH, Fmoc-L-p3-Leu-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-Rink-amide-MBHA resin, N-methyl-2-pyrrolidone (NMP), dichloromethane (DCM), Et20, N, N-diisopropylethylamine (DIPEA) and piperidine were purchased from Iris Biotech GmbH (Germany). Fmoc-p-Ala-OH and N,N'-diisopropylcarbodiimide (DIG) were purchased from Novabiochem- Merck Millipore (Germany). Fmoc-frans-(1s,2s)-ACPC-OH was purchased from Chem-lmpex International (USA). 2-(1 H-Benzotriazol-1-yl)-1,1 ,3,3-tetramethyluronium-hexafluorphosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) were purchased from Biosolve (The Netherlands). 1 ,2-Ethanedithiol (EDT), thioanisole (TIA), and 5,6- carboxyfluorescein (FAM) were purchased from Fluka (Germany). Trifluoroacetic acid (TFA) for HPLC was purchased from Alfa Aesar (Thermo Fisher Scientific, Germany). Triisopropylsilane (TIS), acetic anhydride, acetonitrile for HPLC (ACN), MeOH, and N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich (Merck, Germany). D2O and deuterated solvents were purchased from Armar GmbH (Germany).
The solid-phase peptide syntheses of 1b, 1_ALA, and 1a were carried out manually. The Fmoc-Rink-amide-MBHA resin was swollen in 500 pL DMF/NMP (80:20 v/v) for 30 min, then the solvent was sucked off. The Fmoc-protecting group was cleaved by adding 500 pL 40% piperidine in DMF/NMP (80:20 v/v), shaking for 3 min, sucking the solution off, then adding 500 pL 20% piperidine in DMF/NMP (80:20 v/v), shaking for 12 min, sucking the solution off. The resin was washed with DMF five times. For the double coupling, each Fmoc-amino acid (3 equiv. for the p-amino acid, 5 equiv. for the a-amino acid) was dissolved in DMF/NMP (80:20 v/v) containing 3 equiv. or 5 equiv. HOBt, added to the resin, followed by 2.8 equiv. or 4.8 equiv. HBTU, and 6 equiv. or 10 equiv. DIPEA, then the suspension was shaken for 1 h. The reaction mixture was sucked off, and the resin was washed with DMF. The coupling procedure was repeated. The acetylation reaction was performed at the end of the chain elongation using 10 equiv. DIPEA and 10 equiv. acetic anhydride in 600 pL DMF/NMP (80:20 v/v). For FAM labeling, the N-terminus of the resin-bound peptide was acylated with 5 equiv. FAM in the presence of HOBt/DIC (5 equiv. each) for 45 min. The acylation procedure was repeated, followed by treatments with 20% piperidine in DMF (2x30 min). Finally, the resin was washed with DMF, DCM, Et20 (five times each). For the cleavage, 800 pL of the mixture TIS/EDT/TIA/H2O/TFA (3:3:3: 1 :90 v/v) were added to the peptide-resin and the suspension was shaken for 3.5 h. The resin was removed by filtration, then ice-cold Et20 was added to the filtrate to precipitate the peptide. After 10 min at -20 °C, the suspension was centrifuged for 5 min at 8 °C, then the ether was removed by decantation. The washing with ice-cold ether and the centrifugation steps were repeated three times. The precipitate was dried under a nitrogen flow. The peptide homogeneity (85-90%) and identity were confirmed by reversed phase HPLC (Thermo Fisher Scientific Dionex, model UltiMate 3000, with a Syncronis C18 column, 100 A, 5 pm, 250x4.6 mm) and MALDI-TOF-MS (Bruker Daltonics, model Autoflex Speed) using o-cyano-4-hydroxycinnamic acid as matrix (Table 1).
Table 1. Analytical characterization of the in-house synthesized peptides.
Figure imgf000034_0001
NMR spectroscopy
The NMR spectra were recorded on a Broker (Germany) AVANCE III HD 600 MHz spectrometer equipped with a QXI (1H/13C/15N/31P) probe. The NMR peptide samples (1.1-1 .8 mM in water with 7% D2O, 500 JJ.L) were measured in standard 5-mm TA tubes (Armar, Germany) at 298 K. The spectra were 1H calibrated using the Broker standard sample of 2 mM sucrose-0.5 mM DSS (4, 4-di methyl-4-silapentane- 1 -sulfonic acid). 13C frequencies were calibrated indirectly using the recommended scaling factor of 0.25144953. The following 2D NMR experiments were performed: 1H-1H TOCSY (4 scans, 1024x256 complex points, 12 ms and 120 ms mixing time, 8.33 ppm F1 spectral widths, 1 s recycle delay); 1H-1H ROESY (80 scans, 1024x350 complex points, 200 ms mixing time, 8.33 ppm F1 spectral widths, 1.2 s recycle delay); 1H-13C HSQC (128 scans, 512x128 complex points, 77 ppm F1 spectral widths, 1.5 s recycle delay); 1H-13C HSQCaro (128 scans, 512x50 complex points, 44.2 ppm F1 spectral widths, 1.5 s recycle delay); 1H-13C HMBC (80 scans, 1024x128 complex points, 222.4 ppm F1 spectral widths, 2 s recycle delay); 1H-13C HSQC-TOCSY (80 scans, 1024x128 complex points, 55.2 ppm F1 spectral widths, 2 s recycle delay). Bruker TopSpin version 3.6.1 was used to process the NMR data. TopSpin and Sparky (T. D. Goddard and D. G. Kneller, SPARKY 3; University of California, San Francisco) were used to analyze and assign NMR data (Tables 2-11).
Table 2. 13C assignments of 1b in water.
Figure imgf000035_0001
Table 3. 1H NMR assignments of 1b in water.
Figure imgf000035_0002
Table 4. 13C NMR assignments of 1_ALA in water.
Figure imgf000036_0001
Table 5. 1H NMR assignments of 1_ALA in water.
Figure imgf000036_0002
Table 6. 13C NMR assignments of 1a in water.
Figure imgf000036_0003
Table 7. 1H NMR assignments of 1a in water.
Figure imgf000037_0004
Table 8. Sequential and short-range NOE cross-peaks of 1b in water.
Figure imgf000037_0001
Table 9. Sequential NOE cross-peaks of 1_ALA in water.
Figure imgf000037_0002
Table 10. Sequential NOE cross-peaks of 1a in water.
Figure imgf000037_0003
Table 11. 3JHNHP Couplings at 25 °C, temperature coefficients and amide proton chemical shifts of 1_ALA (1.1 mM in water). *Estimates suffering from overlap
Figure imgf000038_0001
CD spectroscopy
The circular dichroism (CD) measurements were recorded on a Chirascan-plus CD spectrometer (Applied Photophysics, United Kingdom) at 23 °C using a 1 mm quartz cell (Hellma Analytics, Germany). The peptides were dissolved in water, TFE (2,2,2-trifluoroethanol, Iris Biotech GmbH), MeOH, ACN, and 10 mM phosphate buffer (pH 7.3) without or with POPC (1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, Sigma-Aldrich) vesicles. The latter were prepared as follows: dried POPC was suspended in phosphate buffer, incubated for 1 h at room temperature, then vortexed vigorously to obtain a milky, uniform POPC suspension. Successively, the suspension was sonicated until the suspension changed from milky to nearly clear in appearance (max. 30 min sonication time). The peptide concentrations were determined spectrophotometrically by using a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Germany) and the UV absorbance of tyrosine at about 280 nm with the molar extinction coefficient 1480 M’1 cm 1 (H. Mach, C. R. Middaugh, R. V. Lewis, Anal Biochem 1992, 200, 74-80). The peptide concentrations were as follows: 1b: 0.19 mM in water, 0.12 mM in MeOH; 1_ALA: 0.23 mM in water, 0.14 mM in MeOH; 1a: 0.39 mM in water, 0.34 mM in MeOH. For each CD spectrum, three scans were accumulated using a step resolution of 1 nm, a bandwidth of 1 nm, and a time-per-point of 1 s. The CD spectrum of the solvent was subtracted, and the difference spectrum was normalized to express the ellipticity in mean-residue molar ellipticity, divided by 103 and represented in the graphs as [0]R X 103 (deg cm2 dmol 1).
Secondary structure
The secondary structure of the p-peptides in Scheme 2 was investigated in water and in MeOH using CD spectroscopy (Figure 1). The two p-peptides with the register cyclic-acyclic show a maximum at 223 nm, a minimum at 204 nm and a zero crossing at 215 nm. The ratio min./max. is 4.6 for the shorter p-peptide 1b and 7.6 for the longer p-peptide 1_ALA. This CD signature is the mirror image of the CD signature for the left-handed 12(=2.5i2)- helix and suggests that the cyclic building block takes precedence over the acyclic building block and that the preferred secondary structure for 1b and 1_ALA is predominantly the right-handed 12(=2.5i2)-helix. The p-peptide with the register acyclic-cyclic-acyclic (1a) shows a maximum at 222 nm, a minimum at 204 nm and a zero crossing at 214 nm. The ratio min. /max. is 3.5. Compared to the CD spectra of 1b and 1_ALA, the maximum of 1a, and thus also the zero crossing, is shifted to shorter wavelengths. Since a shift to shorter wavelengths was observed for shorter and therefore less stable 12(=2.5i2)-helices, we hypothesize that 1a adopts a less well-defined or distorted 12(=2.5i2)-helix. However, other secondary structures cannot be ruled out, as it has been reported that p-peptides composed of mixed p2-amino acids and p3-amino acids can form a 10/12-helix with the CD signature showing a broad maximum at 205 nm (D. Seebach, S. Abele, K. Gademann, G. Guichard, T. Hintermann, B. Jaun, J. L. Matthews and J. V. Schreiber, Helv Chim Acta, 1998, 81, 932-982; D. Seebach, J. V. Schreiber, S. Abele, X. Daura and W. F. van Gunsteren, Helv Chim Acta, 2000, 83, 34-57). Furthermore, it has been reported that short oligomers of trans-ACBC tend to adopt an 8-helix (E. Gorrea, G. Pohl, P. Nolls, S. Celis, K. K. Burusco, V. Branchadell, A. Perczel and R. M. Ortuno, J Org Chem, 2012, 77, 9795-9806; E. Torres, E. Gorrea, E. Da Silva, P. Nolls, V. Branchadell and R. M. Ortuno, Org Lett, 2009, 1 , 2301-2304). In water, the CD spectra of the p-peptides show curves similar to those in MeOH, but with low intensity. Again, the CD spectrum of 1a differs from the CD spectra of 1b and 1_ALA (shift to the left, as already observed in MeOH). It is believed that the p-peptides are less structured in water than in MeOH. This suggests that these peptides adopt a more defined structure in a less polar environment (e.g. in the cell membrane).
Accordingly, the p-peptides with register cyclic-acyclic (examples 1b and 1. _ALA) are prone to adopt a right-handed 12(=2.5i2)-helix. This is particularly preferred in a less polar environment. The p-peptides with acyclic-cyclic-acyclic register (examples 1a and 1aR) tend to adopt a right-handed 12(=2.5i2)-helix, which is less well-defined, likely distorted and/or in equilibrium with other secondary structures (due to the less negative CD signal below 218 nm, it is suggested that a right-handed 10/12-helix might be in equilibrium with the right-handed 12-helix). The formation of these secondary structures is particularly favored in a less polar environment.
Cellular studies
The following materials were used in the cellular studies described herein: Ethanol (VWR, Austria), Hoechst 33342 (Fluka), Annexin V-APC (Immunotools), Lumit™ uman IL-1 p Immunoassay (Promega), CellTiter-Glo® 3D (Promega), LDH-Cytox™ (Lactate dehydrogenase) Assay Kit (Biolegend), ROS, 20% triton-X, Doxorubicin (Selleckchem), formaldehyde (Merck), glutaraldehyde (Fisher Scientific), citric acid (Merck), hexacyano-ferrate (Merck), magnesium chloride (Merck), sodium chloride (Merck), X-gal (Invitrogen), FLICA 660 Caspase-1 Assay it® (Biorad), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), RNase, Accutase® solution, propidium iodide, fetal bovine serum, penicillin— streptomycin and L-glutamine were purchased from Sigma-Aldrich (Austria).
Dulbecco's modified Eagle's medium (DMEM)-high glucose and Roswell Park Memorial Institute (RPMI) 1640 medium and 1X Dulbecco's Phosphate Buffered Saline (DPBS) were purchased from Szabo-Scandic (Austria). DMSO (dimethyl sulfoxide) was from VWR (Austria). The primary human lung fibroblasts were a gift from Prof. Dr. Jutta Horejs-Hock, University of Salzburg (Austria). The cell line MCF-7 was a gift from Prof. Dr. Barbara Krammer, University of Salzburg (Austria). The cell lines HT1975, A427 and SKLU-1 were a gift from Prof. Dr. Emilio Casanova, Medical University of Vienna (Austria). The cell lines A549 (ATCC: CLL-185), H460 (ATCC: HTB-177), H520 (ATCC: HTB-182) and HCC827 (ATCC: CRL-2868) were purchased from ATCC. The cisplatin resistant A549 (ddA549) cell line was purchased from Szabo Scandic. MitoView™720 and MemBrite™ Fix 660/680 were purchased from Biotium, Dihydrorhodamine 123 (DHR 123) was from Sigma.
Cell culture
Human primary lung fibroblasts and MCF-7 cell line were cultured in DMEM-high glucose supplemented with 10% fetal bovine serum, 1 % penicillin— streptomycin and 1 % L-glutamine. All other cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin— streptomycin and 1 % L-glutamine. Cells were grown in a humidified atmosphere at 37 °C in 5% CO2. For all experiments, 80% confluent cells were used.
Viability assay
Cells (1x104 cells/well) in complete growth medium were seeded into 96-well culture plates. The day after, the medium was changed to a serum free medium with peptides at various concentrations. After an additional incubation period of 24 h, the cell viability was determined by adding 10 pL MTT solution (5 mg/mL in DPBS) to the treated and non-treated control cells for 2 h at 37 °C in the dark. Then the medium was aspirated, cells were lysed with 100 pl DMSO and the absorbance of the resulting product formazan in viable cells was measured at 550 nm with a GloMax® multimode microplate reader. At least three independent experiments (with each sample in triplicate) were performed and cell viability was normalized to the untreated control.
Cellular uptake and colocalization of peptide 1aR
Cells (3x104/chamber) were grown overnight in 8 well chamber pi-Slides (I bidi). The day after, the medium was changed to a serum and phenol red free medium (control) or 5 piM FAM-1aR. The cell membrane was stained with MemBrite™ Fix 660/680 (Biotium), whereas the nucleus was stained with 1 pig/mL Hoechst 33342 (Fluka). For colocalization with the mitochondria, 100 nM MitoView™ 720 (Biotinum) for 15 min at 37 °C were added. The colocalization images were performed with an Olympus IX73 inverted microscope, whereas live cell imaging was performed with an LSM 700 laser scanning confocal microscope from Carl Zeiss. The FITC channel was used for FAM-1 aR (excitation = 494 nm, emission = 518 nm), the CY7 channel for the MitoView™720 (excitation = 550 nm, emission = 565 nm), MemBrite™ Fix 660/680 (excitation = 663 nm, emission = 682 nm), whereas the DAPI channel was used for Hoechst 33342 (excitation = 345 nm, emission = 455 nm).
Lactate dehydrogenase assay
Cells (1x104 cells/well) in complete growth medium were seeded into 96-well culture plates. The day after, the medium was changed to a serum free medium with 20 piM 1aR. After an additional incubation period of 1 h, 2 h, 4 h and 24 h, the plate was centrifuged for 5 min with 300 rpm to avoid any cellular debris in the supernatant. Afterwards, 50 piL of the supernatant was transferred to a new 96 well plate, and 50 piL LDH working solution were added. The plate was incubated at room temperature for 30 min in the dark. To stop the reaction, 25 piL stop solution were added, and the absorbance of the LDH release from the cells was measured at 490 nm with a GloMax® multimode microplate reader. At least three independent experiments (with each sample in triplicate) were performed. The cytotoxicity was calculated by the following equation: Cytotoxicity(%)=((peptide-low control)/(high control-low control))*100 Propidium iodide (PI) staining for observation of peptide-induced cell membrane damage
Cells (3x104/chamber) were grown overnight in 8-well chamber pi-Slides (Ibidi). The day after, the medium was changed to a serum free medium (control) or 10 piM 1aR. The cells were incubated for 1 h and 24 h and subseguently stained with propidium iodide (PI) and Hoechst 33342 for 10 min. After two washing steps with DPBS, the fluorescence images were taken with an Olympus IX73 inverted microscope, using the CY3 channel for the PI staining (excitation = 550 nm, emission = 565 nm), and the DAPI channel for the cell nuclei marker Hoechst 33342 (excitation = 345 nm, emission = 455 nm).
Annexin V/PI assay
Cells (1x105 cells/well) in complete growth medium were seeded into 12-well culture plates. The day after, the medium was changed to a serum free medium with peptides at various concentrations. After an additional incubation period of 1 h and 24 h, the supernatants were harvested and transferred into 2 mL Eppendorf tubes. Cells were detached with 500 pL Accutase® solution and added to the respective supernatant, the suspension was then centrifuged for 5 min at 1500 rpm. After removal of the supernatant, the cell pellet was washed twice with 1 mL DPBS and resuspended in 98.5 piL of 1x annexin V buffer with 1 pL annexin V-APC and 0.5 pL of 1 mg/mL PI. After 15 min incubation at room temperature in the dark, 200 pL of 1x annexin V buffer were added and subseguently measured with the CytoFLEX flow cytometer. Analyses from at least three independent experiments were performed with the Kaluza 1 ,5a flow cytometer analysis software (Beckman Coulter).
Cell-cycle analysis
Cells (5x105 cells/well) in complete growth medium were seeded into 35 mm cell culture dishes. The day after, the medium was changed to a serum free medium with peptides at various concentrations. After an additional incubation period of 24 h, the supernatants were harvested and transferred into 2 mL Eppendorf tubes. Cells were detached with 500 pL Accutase® solution and added to the respective supernatant, the suspension was then centrifuged for 5 min at 1500 rpm. After removal of the supernatant, the cell pellet was resuspended in 200 pL DPBS. For fixation, 2 mL of ice-cold 70% ethanol was added dropwise to the suspension under gentle agitation. After freezing for at least 1 h at -20 °C, the cell suspension was centrifuged for 5 min at 1500 rpm and washed twice with 2 mL DPBS. The cells were then stained with a solution of 25 pL 0.4 mg/mL PI, 25 pL of 1 mg/mL RNase solution and 450 pL DPBS. After incubation for 15 min at 37 °C in the dark, the fluorescence of PI bound to DNA was measured with the CytoFLEX flow cytometer. Cell-cycle analyses from at least three independent experiments were performed with the Kaluza 1 ,5a flow cytometer analysis software (Beckman Coulter).
Dihydrorhodamine 123 (DHR 123) assay
Cells (1x104 cells/well) in complete growth medium were seeded into 96-well culture plates. The day after, the medium was changed to a serum free medium, and cells were stained with 10 piM DHR 123 for 30 min. After treatment with 10 piM 1aR, ROS induction was measured with a GloMax® multimode microplate reader.
Caspase-1 assay
Cells (1x105 cells/well) in complete growth medium were seeded into 12-well culture plates. The day after, the medium was changed to a serum free medium with peptides at various concentrations. After an additional incubation period of 1 h, 4 h and 24 h, the supernatants were harvested and transferred into 2 mL Eppendorf tubes. Cells were detached with 500 pL Accutase® solution and added to the respective supernatant, the suspension was then centrifuged for 5 min at 1500 rpm. After removal of the supernatant, the cell pellet was stained with 1.6 piL FLICA working solution in 98.4 piL medium. After 20 min incubation at 37 °C in the dark, 0.5 piL of 1 mg/mL PI were added, followed by incubation for additional 10 min. Cells were washed twice with 2 mL FLICA wash solution, subsequently resuspended in 300 piL FLICA wash solution and measured with the CytoFLEX flow cytometer. Analyses from at least three independent experiments were performed with the Kaluza 1 ,5a flow cytometer analysis software (Beckman Coulter).
IL1-8 assay
Cells (1x104 cells/well) in complete growth medium were seeded into 96-well culture plates. The day after, the medium was changed to a serum free medium with 10 piM 1aR for 24 h. The release of IL1-p was measured according to the manufacturer's protocol. Analyses from at least three independent experiments were performed.
Scanning electron microscopy
The cells were fixed with 2.5% glutaraldehyde in 0.1 M PBS for 30 min. Afterwards, the cells were washed 3x for 10 min in 0.1 M PBS. Samples were dehydrated in a graded ethanol series. Once in 100% ethanol, the cells were dried with a critical point dryer (EM-CPD300, Leica Microsystems) and subsequently coated with 5 nm platinum using a sputter coater (EM-ACE600, Leica Microsystems). The cover slips with the dried cells were finally imaged with a scanning electron microscope (FE-SEM Merlin compact VP, Carl Zeiss) at 5 kV using a secondary electron detector.
Spheroid generation
1x104 cells were seeded in a BIOFLOAT FLEX (faCellitate) coated, non-TC treated U-bottom 96-well plate. Subsequently, the plate was centrifuged for 5 minutes at 1500 rpm, to allow the cells to gather in the middle of the well to facilitate spheroid formation. After an incubation period of 4 days, the spheroids were treated with increasing concentrations of the compounds in medium without FCS. To measure cell viability with the Cel ITiter-G/o® 3D Cell Viability Assay (Promega), the spheroids were transferred to a white opaque 96-well plate after 48 h and as described in manufacturer's protocol.
Senescence induction
The human lung adenocarcinoma cells A549 were seeded in flasks for senescence induction at a concentration of 1x105/mL in fully supplemented RPM1 1640 medium containing 0.1 pig/mL doxorubicin. Medium change was done every 48 h with RPM1 1640 with 0.1 pig/mL doxorubicin until 60% confluence was reached.
Senescence-associated 8-galactosidase activity assay
A549 cells were seeded in 6-well plates at a concentration of 1.25x105/mL and incubated for 18 h at 37 °C. Subsequently, cells were washed with PBS and fixed (2% formaldehyde and 0.25% glutaraldehyde) for 2 min at room temperature. After a second wash step with PBS, cells were incubated with a freshly prepared staining solution (40 mM citric acid, 5 mM hexacyano-ferrate, 2 mM magnesium chloride, 150 mM sodium chloride and 1 mg/mL X-gal (5-bromo-4-chloro-3-indolyl-p-galactopyranoside), pH 6.0) in the dark at 37 °C for 18 h. Afterwards, the staining solution was aspirated, cells were washed with PBS and incubated in methanol for 1 min, prior observing them under an inverse bright field microscope.
Cellular toxicity
The p-peptides in Scheme 2 were tested on cancer cell lines. p-Peptides with the register acyclic-cyclic-acyclic (1a and 1aR) exhibit various biological effects, with 1aR being considerably more effective than 1a. In contrast, the p- peptides with the register cyclic-acyclic (1b and 1_ALA) have only a weak cytotoxic effect (Table 12).
Since these two groups of p-peptides contain the same building blocks but different registers, it is reasonable to assume that the biological effect of compounds 1a and 1aR is ascribed to the unique secondary structure properties of the hydrophobic p-peptide part. Accordingly, the two groups of p-peptides have different CD signatures. Furthermore, the amphiphilic character of the p-peptides 1a and 1aR alone cannot explain their effect because the non-effective p-peptides 1b and 1_ALA are also amphiphilic.
Table 12. Cellular toxicity of the p-peptides, as determined by the MTT viability assay after 24 h incubation at different concentrations of the p-peptides (n.r., not reached).
Figure imgf000043_0001
Further cellular studies
Highly effective and specific anticancer properties of the peptide 1aR of the present invention have been demonstrated as follows.
The effect of the peptide on the viability of various human cancer cell lines was analyzed with the MTT assay and annexin V/PI staining. The IC50 values for various human lung cancer cell lines after 24 h of peptide treatment were 3.5- to 12-fold lower than those for the healthy lung fibroblast cell line MRC-5 and the healthy human bronchial epithelial cell line BEAS-2B. The corresponding IC50 values are summarized in Table 13.
Table 13. Effect of 1aR on the viability of human cell lines after 24 h treatment with 1aR. I C50 values were calculated from at least three independent experiments using GraphPad Prism 6.
Figure imgf000043_0002
Figure imgf000044_0001
To further determine the effect on cell death, the annexin V/ propidium iodide (PI staining was performed. Upon treatment with 20 piM 1aR, 94.02% (lung adenocarcinoma, A549), 91.43% (lung adenocarcinoma with EGFR mutation AE746-A750, HCC827) and 92.12% (squamous cell carcinoma (SCO) of the lung, H520) underwent cell death (% cells not double negative for annexin V and PI). By contrast, untreated control cultures only contained 4.56 - 26.09% dead cells. In addition, the healthy lung fibroblast cell line MRC-5 was largely insensitive to the treatment, showing cell-death induction of only 8.15% compared to untreated cells (Figure 2A). Interestingly, 63.46% dead cells occurred already 1 h after treatment with the majority of dead cells double positive for annexin V and PI (Figure 2B). This indicates that the mode of induced cell death is not apoptosis but rather necrosis or pyroptosis.
The peptide 1aR possesses good cell permeability, as shown by the cellular uptake of a fluorescence-labeled analog bearing 5,6-carboxyfluorescein in place of the N-terminal acetyl group (FAM-1aR) that was found well distributed along the mitochondrial network. As shown in Figure 3, the green fluorescence of FAM-1aR overlapped with the red fluorescence of the mitochondrial probe MitoView™ 720 in A549 cells. In addition, peptide uptake is energy independent, as peptide accumulation within the cells appeared upon incubation at 37 °C as well as at 4 °C (Figure 4). The specificity of 1aR for cancer cells does not seem to be due to inferior uptake of the peptide (Figure 5). Furthermore, 1aR induces significant morphological changes on cancer cell lines. Cells became swollen and displayed pronounced large bubbles budding from the plasma membrane, which is characteristic of pyroptosis (Figures 6, 14 and 15). In contrast, the healthy lung fibroblast cell line MRC-5 did not undergo morphological changes (Figure 6).
Peptide 1aR can also induce pyroptosis. Pyroptosis refers to the process of gasdermin (GSDM)-mediated programmed cell death. The GSDM-family members (GSDMA-E) can be cleaved by inflammatory caspases (caspase-1, -4, -5 and -11) as well as by apoptosis-related caspases (caspase-3, -6, -7 and -8). After cleavage, the N-terminal domain (GSDM-N) executes pyroptosis by the formation of pores within the cell membrane, resulting in characteristic morphological changes and the release of proinflammatory mediators like interleukin-18 (IL-18) and interleukin- 1 p (IL-113) into the extracellular environment, thus amplifying the systemic immune response. To confirm membrane leakage, lactate dehydrogenase (LDH) assay was performed. LDH is abundant in the cytoplasm and can only be released and detected if the cell membrane is disrupted. Indeed, LDH was increased up to ~120-fold over the untreated control in A549 cultures after 1aR treatment, whereas no increase was detectable in the noncancer cell line MRC-5 neither after 1 h nor after 24 h treatment (Figure 7). These observations were compatible with PI staining (as shown in Figure 8) after 30 min of treatment (10 piM 1aR), indicating rapid cell membrane damage, as PI is only able to enter cells when cell membranes become permeable. In MRC-5 cells no red fluorescence was detectable neither after 30 min nor after 24 h treatment, suggesting the membrane of these cells was still intact. A further characteristic of pyroptosis is that the nucleus remains intact, which was still the case after 24 h treatment (Figures 3, 6, 8, 11, 14 and 15). To further demonstrate disruption of the cellular membrane, scanning electron microscopy was performed, showing pronounced membrane perforation in A549 cells after 10 piM 1aR treatment for 1 h, whereas MRC-5 cells showed no alteration neither after 1 h nor after 24 h (Figure 9).
Pyroptosis induction was further confirmed by the human IL-1 p as well as by the caspase 1 assay, showing a significant induction of active caspase 1 and IL-1 p in A549 treated cells compared to control and healthy MRC-5 cells, respectively (Figure 10).
Cleaved GSDMD and GSDME are able to form pores within the mitochondrial membrane leading to their permeabilization, dissipation of mitochondrial membrane potential, fragmentation of the mitochondrial network and as a result to the production of mitochondrial reactive oxygen species (ROS) (doi: 10.1073/pnas.1414859111 , https://doi.org/10.1038/s41467-019-09397-2, doi: 10.1080/23723556.2019.1621501). Therefore, the effect of 1aR on the mitochondria was analyzed. Indeed, treatment of A549 cells led to mitochondrial membrane depolarization (Figure 11) and a ~18-fold increase in ROS production (Figure 12).
Therapy induced senescence (TIS) has been associated with reduced tumor growth and is contribute to chemoresistance of cancer cells. However, TIS has been associated with reduced tumor growth, TIS and spontaneously senescent cancer cells are able to reactivate their proliferative potential leading to cancer recurrence accompanied by increased aggressiveness. Thus, improved treatments are needed that either avoid induction of TIS or enable targeting of senescent or otherwise chemoresistant cancer cells. Recently, studies have shown that the induction of pyroptosis remains effective on cisplatin resistant A549 (ddA549) cancer cells with an I C50 value of 7.91 ± 0.9ln line with this, preliminary experiments, showed up to -80% cell death of senescent A549 cancer cells after treatment with 1aR (Figure 13).
The results of further relevant cellular experiments are shown in Figures 14 - 16.
Further peptide analogues of 1aR
The chemical diversity of the R1 group of the building block "A” (as in formulae (I) and (II)) was explored by the synthesis of the 1aR analogues reported in Scheme 3. In particular, the following R1 groups were chosen to replace the isopropyl and isobutyl groups of 1aR:
• Methyl (alkyl group, much less hydrophobic and much less sterically demanding than isopropyl and isobutyl).
• Allyl (unbranched alkenyl group, less hydrophobic and less sterically demanding than isobutyl).
• Propargyl (unbranched alkynyl group, less hydrophobic and less sterically demanding than isobutyl). • sec-Butyl (branched alkyl group, structural isomer of isobutyl).
• n-Butyl (unbranched alkyl group, structural isomer of isobutyl, more hydrophobic than isopropyl and isobutyl).
• 2-Phenylethyl (aromatic group, more hydrophobic and more sterically demanding than isopropyl and isobutyl).
• Benzyloxymethyl (aromatic group, more hydrophobic and more sterically demanding than isopropyl and isobutyl).
• 4-(Trifluoromethyl)-phenylmethyl (aromatic and fluorinated group, more hydrophobic and more sterically demanding than isopropyl and isobutyl).
As mentioned above in the "Synthesis” section, the 1aR peptide analogues in Scheme 3 have been prepared at GenicBio (China) by Fmoc-based solid-phase chemistry with standard protocols.
Scheme 3. Chemical structures of the 1aR peptide analogues (the synthesis, purification and analytical characterization by HPLC and MS were performed at GenicBio, China).
Figure imgf000047_0001
Chemical Formula: C75H117N21O14
Exact Mass: 1535.91
MH+ found: 1536.86
Purity (HPLC): 97.65%
Figure imgf000048_0001
MH+ found: 1434.77
Purity (HPLC): 95.87% The R1 group combinations in the synthesized peptides, including the lead peptide 1aR, are as follows ("pos.” = position/s) (see Table 14 for the chemical structure of each one of the selected R1 group):
• R1 in 1aR: isopropyl (pos. 3), isobutyl (pos. 5, 6, 8). • R1 in 1aR-Me3,(CF3)8: methyl (pos. 3), isobutyl (pos. 5, 6), 4-(trifluoromethyl)-phenylmethyl (pos. 8).
• R1 in 1aR-iBu3: isobutyl (pos. 3, 5, 6, 8).
• R1 in 1aR-mix1: allyl (pos. 3); butyl (pos. 5), benzyloxymethyl (pos. 6), methyl (pos. 8).
• R1 in 1aR-mix2: propargyl (pos. 3); butyl (pos. 5); benzyloxymethyl (pos. 6); methyl (pos. 8).
• R1 in 1aR-Me3,(CF3)5: methyl (pos. 3); 4-(trifluoromethyl)-phenylmethyl (pos. 5), isobutyl (pos. 6, 8). • R1 in 1aR-mix3: methyl (pos. 3), 2-phenylethyl (pos. 5), butyl (pos. 6), sec-butyl (pos. 8).
• R1 in 1aR-Me3,5: methyl (pos. 3, 5), isobutyl (pos. 6, 8).
• R1 in 1aR-Me6,8: isopropyl (pos. 3), isobutyl (pos. 5), methyl (pos. 6, 8).
Table 14. Chemical structure of the R1 groups in 1aR and its peptide analogues ("pos.” = position/s; “m=1” refers
Figure imgf000049_0001
Figure imgf000050_0001
The peptides embodying the combinations of the selected R1 groups have been characterized with respect to the following properties: • IC50 values at the A549 cancer cells.
• I C50 values at the MRC-5 cells.
These data have been evaluated based on their ratio with the IC50 values of the lead peptide 1aR (Table 15). The IC50 values of the new peptides at the A549 cancer cells can be classified as follows:
• comparable to 1aR (up to 1 :2)
• slightly higher than 1aR (up to 1 :4)
• moderately higher than 1aR (up to 1 :12) • considerably higher than 1aR (up to 1 :28) Table 15. Comparison of the IC50 values of 1aR and its analogues for the A549 cancer cells and the MRC-5 cells
(see also Table 16 for the IC50 values).
Figure imgf000051_0001
Importantly, the IC50 values at the A549 cancer cells of all peptides (including the lead peptide 1aR) fall in a narrow range (4-111 mM), which indicates a good up to excellent tolerance of the R1 group for chemical moieties with different degree of hydrophobicity and steric demand. However, the following considerations can be made:
Overall hydrophobicity and steric demand of the peptide chain: a balance of the hydrophobicity and steric demand among the R1 groups seems to be correlated with better IC50 values, as suggested by the following observations:
• Drastic reduction of the overall hydrophobicity and steric demand of the lead peptide 1aR correlates with the IC50 values classified as considerably higher than that of 1aR (up to 1 :28), as shown by the peptides 1aR-Me3,5 and 1aR-Me6,8.
• Moderate increase in the overall hydrophobicity and steric demand of the lead peptide 1aR correlates with the IC50 values classified as comparable to that of 1aR (up to 1 :2), as shown by the peptide 1aR-iBu3 that contains the moderately more hydrophobic and more sterically demanding isobutyl group than the isopropyl group in position 3.
• In contrast, the balance of reduction/increase in both hydrophobicity and steric demand at the more external positions 3 and 8 of the lead peptide 1aR correlates with the IC50 values classified as comparable to that of 1aR (up to 1 :2), as shown by the peptide 1aR-Me3,(CF3)8.
• However, the balance of reduction/increase in both hydrophobicity and steric demand is not fully independent from the position along the peptide chain, as shown by the peptide 1aR-Me3,(CF3)5, whose IC50 value is classified as moderately higher than that of 1aR (up to 1 :12). Moderate sensitivity of position 5: Significant increase in both hydrophobicity (and/or aromaticity) and steric demand at position 5 of the lead peptide 1aR correlates with the IC50 values classified as moderately higher than that of 1aR (up to 1 : 12), as shown by the peptides 1aR-Me3,(CF3)5 and 1aR-mix3.
• Slight sensitivity of position 6: Significant increase in both hydrophobicity (and/or aromaticity) and steric demand at position 6 of the lead peptide 1aR correlates with the IC50 values classified as slightly higher than that of 1aR (up to 1 :4), as shown by the peptides 1aR-mix1 and 1aR-mix2.
The IC50 values of the 1aR peptide analogues for the A549 and MRC-5 cell lines after peptide treatment at concentrations up to 200 piM for 24 h are shown in the following Table 16.
Table 16. IC50 values of the 1aR peptide analogues for the A549 and MRC-5 cell lines after treatment at concentrations up to 200 piM for 24 h
Figure imgf000052_0002
Further peptide analogues of 1aR with Z subunit characterized by m = 0
A variant of the lead peptide 1aR containing the cyclobutane-based building block “Z” is suggested as follows:
Figure imgf000052_0001
1aR-Cbu displays the following substitutions with respect to the lead peptide 1aR: • The R1 group iso-propyl in pos. 3 of 1aR is replaced by the moderately more hydrophobic and moderately more sterically demanding R1 group iso-butyl.
• The cyclopentane-based building block "Z” (m = 1) in pos. 4 and 7 of 1aR is replaced by the moderately less hydrophobic and moderately less sterically demanding cyclobutane-based building block “Z” (m = 0).
As mentioned herein, homooligomers of the cyclic p-amino acids trans-2-aminocyclopentanecarboxylic acid ([trans- ACPC]n=68) (D. H. Appella, L. A. Christianson, D. A. Klein, D. R. Powell, X. L. Huang, J. J. Barchi and S. H. Gellman, Nature, 1997, 387, 381-384) and trans-2-aminocyclobutanecarboxylic acid ([trans-ACBC]n=68) (C. Fernandes, S. Faure, E. Pereira, V. Thery, V. Declerck, R. Guillot and D. J. Aitken, Org Lett, 2010, 12, 3606-3609) are known to form so-called 12(=2.5i2)-helices in solution as well as in crystals (R. P. Cheng, S. H. Gellman and W. F. DeGrado, Chem Rev, 2001 , 101, 3219-3232) (Scheme 1).
The substitution of the cyclic subunit “Z” in the formulae (I) and (II), which is based on the cyclopentane ring (m = 1) in the lead peptide 1aR as well as in its peptide analogues shown in Scheme 3, with the cyclic homolog based on the cyclobutane ring (m = 0) is considered to be tolerated.
The cyclobutane-based building block “Z” (m = 0) is moderately less hydrophobic and moderately less sterically demanding than the cyclopentane-based building block “Z” (m = 1), which would result in decreased hydrophobicity of the -[A-Z-A]- moiety.

Claims

1 . A compound comprising a moiety of formula (I):
-[A-Z-A]n-
(I) wherein: each A is independently a group according to the following formula:
Figure imgf000054_0001
each R1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)-carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-OH, -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci-6 alkyl), -S(Ci-6 alkylene)-SH, -S(Ci.6 alkylene)-S(Ci.6 alkyl), -NH-OH, -N(CI.6 alkyl)-OH, -NH-O(CI.6 alkyl), -N(CI.6 alkyl)-O(Ci-6 alkyl), halogen, C1-6 haloalkyl, -O-(Ci-6 haloalkyl), -CF3, -CN, -NO2, -CHO, -C0-(Ci-6 alkyl), -C0-0-(Ci-6 alkyl), -O-CO-(Ci.6 alkyl), -CO-NH2, -CO-NH(CI.6 alkyl), -CO-N(CI.6 alkyl)(Ci.6 alkyl), -NH-CO-(CI.6 alkyl), -N(CI.6 alkyl)-CO-(Ci.6 alkyl), -NH-CO-O-(CI.6 alkyl), -N(CI.6 alkyl)-CO- O-(Ci.6 alkyl), -O-CO-NH-(CI.6 alkyl), -O-CO-N(CI.6 alkyl)-(Ci.6 alkyl), -SO2-NH2, -SO2-NH(CI.6 alkyl), -SO2-N(CI.6 alkyl)(Ci.6 alkyl), -NH-SO2-(CI.6 alkyl), -N(CI.6 alkyl)-SO2-(Ci.6 alkyl), -SO2-(Ci.6 alkyl), -SO-(Ci-6 alkyl), cycloalkyl, and heterocycloalkyl, and wherein one -CH2- unit in the Co-6 alkylene moiety in said -(Co-6 alkylene)-carbocyclyl or in said -(Co-6 alkylene)-heterocyclyl is optionally replaced with -O-, -S-, -NH- or -N(CI-6 alkyl)-; each Z is independently a group according to the following formula:
Figure imgf000054_0002
each m is independently 0 or 1 ; and n is 2, 3 or 4; or a pharmaceutically acceptable salt thereof.
2. The compound of claim 1 , wherein each R1 is independently C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -(Co-6 alkylene)-carbocyclyl or -(Co-6 alkylene)-heterocyclyl, wherein the carbocyclyl moiety in said -(Co-6 alkylene)- carbocyclyl and the heterocyclyl moiety in said -(Co-6 alkylene)-heterocyclyl are each optionally substituted with one or more groups independently selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, -OH, -O(Ci-6 alkyl), -O(Ci-6 alkylene)-OH, -O(Ci-6 alkylene)-O(Ci-6 alkyl), -SH, -S(Ci-6 alkyl), -S(Ci-6 alkylene)-SH, -S(Ci-6 alkylene)-S(Ci.6 alkyl), -NH-OH, -N(CI.6 alkyl)-OH, -NH-O(CI.6 alkyl), -N(CI.6 alkyl)-O(Ci.6 alkyl), halogen, C1.6 haloalkyl, -O-(Ci.6 haloalkyl), -CF3, -CN, -NO2, -CHO, -CO-(Ci.6 alkyl), -CO-O-(Ci.6 alkyl), -O-CO-(Ci.6 alkyl), -CO-NH2, -CO-NH(CI.6 alkyl), -CO-N(CI.6 alkyl)(Ci.6 alkyl), -NH-CO-(CI.6 alkyl), -N(CI.6 alkyl)-CO-(Ci.6 alkyl), -NH-CO-O-(CI-6 alkyl), -N(CI.6 alkyl)-CO-O-(Ci.6 alkyl), -O-CO-NH-(CI.6 alkyl), -O-CO-N(CI.6 alkyl)-(Ci.6 alkyl), -SO2-NH2, -SO2-NH(CI.6 alkyl), -SO2-N(CI.6 alkyl)(Ci.6 alkyl), -NH-SO2-(CI.6 alkyl), -N(CI.6 alkyl)-SO2-(Ci-6 alkyl), -SO2-(Ci-6 alkyl), -SO-(Ci-6 alkyl), cycloalkyl, and heterocycloalkyl
3. The compound of claim 1 or 2, wherein n is 2.
4. The compound of any one of claims 1 to 3, wherein each R1 is independently C2-5 alkyl, preferably wherein each R1 is independently C3-4 alkyl, more preferably wherein each R1 is independently isopropyl or isobutyl.
5. The compound of any one of claims 1 to 4, wherein m is 1 .
6. The compound of any one of claims 1 to 5, wherein:
- each A has the following configuration:
- each C has the following configuration:
Figure imgf000055_0001
7. The compound of any one of claims 1 to 6, wherein the compound is a compound of formula (II):
X-[Z]p-[A-Z-A]n-Y
(II) wherein:
A, Z and n are as defined in any one of claims 1 to 6; p is 0 or 1 ; X is an amino acid sequence of 1 to 5 hydrophobic amino acid residues, wherein X is attached via its C-terminus to the rest of the compound of formula (II), optionally wherein X has an alkanoyl group at its N-terminus; and
Y is an amino acid sequence of 1 to 5 polar amino acid residues, which are preferably basic amino acid residues or a combination of basic and polar neutral amino acid residues, wherein Y is attached via its N-terminus to the rest of the compound of formula (II), optionally wherein the C- terminal COOH group of Y is replaced by -CONH2 or CH2OH; or a pharmaceutically acceptable salt thereof.
8. The compound of claim 7, wherein said hydrophobic amino acid residues are each independently selected from Gly, Ala, Vai, Leu, lie, Pro, Phe, Tyr, Met and Trp, preferably from Vai, Leu, lie, Phe, Tyr and Trp, more preferably from Phe, Tyr and Trp.
9. The compound of claim 7 or 8, wherein said polar amino acid residues are each independently selected from Arg, Lys and His, preferably from Arg and Lys.
10. The compound of any one of claims 7 to 9, wherein p is 1.
11. The compound of any one of claims 7 to 10, wherein X is alkanoyl-Tyr-, preferably wherein X is alkanoyl-(L- Tyr)-, and/or wherein Y is -Arg-Arg-NH2 or -Arg-Arg-Arg-NH2, preferably wherein Y is -(D-Arg)-(D-Arg)-NH2 or -(D-Arg)- (D-Arg)-(D-Arg)-NH2.
12. The compound of any one of claims 1 to 11 , wherein the compound is amphipathic.
13. The compound of claim 1 or 7, wherein said compound is selected from any one of the following compounds:
Figure imgf000056_0001
and
Figure imgf000057_0001
or a pharmaceutically acceptable salt of any of these compounds.
14. The compound of claim 1 or 7, wherein said compound is selected from any one of the following compounds:
Figure imgf000057_0002
Figure imgf000058_0001
or a pharmaceutically acceptable salt of any of these compounds.
A pharmaceutical composition comprising the compound of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.
16. The compound of any one of claims 1 to 14 or the pharmaceutical composition of claim 15 for use as a medicament.
17. The compound of any one of claims 1 to 14 or the pharmaceutical composition of claim 15 for use in treating or preventing cancer.
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