AMINO ALCOHOL

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of Singapore patent application No. 10201806551S filed on 31 July 2018, the content of which is incorporated herein by reference in its entirety for all purposes.

TECHNICAL FIELD

[0002] Various embodiments relate to a solid- supported hydroxy amino acid and a solid- supported amino alcohol, and a method of synthesizing the solid- supported hydroxy amino acid and the solid- supported amino alcohol. The solid- supported hydroxy amino acid and solid- supported amino alcohol may be applied in peptide synthesis.

BACKGROUND

[0003] Solid-supported hydroxy amino acids and amino alcohols are important building blocks for several peptides and peptide alcohols. Peptide alcohols (PA) are peptides in which the carboxyl terminus of at least one amino acid is reduced to an alcohol, the thus reduced amino acid being termed as an amino alcohol. Such amino alcohols are typically obtained from the natural amino acid, eg, the amino alcohol from alanine is alaninol (Ala-ol or Alaol). Peptaibols (a class of peptide alcohols) are linear amphipathic peptides and have a molecular weight around 2000 Da. In nature, they are produced by a wide spread soil fungi such as Trichoderma, Hypocrea or Stilbella. Due to the ability to modify cell membranes and induce a characteristic voltage-dependent ion-conductivity, which causes cell lysis at high concentration, they possess a range of biological activities, including antibiotic properties. Additionally, some antimicrobial PAs from Trichoderma induce programmed cell death in plant fungal pathogens.

[0004] For example, one of the most important antibiotic PAs is Alamethicin which has a Phenylalaninol (Phe-ol) at the C-terminus. It is one of the peptaibols that induces characteristic voltage dependent ion-conductivities in lipid bilayer membranes which cause cell lysis at high concentrations. Structurally, Alamethacin, is a peptaibol characterized by a number of common features. In addition to proteinogenic amino acids, it contains an unusually high proportion (about 50%) of a-aminoisobutyric acid (Aib), the N-terminus is N-acetylated whereas the C- terminus is phenylalaninol (Pheol). A further and very important peptide alcohol is named Octreotide (trade name Sandostatin, among others), which mimics the natural somatostatin pharmacologically, and is a metabolically stable somastatin analogue. It was first synthesized in 1979 by the chemist Wilfried Bauer. Octreotide has potent and unique biological activity and is therefore used clinically for the diagnosis and treatment of various neuroendocrine tumors and gastrointestinal disorders. It is a more potent inhibitor of growth hormone glucagon, and insulin than the natural hormone somastatin. Octreotide is used for the treatment of growth hormone producing tumors, bleeding esophageal varices, acromegaly and radiolabelling in nuclear medicine imaging.

[0005] Due to the potent biological activities reported for the peptide alcohols, there is therefore a need for a robust method of synthesizing peptide alcohols. Not too many methods for the synthesis of PAs are reported. However, some examples are presented herein. The Alamethicin F30 sequence is characterized as follows: Ac- Alb-Pro- Alb- Ala- Aib-Aia-Gln-Alb- Val-Aib-Gly-Leu- Alb-Pro- Val- Alb- Aib-Glu-Gln-Pheol. In one known method for the synthesis of Alamethicin, a commercial 2-chlorotrityl resin was loaded with Fmoc- phenylalaninol using a binary mixture of DCM and DMF with pyridine as base. Loadings of about 0.24 mmol/g were obtained after 6 hours (h) for this Fmoc-amino alcohols. The remaining synthesis was performed in a conventional way.

[0006] Octreotide was synthesized previously by loading the first Fmoc amino alcohol (threoninol) to a 2-chlorotrityl resin. This step in this case took 21 h and was performed using the same conditions previously reported.

[0007] An additional approach for the synthesis of peptide alcohols is based on O-N acyl transfer. The first Fmoc amino alcohol is attached to a 2-chlorotrityl resin in the presence of 2% DBU. Under this condition the amino group of the Fmoc amino alcohol was attached to the resin. The second step is an ester formation that in many cases can lead to high racemization. However, several important bioactive peptide alcohols were synthesized including octreotide, a fragment of gramicidin A and the antimicrobial peptide trichogin GA IV. One other synthesis of peptide alcohols was described by G. Kokotos, which consists of the reduction of the active ester after the addition of ethyl chloroformate using sodium borohydride in THF.

[0008] In one other method reported by Wenschuh et al, 2-chlorotrityl resin was loaded with the first amino acid. This reaction is not general and takes more than six hours to be completed. Tailhades et al reported a synthesis of PAs by an O-N acyl transfer reaction using the 2-chloro- trityl resin. However, similarly, this is not a general method and it has several disadvantages, for example, the incorporation of the second amino acid will render an ester. This reaction is usually catalysed by dimethylamino pyridine (DMAP), which always promotes racemization. A high degree of racemization is observed for the incorporation of cysteine. In order to minimize this problem, a Mitsunobu reaction is performed at this step. One other disadvantage is that after the synthesis, an additional step is necessary. In order to get the final peptide, special conditions are needed to promote the O-N acyl transfer. As a further application, PAs serve as precursors for the synthesis of peptide aldehydes, an important class of protease inhibitors and versatile synthetic intermediates.

[0009] Hence, there remains a need for improved solid- supported hydroxy amino acids and amino alcohols and methods to synthesize peptide alcohols that address or at least alleviate one or more of the above-mentioned problems.

SUMMARY

[0010] In a first aspect, a solid- supported hydroxy amino acid or a solid-supported amino alcohol is provided. The solid- supported hydroxy amino acid or the solid- supported amino alcohol may have the following Formula (I)

wherein is a solid support,

X and Y are independently selected from the group consisting of a bond, -O-, -S-, -NH-, -COO- , -OOC-, -CONH-, -NHCO, an optionally substituted C _1-20 alkyl, an optionally substituted phenyl, an optionally substituted C _1-20 alkoxy, an optionally substituted C _2-20 alkenyl and an optionally substituted C _2-20 alkynyl, Z is selected from the group consisting of hydrogen, a bond, -O-, -S-, -NH-, -COO-, -OOC-, an optionally substituted C _1-20 alkyl, an optionally substituted C _1-20 alkoxy, an optionally substituted C _2-20 alkenyl and an optionally substituted C _2-20 alkynyl, is an optional bond,

at least one of Y and Z is -O- , the carbon atom marked with an asterisk * is in the stereo configuration (R) or (S),

Ri is selected from the group consisting of hydrogen, an optionally substituted phenyl, an optionally substituted C _1-20 alkyl, an optionally substituted C _2-20 alkenyl, an optionally substituted C _2-20 alkynyl and an optionally substituted C _1-20 alkoxy,

wherein the optionally substituted phenyl may optionally be covalently connected to Z,

R ₂ , R ₄ , R ₅ and R ₆ are independently selected from the group consisting of hydrogen, halogen, an optionally substituted C _1-20 alkyl, an optionally substituted C _1-20 alkoxy, an optionally substituted C _2-20 alkenyl and an optionally substituted C _2-20 alkynyl,

R ₃ is hydrogen or an amino-protecting group;

wherein either

A is an optionally protected amino acid residue and B is C _1-5 -alkyl, or

A is -(CH ₂ ) _m -C(O)R ₇ and B is selected from the group consisting of -CH ₂ -, -CH(CH ₃ )-, and - CH ₂ -C ₆ H ₄ -,

R ₇ is -O- C _1-20 alkyl, -O-C _2-20 alkenyl, -O-C _2-20 alkynyl, -O- C _6-12 aryl or NH ₂ ,

m and n are independently integers selected from 0 to 5,

or an enantiomer, a diastereomer, a salt or a solvate thereof.

[0011] In a second aspect, a method for the synthesis of a solid-supported hydroxy amino acid as described above is provided. The method may comprise a reaction step wherein a first reactant of the Formula (IV)

is reacted with a protected hydroxy amino acid or a protected amino alcohol of the Formula (V), or an enantiomer thereof,

to give a product of the Formula (VI), or an enantiomer thereof,

wherein

the carbon atom marked with an asterisk R _{1 2} , R ₄ R ₅ , R ₆ A, B

and n are as defined herein before,

LG is a leaving group; R ₈ is an amino-protecting group;

or an enantiomer, a diastereomer, a salt or a solvate thereof, and may further comprise optionally deprotecting the amino-protecting group R ₈ .

[0012] In a third aspect, a method of synthesizing a peptide alcohol is provided. The method may comprise

(i) Providing a solid- supported hydroxy amino acid or a solid-supported amino alcohol as described above, and (ii) Carrying out at least one peptide coupling step with an amino acid or an amino acid derivative.

[0013] In a fourth aspect, a peptide alcohol synthesized by the method as described above is provided.

[0014] In a fifth aspect, a stapled peptide alcohol synthesized by one embodiment of the method as described above is provided.

[0015] In a sixth aspect, a stapled peptide alcohol is provided. The stapled peptide alcohol may have the following Formula (VIII)

wherein

A, B and the carbon atom marked with an asterisk * are as defined herein before, P ¹ and P ² are each independently an oligopeptide chain or a polypeptide chain, each q is independently an integer between 0 and 12; r is an integer between 1 and 8;

C is an amino acid or an amino acid derivative; and is a carbon-carbon single bond that is attached to a carbon atom of the double bond such that the compound of Formula (VIII) is in either the (^-configuration or the (Z) configuration or is a mixture of these; or an enantiomer, a diastereomer, a salt or a solvate thereof. [0016] In a seventh aspect, a method of preventing or treating cancer is provided. The method may comprise administering an effective amount of the stapled peptide alcohol as described herein to a mammal.

[0017] In an eighth aspect, a stapled peptide alcohol as described herein is provided for use in therapy.

[0018] In a ninth aspect, use of a stapled peptide alcohol as described herein is provided in the manufacture of a medicament for the prevention or treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0020] FIG. 1 is a schematic drawing illustrating various embodiments of the disclosure.

[0021] FIG. 2 is a High Performance Liquid Chromatography (HPLC) graph showing the elution peak of LARGY-Phe-ol using the Fmoc Phenylalaninol Ramage-PS Resin, as shown in Example 5. LARGY, in this context, stands for a peptide having the following sequence: Leu-Ala- Arg-Gly-Tyr. PS, in this context, stands for polystyrene. The retention time (RT) is at 11.335 min.

[0022] FIG. 3A is a Liquid chromatography mass spectrometry (LCMS) graph showing the identified molecular weight of ASTP7041-ol on Fmoc-Alaninol-Ramage-PS and the trifluoroacetic acid (TFA) adducts (Example 6).

[0023] FIG. 3B a HPLC graph showing the elution peak of ASTP704l-ol, measured at a gradient: 50:90:50 on a phenomenex Jupiter 5 mm C4 300 Å 100x 4.6 mm (Example 6).

[0024] FIG. 4A is an LCMS graph showing the identified molecular weight of ASTP7041- peptaibol-like peptide using the Fmoc Alaninol-Ramage-PS resin (Example 7).

[0025] FIG. 4B is a HPLC graph showing the elution peak of ASTP704l-peptaibol-like peptide, measured at a gradient: 50:90:50 (Example 7)

[0026] FIG. 5A is a HPLC graph of LARGYG-Phe-ol using the chlorinated resin of Example

9 (Gradient 5:45:5 20min, retention time (RT) 11.185min).

[0027] FIG. 5B is an LCMS graph showing the identified molecular weight of LARGYG-Phe- ol using the chlorinated resin of Example 9.

[0028] FIG. 6 is a HPLC graph of LARGYG-Phe-ol using the chlorinated resin of Example

10 (Gradient 5:45:5 20min RT l l.l85min). [0029] FIG. 7 is a HPLC graph of LARGY-Phe-ol of Example 12, indicating a diastereomeric mixture.

[0030] FIG. 8 is a HPLC graph of LARGY-Phe-ol of Example 12 showing a single diastereomer.

[0031] FIG. 9A is a HPLC graph of the reduced form of octreotide (Example 13).

[0032] FIG. 9B is an LCMS graph showing the identified molecular weight of the reduced form of octreotide (Example 13).

[0033] FIG. 10 is an LCMS graph showing the identified molecular weight of trichogin (Example 14).

[0034] FIG. 11A is an LCMS graph showing the identified molecular weight of Alamethacin, along with two other major peaks, identified for the masses 775.76 and 1204.12 (Example 15).

[0035] FIG. 11B shows the amino acid sequence for Alamethacin and indicates hydrolysis of the Aib-Pro-bond, and the mass fragments which would be obtained after such hydrolysis.

[0036] FIG. 11C is an LCMS graph showing the molecular weight of a segment of Alamethicin A30.

[0037] FIG. 12 shows the retention times for PM2-ENF-amide and PM2-ENF-ol Phenomenex Jupiter 4u Proteo 90A 150x4.6mm. 50-90-50 l5min.

[0038] FIG. 13A shows the K _d for some of the peptide amides and some of the Stapled Peptide Alcohols (SPALs), as well as their molecular weight and their sequence.

[0039] FIG. 13B shows the K _d for Mdm2 and Mdm4 for some of the peptide amides and some of the Stapled Peptide Alcohols (SPALs).“SD” in this context refers to the standard deviation.

[0040] FIG. 13C shows the K _d for Mdm2 and Mdm4 for some of the peptide amides and some of the Stapled Peptide Alcohols (SPALs).

[0041] FIG. 13D shows the K _d in a Fluorescent Probe FP Assay, which measures the affinity of the peptide to displace 5(6)-Carboxyfluorescein (FAM) peptide from Mdm2.

[0042] FIG. 13E is a Table illustrating the peptide sequence of some of the stapled peptide alcohols and their molecular weight, as well as their diastereomeric purity.

[0043] FIG. 14A shows the half maximal effective concentration (EC50)s for some of the peptide amides and SPALs.

[0044] FIG. 14B shows the EC50s for some of the peptide amides and SPALs.

[0045] FIG. 15A shows Lactate Dehydrogenase (LDH) release for some of the peptide amides in AML2. [0046] FIG. 15B shows LDH release for some of the SPALs in AML2. AML2 is known to be very sensitive to drug treatment. So treatment with the -ol peptides seems to trigger some membrane damage as seen from the high LDH release in VIP82ol and VIP1 l5ol.

[0047] FIG. 15C shows LDH release for some of the SPALs in HL60 at 4h. The more interesting peptides in the list were tested on HL60 which is a p53 null cell line. Most of them showed minimal LDH release.

[0048] FIG. 16A shows three graphs illustrating the T22 cell reporter assay p53 activation for the peptide amides and for SPALs.

[0049] FIG. 16B is a Table comparing the Mean EC50s of the peptide amides and for SPALs in the T22 cell reporter assay p53 activation.

[0050] FIG. 17A shows the Western Blot for SPAMs vs SPALs Mdm2 Antagonists (AML2, 20uM in 10% FCS, 24h).

[0051] FIG. 17B shows the Western Blot for SPAMs vs SPALs Mdm2 Antagonists (AML3, 20uM in 10% FCS, 24h). AMF3 is quite resistant to drug treatment. So based on the blots, 24 hour treatment may not be enough for significant p53 activation. However, the cells seem to get arrested as seen from the upregulation of p2l.

[0052] FIG. 17C shows the Western Blot for SPAMs vs SPALs Mdm2 Antagonists (AMF2, 20uM in 10% FCS, 24h). PARP is involved in DNA damage repair but is cleaved by the caspases in cells when the cells undergo programmed cell death. So a stronger cleaved band (Cl.) indicates that more cells are undergoing apoptosis. p2l is linked to cell cycle arrest. Based on the blots, the PM2-ols do not seem to perform significantly better than the PM2 amide peptides. VIP82 and VIPI I60I seem to be killing more cells than VIP82 and VIP116 as seen from the strong Cl. PARP bands. But since the Mdm2 and p53 bands for VIP82ol and VIP1160I are not significantly stronger than their amide counterparts, more investigation is required to determine if the apoptosis is triggered in a p53 specific manner or due to cell membrane damage.

[0053] FIG. 17D shows the Western Blot for SPAMs vs SPALs Mdm2 Antagonists (AMF2, 20uM in 10% FCS, 24h).

[0054] FIG. 17E shows the Western Blot for SPAMs vs SPALs Mdm2 Antagonists (HF60, 20uM in 10% FCS, 24h). None of the alcohol peptides show signs of cell death as the cleaved PARP bands remain at basal levels, while Doxorubicin, a non-p53 specific drug shows p53 activation by a different pathway in which Mdm2 is not related.

[0055] FIG. 18 shows the difference in structure of the SPAMs (shown on the left) and the SPALs (shown on the right). [0056] FIG. 19A is an LCMS graph showing the identified molecular weight of the peptide as synthesized in Example 16.

[0057] FIG. 19B is a HPLC graph of the peptide as synthesized in Example 16.

[0058] FIG. 20A is an LCMS graph showing the identified molecular weight of the peptide as synthesized in Example 17.

[0059] FIG. 20B is a HPLC graph of the peptide as synthesized in Example 17.

[0060] FIG. 21A is an LCMS graph showing the identified molecular weight of the peptide as synthesized in Example 18.

[0061] FIG. 21B is a HPLC graph of the peptide as synthesized in Example 18.

[0062] FIG. 22 illustrates that PM2, ENF-ol, VIP82-ol and VIP116-oI show good upregulation of Mdm2 and p2l at the RNA level. PM2-ENF-ol is also termed as VIP65-ol and VIP65 SPAL. VIP82-ol is also termed as VIP82 SPAL and VIPI I6-oI is also termed as VIP116 SPAL. All show good upregulation compared to the amide versions (known also as SPAMs) of VIP65, VIP82 and VIP116. The bars signify from left to right the result from DMSO, Dox, Nutlin, PM2, PM2 ENF, PM2 ENFol, PM2 ENY, PM2 ENY-ol, VIP115, VIP115-ol, VIP116, VIP116 Hex, VIP Pro, VIP116 Sc, VIP116-oI, VIP116-oI Sc, VIP82, VIP82-ol, each for Mdm2, NOXA, p21 and PUMA.

[0063] FIG. 23 shows performance of the SPAMs vs the SPALs as Mdm2 Antagonists on HL60 (p53 null cell line) 20uM in 10% FCS, 24h. This Figure illustrates that there is no upregulation of Mdm2 at the RNA level. Although there seems to be upregulation of p2l at the RNA level, there does not seem to be an upregulation of p2l at the protein level. So these peptides do seem to act in a p53 specific manner. The bars signify from left to right the result from DMSO, Dox, Nutlin, PM2, PM2 ENF, PM2 ENFol, PM2 ENY, PM2 ENY-ol, VIP115, VIP115-01, VIP116, VIP116 Hex, VIP Pro, VIP116 Sc, VIP116-oI, VIP116-oI Sc, VIP82, VIP82-ol, each for Mdm2, NOXA, p21 and PUMA.

[0064] FIG. 24 shows a flow cytometry analysis to determine differentiation of AML2 cells exposed to All Trans Retinoic acid (ATRA), using myeloid lineage for cell differentiation markers. CD14 is a marker for macrophages, CDl lb for neutrophils, CD15 and CD66b for granulocytes. For the four graphs, the graph nearest to the baseline shows exposure of 0 hours, and the graphs successively stacked on from there show exposure of 24h, 48h, 72h, and 96h.

[0065] FIG. 25 shows a flow cytometry analysis to determine differentiation of AML2 cells exposed to Nutlin 3, using myeloid lineage for cell differentiation markers. CD14 is a marker for macrophages, CDl lb for neutrophils, CD15 and CD66b for granulocytes. For the four graphs, the graph nearest to the baseline shows exposure of 0 hours, and the graphs successively stacked on from there show exposure of 24h, 48h, 72h, and 96h.

[0066] FIG. 26 shows flow cytometry analysis to determine differentiation of AML2 cells exposed to Doxorubicin, using myeloid lineage for cell differentiation markers. CD14 is a marker for macrophages, CDl lb for neutrophils, CD15 and CD66b for granulocytes. For the four graphs, the graph nearest to the baseline shows exposure of 0 hours, and the graphs successively stacked on from there show exposure of 24h, 48h, 72h, and 96h.

[0067] FIG. 27 shows a flow cytometry analysis to determine differentiation of AML2 cells exposed to PM2, using myeloid lineage for cell differentiation markers. CD 14 is a marker for macrophages, CDl lb for neutrophils, CD15 and CD66b for granulocytes. PM2 Ac- TSF(R8)EYWALL(S5)-amide. All hydrocarbon staple between R8 and S5. For the four graphs, the graph nearest to the baseline shows exposure of 0 hours, and the graphs successively stacked on from there show exposure of 24h, 48h, 72h, and 96h.

[0068] FIG. 28 shows a flow cytometry analysis to determine differentiation of AML2 cells exposed PM2-ENF (also known as VIP65 SPAM, using myeloid lineage for cell differentiation markers. CD14 is a marker for macrophages, CDl lb for neutrophils, CD15 and CD66b for granulocytes. For the four graphs, the graph nearest to the baseline shows exposure of 0 hours, and the graphs successively stacked on from there show exposure of 24h, 48h, 72h, and 96h.

[0069] FIG. 29 shows a flow cytometry analysis to determine differentiation of AML2 cells exposed PM2-ENF (also known as VIP65-ol SPAL), using myeloid lineage for cell differentiation markers. CD14 is a marker for macrophages, CD11b for neutrophils, CD15 and CD66b for granulocytes. For the four graphs, the graph nearest to the baseline shows exposure of 0 hours, and the graphs successively stacked on from there show exposure of 24h, 48h, 72h, and 96h.

[0070] FIG. 30 shows a flow cytometry analysis to determine differentiation of AML2 cells using CD11b marker for neutrophils exposed to different drugs.

[0071] FIG. 31 shows a flow cytometry analysis to determine differentiation of AML2 cells using CD14 marker for macrophages exposed to different drugs.

[0072] FIG. 32 shows flow cytometry analysis to determine differentiation of AML2 cells using CD 15 marker for granulocytes exposed to different drugs.

[0073] FIG. 33 shows flow cytometry analysis to determine differentiation of AML2 cells using CD66b marker for granulocytes exposed to different drugs.

[0074] FIG. 34 shows a flow cytometry analysis to determine differentiation of AML3 cells using CD11b marker for neutrophils exposed to different drugs. [0075] FIG. 35 shows a flow cytometry analysis to determine differentiation of AML3 cells using CD14 marker for macrophages exposed to different drugs.

[0076] FIG. 36 shows flow cytometry analysis to determine differentiation of AML3 cells using CD 15 marker for granulocytes exposed to different drugs.

[0077] FIG. 37 shows flow cytometry analysis to determine differentiation of AML3 cells using CD66b marker for granulocytes exposed to different drugs.

[0078] FIG. 38 shows a flow cytometry analysis to determine differentiation of AML5 cells using CD11b marker for neutrophils exposed to different drugs.

[0079] FIG. 39 shows a flow cytometry analysis to determine differentiation of AML5 cells using CD 14 marker for macrophages exposed to different drugs.

[0080] FIG. 40 shows a flow cytometry analysis to determine differentiation of AML5 cells using CD 15 marker for granulocytes exposed to different drugs.

[0081] FIG. 41 shows a flow cytometry analysis to determine differentiation of AML5 cells using CD66b marker for granulocytes exposed to different drugs.

[0082] FIG. 42 shows a flow cytometry analysis to determine differentiation of Myelogenous Leukemia cells (ML2) using CD11b marker for neutrophils exposed to different drugs.

[0083] FIG. 43 shows a flow cytometry analysis to determine differentiation of Myelogenous Leukemia cells (ML2) using CD 14 marker for macrophages exposed to different drugs.

[0084] FIG. 44 shows a flow cytometry analysis to determine differentiation of Myelogenous Leukemia cells (ML2) using CD 15 marker for granulocytes exposed to different drugs.

[0085] FIG. 45 shows a flow cytometry analysis to determine differentiation of Myelogenous Leukemia cells (ML2) using CD66b marker for granulocytes exposed to different drugs.

[0086] FIG. 46 shows a flow cytometry analysis to determine differentiation of Human promyelocytic Leukemia cells (HL60, p53 null) using CD11b marker for neutrophils exposed to different drugs.

[0087] FIG. 47 shows a flow cytometry analysis to determine differentiation of Human promyelocytic Leukemia cells (HL60, p53 null) using CD 14 marker for macrophages exposed to different drugs.

[0088] FIG. 48 shows a flow cytometry analysis to determine differentiation of Human promyelocytic Leukemia cells (HL60, p53 null) using CD15 marker for granulocytes exposed to different drugs.

[0089] FIG. 49 shows a flow cytometry analysis to determine differentiation of Human promyelocytic Leukemia cells (HL60, p53 null) using CD66b marker for granulocytes exposed to different drugs. DETAILED DESCRIPTION

Various embodiments disclosed herein are directed to a solid- supported hydroxy amino acid or a solid-supported amino alcohol. The solid-supported hydroxy amino acid or the solid- supported amino alcohol may have the following Formula (I)

wherein is a solid support,

X and Y are independently selected from the group consisting of a bond, -O-, -S-, -NH-, -COO- , -OOC-, -CONH-, -NHCO, an optionally substituted C _1-20 alkyl, an optionally substituted phenyl, an optionally substituted C _1-20 alkoxy, an optionally substituted C _2-20 alkenyl and an optionally substituted C _2-20 alkynyl,

Z is selected from the group consisting of hydrogen, a bond, -O-, -S-, -NH-, -COO-, -OOC-, an optionally substituted C _1-20 alkyl, an optionally substituted C _1-20 alkoxy, an optionally substituted C _2-20 alkenyl and an optionally substituted C _2-20 alkynyl, is an optional bond,

at least one of Y and Z is -O- , the carbon atom marked with an asterisk * is in the stereo configuration (R) or (S), Ri is selected from the group consisting of hydrogen, an optionally substituted phenyl, an optionally substituted C _1-20 alkyl, an optionally substituted C _2-20 alkenyl, an optionally substituted C _2-20 alkynyl and an optionally substituted C _1-20 alkoxy,

wherein the optionally substituted phenyl may optionally be covalently connected to Z,

R ₂ , R ₄ , R5 and R ₆ are independently selected from the group consisting of hydrogen, halogen, an optionally substituted C _1-20 alkyl, an optionally substituted C _1-20 alkoxy, an optionally substituted C _2-20 alkenyl and an optionally substituted C _2-20 alkynyl,

R ₃ is hydrogen or an amino-protecting group;

wherein either

A is an optionally protected amino acid residue and B is C _1-5 -alkyl; or

A is -(CH ₂ ) _m -C(O)R ₇ and B is selected from the group consisting of -CH ₂ -, -CH(CH ₃ )-, and - CH ₂ -C ₆ H ₄ -,

R ₇ is -O- C _1-20 alkyl, -O-C _2-20 alkenyl, -O-C _2-20 alkynyl, -O- C _6-12 aryl or NH ₂ ,

m and n are independently integers selected from 0 to 5,

or an enantiomer, a diastereomer, a salt or a solvate thereof.

[0090] For clarity in the following discussion, as the linker system, the hydroxy amino acid or amino alcohol, and the carbon at which substitution is taking place, the following chemical entities of Formula (I) are meant, as indicated in the square box:

In the Examples section, this linker system is also commonly termed as the“linker” (optionally with a leaving group attached to the carbon atom at which substitution is taking place).

[0091] Advantageously, the solid- supported hydroxy amino acid or the solid- supported amino alcohol is of a formula wherein at least one of Y and Z is an oxygen (-O-). In other words, at least one of the two phenyl rings of the compound of Formula (I) is bonded directly to an oxygen (-O-), thereby representing an ether moiety conjugated to a phenyl ring. The solid- supported hydroxy amino acid or the solid-supported amino alcohol comprises therefore the following entity: -Ph-O-, with Ph being a phenyl ring _. Phenyl, in this context, is understood to mean a C ₆ H ₆ - aromatic ring, wherein one or more hydrogen atoms may be replaced through substitution.

[0092] Without being bound to theory, it is believed that the ether moiety which is in conjugation with at least one of the two phenyl rings has an electron-donating effect on the Ph. Both of the phenyl rings are furthermore bonded to the carbon atom at which substitution is taking place. Due to the Ph being bonded to the carbon atom at which substitution is taking place, the Ph-O- entity may have a facilitating effect in the substitution reaction. Hence, the conjugation system of the phenyl ring is sufficiently strong to transfer the electron-donating effect of the ether moiety to the carbon atom at which substitution is taking place. The substitution pattern of the ether moiety with respect to the carbon atom at which substitution is taking place may be such that the positioning of the ether moiety is in ortho or para position to the carbon atom at which substitution is taking place. Hence, the ether moiety may have a positive mesomeric effect (+M effect) on the substitution reaction, thereby facilitating substitution.

[0093] Accordingly, this technology allows the production of solid-supported hydroxy amino acids or solid- supported amino alcohols for a rapid synthesis of peptide alcohols and peptaibols, starting with the incorporation of hydroxy amino acids and amino alcohols to the linker system. More advantageously, by using these solid-supported hydroxy amino acids or solid- supported amino alcohols, the hydroxy amino acids or solid- supported amino alcohols can be attached and manipulated later on without any epimerization occuring on the amino acids. The linker system described herein further has a generalized scope, such that the technology has been tested on at least 10 different amino alcohols.

[0094] Further advantageously, the incorporation of the hydroxy amino acids and amino alcohols to the linker system can be performed in less than one hour, for example in 15 min, using a microwave peptide synthesizer. This is considerably faster than the prior art technologies, wherein 6-12 hours are needed for this reaction.

[0095] Further advantageously, by using the linker system as described above, the steps of attaching the hydroxy amino acid on the linker system and subsequent steps building up the peptide alcohol or peptaibol can be carried out in one pot as a conventional solid phase peptide synthesis. [0096] As evidence to its efficiency, well-known peptide alcohols, for example octreotide, can be synthesized using the solid- supported hydroxy amino acid and solid-supported amino alcohols (commonly termed as“resin” in the Examples section) opening the path for fast and efficient synthesis for structure- activity relationship (SAR).

[0097] By using the solid-supported hydroxy amino acids and solid- supported amino alcohols, subsequent peptide synthesis and ring-closing metathesis, it is furthermore possible to provide stapled peptide alcohols, providing more efficient tools in the search for more potent Mdm2/p53 agonists.

[0098] The term“hydroxy amino acid”, as used herein, includes an amino acid wherein a side chain of the amino acid comprises a hydroxy group. Hence, before covalent bonding to the carbon atom at which substitution is taking place, the hydroxy amino acid has at least one hydroxy functionality, as shown below:

[0099] In this embodiment, B is selected from -CH ₂ -, -CH(CH ₃ )- and -CH ₂ -C6H4-. Hence, this embodiment includes amino acids such as serine, for which B would be -CH ₂ -, threonine, for which B would be -CH(CH ₃ )-, and tyrosine, for which B would be -CH ₂ -C ₆ H ₄ -. The C- terminus of the amino acid may be protected by a group R ₇ , which may either be an ester protection (by esterifying the carboxylic acid), or an amide (by converting the carboxylic acid to an amide).

[00100] The term“amino alcohol”, as used herein, includes an amino acid which has been modified to reduce the carboxy functionality of the C-terminus. Hence, before bonding to the linker system, the amino alcohol includes an amino alcohol as shown below:

This amino alcohol is an amino acid, wherein the carboxylic acid is reduced to a primary alcohol and A is an optionally protected amino acid residue.

[00101] In both embodiments, the hydroxy amino acid and the amino alcohol, the free hydroxy group engages in a substitution reaction with the carbon atom at which substitution is taking place. This reaction results in the hydrogen of the hydroxy group to be cleaved, as well as a leaving group attached to the carbon at which substitution is taking place to be cleaved, thereby creating a new covalent bond, resulting in the solid-supported hydroxy amino acid or solid- supported amino alcohol as described herein.

[00102] The term“solid support”, as used herein, refers to a solid which may be used to attach the linker to it. Solid supports are known in the literature and include gel-type supports, surface-type supports, and composites. They provide physical stability to permit rapid filtration of liquids, while an amino acid, or a peptide, is retained on the solid support. Suitable supports are inert to reagents and solvents used during solid phase peptide synthesis (SPPS), although they may swell in the solvents used to allow for penetration of the reagents, and allow for the attachment of the hydroxy amino acid or amino alcohol. In some embodiments, the solid support may be selected from the group consisting of a polystyrene, a polyethylene glycol and a combination thereof.

[00103] The N-terminus of the hydroxy amino acid may optionally be protected by an amino-protecting group. The term“amino-protecting group”, as used herein, refers to groups commonly employed to keep amino groups from reacting during reactions carried out on other functional groups. Examples of such protecting groups include carbamates, amides, alkyl and aryl groups, imines, as well as many N-heteroatom derivatives which can be removed to regenerate the desired amine group. In some embodiments, R ₃ may be an amino-protecting group selected from the group consisting of Ac (acetyl), trifluoroacetyl, phthalimide, Bn (benzyl), Trt (triphenylmethyl or trityl), benzylidenyl, p-toluenesulfonyl, Pmb (p- methoxybenzyl), Boc (tert-butyloxycarbonyl), Fmoc (9-fluorenylmethyloxycarbonyl) and Cbz (carbobenzyloxy). In further embodiments, R ₃ may be an amino-protecting group selected from the group consisting of a-Nsmoc, Bsmoc and b-Nsmoc, the structures of which are provided below: a-Nsmoc:

Bsmoc:

b-Nsmoc:

Further details pertaining to these protecting groups may be found in the references Carpino et al J. Org. Chem. 1999, 64, 4324-4338 (for Bsmoc) and Carpino et al J. Org. Chem. 2007, 72, 1729-1736 (for a-Nsmoc and b-Nsmoc).

[00104] The term“amino acid residue” (A), as used herein, refers to the side chain of the amino acid. Hence, in some embodiments, A may be an amino acid residue, selected from the group consisting of -CH ₃ (Ala), -(CH ₂ ) ₃ -NH-C(N)-NH ₂ (Arg), -CH ₂ C(O)OH (Asp), -CH ₂ - C(O)NH ₂ (Asn), -CH ₂ SH (Cys), -CH ₂ CH ₂ C(O)OH (Glu), -CH ₂ CH ₂ CONH ₂ (Gln), -H (Gly), - CH(CH )CH ₂ CH (Ile), -CH ₂ CH(CH ) ₂ (Leu), -(CH ₂ ) ₄ -NH ₂ (Lysine), -CH ₂ CH ₂ SCH (Met), - CH ₂ Ph (Phe), -(CH ₂ ) ₃ bridging to the nitrogen (proline), -CH ₂ OH (Ser), -CHCH ₃ OH (Thr), (Trp), and -CH(CH ₃ ) ₂ (Val). The brackets indicate the amino acid from which the side chain is derived and the terminology is known to the person skilled in the art. The side chain, if necessary during the course of the attachment, can be protected with a protecting group.

[00105] Each of these amino acid residues A may be optionally protected, whereby, in some embodiments, the protecting group may be selected from the group consisting of 2, 2, 5, 7, 8- Pmc (pentamethylchroman-6-sulfonyl), Trt (triphenylmethyl or trityl), tBu (tert- Butyl), Acm (Acetamidomethyl), Boc (tert-butyloxycarbonyl), Pbf (2, 2, 4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl), StBu (tert-butylthio), Fmoc (9- fluorenylmethyloxycarbonyl), and DMT (4,4’-dimethoxytrityl).

[00106] The term“optionally substituted” or“substituted or unsubstituted”, as used herein, refers to a group in which none, one, or more than one of the hydrogen atoms have been replaced with one or more groups such as, but are not limited to, alkyl, heteroalkyl, haloalkyl, heterohaloalkyl, cycloalkyl, aryl, arylalkyl, heteroaryl, non-aromatic heterocycle, halogen, cyano, hydroxy, nitro, silyl, or amino group.

[00107] The term“halogen”, as used herein, refers to a member of the halogen family selected from the group consisting of fluorine, chlorine, bromine, and iodine.

[00108] In present context, the term“alkyl”, alone or in combination, refers to a fully saturated aliphatic hydrocarbon. The alkyl may be linear or branched. In certain embodiments, alkyls are optionally substituted. In certain embodiments, an alkyl comprises 1 to 20 carbon atoms, for example 1 to 10 carbon atoms, wherein (whenever it appears herein in any of the definitions given below) a numerical range, such as“1 to 20” or“ C _1-20 ”, refers to each integer in the given range, e.g.“C _1-20 alkyl” means that an alkyl group comprising only 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, 18 carbon atoms, 19 carbon atoms, or 20 carbon atoms. Lower alkyl means 1 to 8, preferably 1 to 6, more preferably 1 to 4 carbon atoms.

[00109] The term“linear”, as used herein, refers to each of the carbon atom backbone chains having no branch point. The term“branched” means a chain of atoms with one or more side chains attached to it. Branching occurs by the replacement of a substituent, e.g. a hydrogen atom, with a covalently bonded substituent or moiety, e.g. an alkyl group.

[00110] In present context, the term“alkenyl”, alone or in combination, refers to an aliphatic hydrocarbon having one or more carbon-carbon double-bonds, such as two or three carbon-carbon double-bonds. The alkenyl may be linear or branched. In certain embodiments, alkenyls are optionally substituted, i.e. substituted or unsubstituted. In certain embodiments, an alkenyl comprises 2 to 20 carbon atoms, such as 2 to 18, or 2 to 12, or 2-6 carbon atoms. “C2-20 alkenyl” means that an alkenyl group comprising only 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, 18 carbon atoms, 19 carbon atoms, or 20 carbon atoms. Lower alkenyl means 2 to 8, 2 to 6 or 2 to 4 carbon atoms. Examples of alkenyls include, but are not limited to, ethenyl, propenyl, butenyl, l,4-butadienyl, pentenyl, hexenyl, 4-methylhex-l-enyl, 4-ethyl-2-methylhex-l-enyl and the like.

[00111] In present context, the term“alkynyl”, alone or in combination, refers to an aliphatic hydrocarbon having one or more carbon-carbon triple-bonds, such as two or three carbon-carbon triple-bonds. The alkynyl may be linear or branched. In certain embodiments, alkynyls are optionally substituted, i.e. substituted or unsubstituted. In certain embodiments, an alkynyl comprises 2 to 20 carbon atoms, such as 2 to 18, or 2 to 12, or 2 to 6 carbon atoms. “C2-20 alkynyl” means that an alkynyl group comprising only 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, 18 carbon atoms, 19 carbon atoms, or 20 carbon atoms. Lower alkynyl means 2 to 8, 2 to 6 or 2 to 4 carbon atoms. Examples of alkynyls include, but are not limited to, ethynyl, propynyl, butynyl, and the like.

[00112] In present context, the term“aryl” refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom. Aryl rings may be formed by five, six, seven, eight, nine, or more than nine carbon atoms. Aryl groups may be optionally substituted. For example, an aryl group may be 5- and 6-membered carbocyclic aromatic rings, such as, phenyl; bicyclic ring systems such as 7-12 membered bicyclic ring systems wherein at least one ring is carbocyclic and aromatic, selected, for example, from naphthalene, indane, and 1, 2,3,4- tetrahydroquinoline; and tricyclic ring systems such as 10-15 membered tricyclic ring systems wherein at least one ring is carbocyclic and aromatic, for example, fluorene.

[00113] In present context, the term“heteroaryl” refers to an aromatic heterocycle. Heteroaryl rings may be formed by five, six, seven, eight, nine, or more than nine atoms. Heteroaryls may be optionally substituted. Examples of heteroaryl groups include, but are not limited to, aromatic C ₅ -C ₁₅ heterocyclic groups comprising one oxygen or sulphur atom or up to four nitrogen atoms, or a combination of one oxygen or sulphur atom and up to two nitrogen atoms, and their substituted as well as benzo- and pyrido-fused derivatives, for example, connected via one of the ring-forming carbon atoms.

[00114] In some embodiments, Y may be -O- . Hence, the carbon atom at which substitution is taking place may be in para position with -O- . Advantageously, when Y is -O- , the electron-donating effect of the ether moiety may further facilitate substitution reaction at the carbon atom at which substitution is taking place.

[00115] In some embodiments, Ri may be hydrogen. In these embodiments, the carbon atom at which substitution is taking place is a secondary carbon atom. Advantageously, if the carbon atom at which substitution is taking place is a tertiary carbon atom, as opposed to a quaternary carbon atom, this may provide stability to the compound of Formula (I). The combination of the ether moiety on the phenyl ring and the tertiary carbon atom in these embodiments may provide a favourable balance between a facilitated substitution reaction and stability of the compound of Formula (I).

[00116] In some embodiments, both Y and Z may be -O- . In these embodiments, the carbon atom at which substitution is taking place may be bonded to at least two phenyl having an ether moiety attached thereto. Advantageously, when the carbon atom at which substitution is taking place is bonded to two optionally substituted phenyl rings, the electron-donating effect of the ether moiety may be more efficient, such that substitution at the carbon atom at which substitution is taking place may be facilitated. [00117] In particular embodiments, the solid-supported hydroxy amino acid or the solid- supported amino alcohol may be according to Formula (II)

wherein the carbon atom marked with an asterisk *, Z, R ₃ , R ₄ , R ₅ , A, B and n are as defined herein before,

X is selected from the group consisting of a bond, -O-, -S-, -NH-, -COO-, -OOC-, an optionally substituted phenyl, an optionally substituted C _1-20 alkyl, an optionally substituted C _1-20 alkoxy, an optionally substituted C _2-20 alkenyl and an optionally substituted C _2-20 alkynyl,

or an enantiomer, a diastereomer, a salt or a solvate thereof.

[00118] In particular embodiments, the solid-supported hydroxy amino acid or the solid- supported amino alcohol may be according to Formula (Ila)

wherein the carbon atom marked with an asterisk *, R ₃ , R ₄ , R ₅ , A, B and n are as defined herein before,

X and Z are independently selected from the group consisting of a bond, -O-, -S-, -NH-, -COO- , -OOC-, an optionally substituted C _1-20 alkyl, an optionally substituted C _1-20 alkoxy, an optionally substituted C _2-20 alkenyl and an optionally substituted C _2-20 alkynyl, or an enantiomer, a diastereomer, a salt or a solvate thereof.

[00119] In particular embodiments, the solid-supported hydroxy amino acid or the solid- supported amino alcohol may be according to Formula (lib)

wherein the carbon atom marked with an asterisk *, X, Z, n, R ₃ , R ₄ and R5 are as defined for Formula (Ila),

A is an optionally protected amino acid residue, or an enantiomer, a diastereomer, a salt or a solvate thereof. [00120] In some embodiments, X may be a bond. In some embodiments, m may be 1. In other embodiments, m may be 0. In some embodiments, n may be 1. In some embodiments, Z may be -O- or C _1-10 alkyl. In some embodiments, R ₄ and R ₅ may be hydrogen.

[00121] In particular embodiments, the solid-supported hydroxy amino acid or the solid- supported amino alcohol may be according to Formula (III)

wherein the carbon atom marked with an asterisk *, X, R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , A, B and n are defined as herein before, or an enantiomer, a diastereomer, a salt or a solvate thereof.

[00122] In particular embodiments, the solid-supported hydroxy amino acid or the solid- supported amino alcohol may be according to Formula (Ilia)

wherein the carbon atom marked with an asterisk *, X, R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and n are defined as herein before,

A is an optionally protected amino acid residue, or an enantiomer, a diastereomer, a salt or a solvate thereof.

[00123] In particular embodiments, the solid-supported hydroxy amino acid or solid- supported amino alcohol may be according to Formula (IIIb)

wherein , the carbon atom marked with an asterisk *, X, R ₂ , R ₃ , R ₄ , R ₅ , R ₆ and n are defined as herein before,

B is selected from the group consisting of -CH ₂ -, -CH(CH ₃ )-, and -CH ₂ -C ₆ H4-,

R ₇ is -O-C _1-10 alkyl or -NH ₂ , or an enantiomer, a diastereomer, a salt or a solvate thereof.

[00124] In some embodiments, X may be an optionally substituted phenyl. In some embodiments, B may be -CH ₂ -. In some embodiments, R ₂ and R4 may be hydrogen. In some embodiments, R5 and R ₆ may be C _1-20 alkoxy.

[00125] According to specific embodiments, the solid- supported hydroxy amino acid or the solid-supported amino alcohol may be selected from the group consisting of wherein and R ₃ are as defined herein before;

A is an optionally protected amino acid residue,

B is selected from the group consisting of -CH ₂ -, -CH(CH ₃ )-, and -CH ₂ -C ₆ H ₄ -,

R ₇ is -O-C _1-10 alkyl or -NH ₂ , or an enantiomer, a diastereomer, a salt or a solvate thereof.

[00126] In a second aspect, there is provided a method for the synthesis of a solid- supported hydroxy amino acid or a solid- supported amino alcohol as described above. The method may comprise a reaction step wherein a first reactant of the Formula (IV)

is reacted with a protected hydroxy amino acid or protected amino alcohol of the Formula (V), or an enantiomer thereof,

to give a product of the Formula (VI), or an enantiomer thereof,

wherein the carbon atom marked with an asterisk *, R ₁ , R ₂ , R ₄ , R ₅ , R ₆ , A, B and n are as defined in claim 1,

LG is a leaving group; R ₈ is an amino-protecting group;

or an enantiomer, a diastereomer, a salt or a solvate thereof, and further comprising optionally deprotecting the amino-protecting group R ₈ .

[00127] The term“leaving group” as used herein refers to a moiety that is released from a molecule it was covalently bonded to by keeping the pair of electrons previously forming the bond. A leaving group can be a single atom, a molecule, or a functional group. These groups can be an anion or a neutral molecule. The leaving group may have a -I effect. The leaving group may be selected from the group consisting of a halogen, a mono-, di or trihalogenated acetate, and a mono-, di-, tri-, tetra- or pentahalogenated phenol. Alternatively, it may include conventional leaving groups such as -OSO2C4F9, -OSO2CF3, -OSO2F, -OTs, and -OMs.

[00128] The choice of leaving group may depend on the hydroxy amino acid being attached to the first reactant of the Formula (IV). For example, in embodiments, wherein an amino acid having an unprotected amino acid is to be reacted with the compound of Formula (IV), a pentahalogenated phenol may be used preferentially. [00129] In particular embodiments of the leaving group, the halide may be a chloride. In other particular embodiments of the leaving group, the mono-, di or trihalogenated acetate may be a mono-, di or trifluorinated acetate. In other particular embodiments of the leaving group, the mono-, di or trifluorinated acetate may be a trifluoroacetate. When the leaving group is trifluoroacetate, the method of making the linker system may be shortened by some reaction steps, as the trifluoroacetate is commonly encountered as an intermediate during the synthesis. Hence, the reaction sequence for the preparation of the first reactant of the Formula (IV) may be considerably shortened by using trifluoroacetate as the leaving group.

[00130] The leaving group may also be a mono-, di-, tri-, tetra- or pentafluorinated phenol. In these embodiments, the mono-, di-, tri-, tetra- or pentafluorinated phenol may be a pentafluorinated phenol (-O-C6F5).

[00131] In some embodiments, the reaction step proceeds in the presence of a base. Bases may include inorganic bases, organic nitrogen bases, or a combination thereof. The inorganic bases may include inorganic carbonate; inorganic phosphate; inorganic acetate; or a combination thereof. Nitrogen bases may include trialkyl amine; dialkyl amine; N,N- dialkylpyridine; N,N-dialkylaniline; or a mixture thereof. In another preferred embodiment the base is selected from the group consisting of diethylamine, di-n-butylamine, pyrrolidine, piperidine and other dialkylamines, triethylamine, tri-n-butylamine, diisopropylethylamine, dicyclohexylmethylamine and other trialkylamines; N-methyl-2,2,6,6,tetramethylpiperidine; 2,2,6,6,tetramethylpiperidine; pyridine; 2,6-dimethylpyridine (lutidine); 2,6-di-tert- butylpyridine; DABCO (l,4-Diazabicyclo[2.2.2]octane); 4-aminopyridine, and a combination thereof. Advantageously, by using a weak nitrogen base, such as 2,6-dimethylpyridine (lutidine), 2,4-dimethylpyridine (2,4 lutidine), 2,4,6-trimethylpyridine (TMP, collidine), 4- methylpyridine, 2-methylpyridene, or a combination thereof, it is possible to attach the hydroxy amino acid or the amino alcohol to the linker system without substantial loss of enantiomeric purity of the hydroxy amino acid or the amino alcohol.

[00132] The reaction step may proceed at a temperature above 50 °C. In some embodiments, the reaction step may proceed at a temperature above 60 °C, or above 70 °C, or above 80 °C, or below 300 °C, or below 200 °C, or below 150°C.

[00133] In some embodiments, the reaction step may be carried out in a microwave. The reaction step may be carried out for a time period of about less than one hour. In particular embodiments, the reaction step may be carried out for a time period of about less than 30 minutes, or of about less than 20 minutes, or of about 15 minutes. The time period required for the attachment of the hydroxy amino acid to the linker system may therefore be considerably shorter than the time reported in the literature, being 6 to 12 hours.

[00134] In a third aspect, a method of synthesizing a peptide alcohol is provided. The method may comprise

(i) Providing a solid- supported hydroxy amino acid or a solid-supported amino alcohol as described before, and

(ii) Carrying out at least one peptide coupling step with an amino acid or an amino acid derivative.

[00135] In this aspect, it is understood that the method further comprises several deprotection and protection steps, as is customary in the art of solid phase peptide synthesis.

[00136] In some embodiments, the method of synthesizing a peptide alcohol may comprise obtaining at least one reaction product from step (ii) according to Formula (VII)

wherein the carbon atom marked with an asterisk *, R ₁ , R ₂ , R ₄ , R ₅ , R ₆ ,

R ₇ , A, B and n are defined as herein before, R ₈ is an amino-protecting group; each R ₉ is independently hydrogen or C _1-20 alkyl; each AA is independently an optionally protected amino acid residue or a terminal C _2-20 alkene, p is an integer selected from 1 to 100;

or an enantiomer, a diastereomer, a salt or a solvate thereof.

[00137] In some embodiments, AA may be an optionally protected amino acid residue. In those embodiments, R ₉ , for the particular amino acid, may be hydrogen. In other embodiments, AA may be a terminal C _2-20 alkene. In those embodiments, R ₉ , for the particular amino acid, may be C _1-20 alkyl, in particular methyl. A terminal C _2-20 alkene, in this context, is meant to include an alkene, wherein the double bond is a terminal moiety. Hence, AA may be -C _0-18 -CH=CH ₂ . In some embodiments, p may be an integer selected from 1 to 80, or from 1 to 60, or from 1 to 40, or from 1 to 20.

[00138] In some embodiments, the method may further comprise a loading of the hydroxy amino acid or the amino alcohol on the solid support to be lower than 0.8 mmol/g, optionally lower than 0.6 mmol/g, or in the range of about 0.1 mmol/g to about 0.5 mmol/g, or about 0.2 mmol/g to about 0.4 mmol/g. In general, the loading obtained for the linker systems using the previously described methods is between 0.5 to 0.6 mmol/g which is a good range to obtain peptide alcohols with no more than 25 residues. However for longer peptide alcohols, advantageously, it may be beneficial to use a low loading resin, in order to avoid entanglement of the peptide chains. Hence, in order to increase the scope of the method, a low loading amino alcohol resin can be obtained starting with a low loading of about 0.1 mmol/g to about 0.3 mmol/g.

[00139] The peptide coupling step may be carried out using suitable peptide coupling reagents. In one embodiments, the at least one peptide coupling step (ii) may comprise reaction with a carbodiimide. The carbodiimide may be selected from the group consisting of dicyclohexylcarbodiimide (DCC), diisoprop ylcarbodiimide (DIC) and 1 -ethyl-3 -(3- dimethylaminopropyl)carbodiimide (EDC). The at least one peptide coupling step (ii) may further comprise an oxime. The oxime may be ethyl cyanohydroxyiminoacetate.

[00140] The method may be carried out in a microwave. In particular embodiments, the peptide coupling step (ii) may be carried out in a microwave peptide synthesizer.

[00141] The method may further comprise a step (iii) of undertaking a ring-closing metathesis (RCM). The RCM of step (iii) may be conducted in the presence of a catalyst and an organic solvent. The catalyst may be a non-anchored catalyst. It may be a non-anchored alkylidene catalyst. It may comprise ruthenium. It may for example be any one of a Grubbs I catalyst, a Grubbs II catalyst, a Ho veyda- Grubbs I catalyst, a Ho veyda- Grubbs II catalyst, a Grubbs Z catalyst or a mixture of any two or more of these. The RCM, step (iii), may use a single aliquot addition of catalyst, or it may use multiple aliquots of fresh catalyst added to the reaction mixture. It may use 2, 3, 4, 5 or more than 5 aliquot additions. The solvent present for the metathesis reaction may be a halogenated alkane. It may be dichloroethane. The metathesis reaction may be conducted at a temperature between about 15 °C and about 30 °C. It may be conducted at a room temperature. It may be conducted at a temperature between about 40 °C and about 60 °C. After RCM, the method may further comprise a step of cleaving the peptide alcohol from the linker system and purifying the peptide alcohol thus obtained.

[00142] In a fourth aspect, a peptide alcohol may be provided. The peptide alcohol may be obtained by the method of the third aspect. In one embodiment, that peptide alcohol may be octreotide.

[00143] In case a RCM has been carried out, it is possible to obtain a stapled peptide alcohol. Hence, in a fifth aspect, a stapled peptide alcohol synthesized by the method described above is obtained.

[00144] In a sixth aspect, a stapled peptide alcohol is provided. The stapled peptide may have the following Formula (VIII)

wherein

A, B and the carbon atom marked with an asterisk * are as defined in claim 1,

P ¹ and P ² are each independently an oligopeptide chain or a polypeptide chain, wherein P ² has an optionally protected terminal amino group,

each q is independently an integer between 0 and 12;

r is an integer between 1 and 8;

C is an amino acid or an amino acid derivative; and is a carbon-carbon single bond that is attached to a carbon atom of the double bond such that the compound of Formula (VIII) is in either the (E) -configuration or the (Z) configuration or is a mixture of these; or an enantiomer, a diastereomer, a salt or a solvate thereof.

[00145] In the compound of Formula (VIII), r may be an integer between 1 and 6, or between 3 and 6, 2 and 5, 3 and 4, 1 and 5 or 2 and 4, e.g., it may be 1, 2, 3, 4, 5 or 6. Each C may independently be a naturally occurring L-a-amino acid, or at least one C may be an unnatural amino acid. R ₉ may both be methyl.

[00146] Both P ¹ and P ² may comprise at least one naturally occurring L- a-amino acid. Both P ¹ and P ² may comprise at least one unnaturally occurring amino acid. The stapled peptide alcohol of Formula (VIII) may have P ¹ , P ² and C groups that independently comprise a single amino acid, an oligopeptide chain, or a peptide chain. Each amino acid in each of the P ¹ , P ² and C groups in each compound may be a natural L-a-amino acid or an unnatural amino acid or a derivative thereof. It is understood by the person skilled in the art that the P ¹ , P ² and C groups are, when they are connected to other parts of the compound of Formula (VIII), they are so connected via a peptide bond. It is also understood that P ² , which has a terminal part, that this terminal part is either a free amino or an amino -protected moiety. The number of amino acid residues in the C group is defined by r. The stapled peptide alcohol of Formula (VIII) may have r between 1 and 6. It may have a chiral centre on that carbon atom to which each of the R9 group is bound. Each chiral centre may independently be either (R) or (S). Each R9 group may for example be independently methyl, ethyl, propyl or pentyl. Each R9 group may be a branched alkyl group, for example isopropyl, sec -butyl, tert-butyl or sec-pentyl. Each R9 group may be a cycloalkyl group, for example cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

[00147] The stapled peptide alcohol of Formula (VIII) may have a combined number of amino acid units (i.e., the sum of the number of amino acid units of P ¹ + P ² + r + 2) that is between 5 and 20, or between 5 and 15, 10 and 20, 5 and 10 or 10 and 15, e.g., it may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[00148] In a seventh aspect, a method of preventing or treating cancer is provided. The method may comprise administering an effective amount of the stapled peptide alcohol as described herein to a mammal.

[00149] In an eighth aspect, a stapled peptide alcohol as described herein is provided for use in therapy.

[00150] In a ninth aspect, use of a stapled peptide alcohol as described herein in the manufacture of a medicament for the prevention or treatment of cancer is provided.

[00151] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00152] Hereinafter, the present disclosure will be described more fully with reference to the accompanying drawings, in which exemplary embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In the drawings, lengths and sizes of layers and regions may be exaggerated for clarity.

EXAMPLES

[00153] The production of Fmoc-amino alcohols resins starts with the incorporation of all commercially available Fmoc protected amino acid alcohols (around 15 Fmoc amino acid alcohols) to two different matrixes:

• polystyrene (PS)

• polyethylene glycol (PEG)

This technology also allows the use of at least three different linkers:

• Rink

• Ramage

• Sieber

The incorporation of the amino alcohols proceeds without epimerization. Also it provides the opportunity for commercializing at least 15 different pre-loaded Fmoc-amino acid per type of resin.

[00154] Example 1: Synthesis of the Rink Chloride resin

The Rink resin was synthesized according to the following reaction sequence:

[00155] To a previously swelled Rink-OH resin in THF (0.280g., 0.25 mmol.) was added triphenylphosphine (0.630g, 2.5mmol.) dissolved in 3 ml of THF and hexachloroethane (0.592 g., 2.5 mmol.) also in 3ml of THF. The mixture was agitated by shaking for 10 to l2h. The resin was filtered and washed with THF and was used in the next step.

[00156] Example 2: Procedure to attach the Fmoc-amino acid alcohol

The amino acid alcohol was attached according to the following reaction

[00157] The Rink-chloride resin (0.2g, 0.10 mmol) was placed in a lOml microwave tube (G10), DMF (2mL) was added, then Fmoc-Phenylalaninol (373.0 mg lmmol) dissolved in 2ml of DMF was added to the resin. Lutidine (2,6-dimethylpyridine, l28ul, 1 lmmol) was added to the mixture. The reaction was placed in a Monowave 300 Anton Paar microwave at 90C for l5min. After the reaction the Fmoc protection was removed using 20% piperidine for 15 min. The resin was washed with DMF 5ml x4. A positive Kaiser test was obtained after the deprotection step.

[00158] Commercially available Fmoc-amino alcohols:

The following commercially available amino alcohols (Advanced Chemtech/Creosalus Louisville KY, USA) can be incorporated using this method without loss of configuration (no racemization).

[00159] Example 3: Synthesis of the peptide model Leu-Ala-Arg-Gly-Tyr-AA-ol Several peptide alcohols models have been synthesized using the following resins:

The following Table shows the peptide alcohols synthesized so far with six different Fmoc- amino alcohols:

[00160] Linkers tested for the synthesis of peptide alcohols (PA): The following linkers on polystyrene matrix were successfully tested for the synthesis of peptide alcohols. The incorporation of the Fmoc-amino acid alcohol was performed in the same manner previously described for the rink-chloride linker.

[00161] The Sieber-Cl

The Sieber chloride and Ramage chloride was obtained using the same protocol previously described for the Rink-Chloride.

[00163] 9-hydroxy-9-(4-carboxyphenyl)xanthene -derived resin

In order to obtain peptide alcohols, an additional xanthene system can be used. 9-hydroxy-9- (4-carboxyphenyl)xanthene can be attached to a tentagel resin and chlorinated later using the same approach. [00164] Fmoc Amino alcohol Ramage-PS Resin:

The linker Ramage was also tested for the production of resins and for the synthesis of peptide alcohols:

LINKER= Ramage

SUPPORT= Polystyrene

[00165] Example 4: Synthesis of Fmoc Phenylalaninol Ramage-PS Resin

The above scheme illustrates the synthetic pathway to the Fmoc Phenylalaninol Ramage-PS Resin.

[00166] Example 5: Synthesis of LARGY-Phe-ol using the Fmoc Phenylalaninol Ramage-PS Resin

FARGYG-Phe-ol was synthesized on the Fmoc-Phe-ol-Ramage-PS resin:

The successful synthetic result is depicted in FIG. 2, showing a single HPLC peak. [00167] Example 6: Synthesis of ASTP704l-ol on Fmoc-Alaninol-Ramage-PS

Fmoc-Ala-ol-Ramage-PS Loading= 0.5-0.6mmol/g

The successful synthetic result is depicted in FIG. 3A, showing the LCMS trace with the desired molecular weight. The following Table illustrates the identified masses therein:

An HPLC graph is depicted in FIG. 3B. The following Table illustrates the identified peaks therein:

[00168] Example 7: ASTP704l-peptaibol-like peptide using the Fmoc Alaninol Ramage-PS Resin

[00169] ASTP7041 is a peptaibol-like peptide having the following structure and molecular weight:

This peptaibol-like peptide was synthetized using the Fmoc Alaninol Ramage-PS resin. The successful synthetic result is depicted in FIG. 4A, showing the LCMS trace with the desired molecular weight, as illustrated in the Table below:

An HPLC graph is depicted in FIG. 4B. The following Table illustrates the identified peaks therein: [00170] Fmoc Amino alcohol Sieber-PS Resin: It was possible to attach an Fmoc- protected amino alcohol to the Sieber-PS resin, as shown in the following example.

[00171] Example 8: Fmoc-Phenylalaninol-Sieber-PS resin

The Fmoc-Phenylalaninol-Sieber-PS resin was obtained according to the following reactions:

The above scheme illustrates the synthetic pathway to the Fmoc-Phenylalaninol-Sieber-PS resin. [00172] Synthesis of LARGY-Phenylalaninol using Fmoc Amino alcohol Sieber-PS Resin:

The peptide model LARGY-Phe-ol was also obtained using this resin.

[00173] Example 9: Matrices (solid supports) tested for the synthesis of peptide alcohols (PA)

In addition of the polystyrene, the polyethylene glycol matrix was successfully tested for the synthesis of peptide alcohols. It was possible to produce Fmoc-amino alcohols resins on the two solid supports used in peptide synthesis, polystyrene (PS) and polyethylene glycol (PEG). Both supports work for the synthesis of Fmoc-amino alcohols resins and subsequently for the synthesis of any peptide alcohol. In order to increase the scope of this technology, an additional linker and solid support was tested. The linker chosen was Ramage (NH ₂ -Suberol), shown below.

[00174] Ramage linker

[00175] The next solid support tested was an amino-methyl-PEG resin (shown below) that can be functionalized with a wide range of linkers. This resin was chosen because it is a 100% polyethylene glycol resin (PEG-resin). If both, polystyrene and a polyethylene glycol (PEG) supports can produce Fmoc-amino alcohols resins, all the solid supports used in solid phase peptide synthesis (SPPS) are covered by the present disclosure.

The PEG solid support is shown below, attached to the Ramage linker:

[00176] The following scheme was used for the synthesis of Fmoc-Phe-ol-PEG solid support resin:

The above scheme illustrates the successfully carried out synthetic pathway to the Fmoc-Phe- ol-PEG solid support resin.

[00177] The resin was tested in a peptide model to make the following peptide:

The peptide was successfully synthesized in an automated microwave peptide synthesizer using the standard protocol (DIC/ethyl cyanohydroxyiminoacetate). The successful synthesis result is depicted in FIG. 5A, showing an HPLC trace and in FIG. 5B showing the LCMS result.

[00178] Example 10

[00179] A second approach was tried bypassing the hydrolysis of the trifluoroacetate resin and the chlorination. The reaction between the trifluoroacetate resin and Fmoc-amino alcohol (Fmoc-Phe-ol) was attempted on the same microwave conditions used before in Example 2. The resin was dried and the synthesis of the same peptide model was performed in an automated microwave peptide synthesizer. The peptide was cleaved from the resin obtaining the same result previously observed using the chlorinated resin.

This scheme illustrates the synthesis of Fmoc-Phe-ol-PEG solid support resin directly from the trifluoroacetate resin. The successful synthesis result is depicted in FIG. 6, showing the HPLC trace. [00180] Example 11: Loading experiments

[00181] In general, the loading obtained for the resins using the previously described methods is between 0.5 to 0.6 mmol/g which is a good range to obtain peptide alcohols with no more than 25 residues. However for longer peptide alcohols is necessary to use a low loading resin. In order to increase the scope of the disclosed method, a low loading amino alcohol resin can be obtained starting with a low loading amino functionalized polystyrene (PS) or Polyethylene glycol (PEG) solid support according to the following reactions (see scheme).

[00182] Starting with 4’-hydroxy-2,4-dimethoxybenzophenone (CAS Number 41351- 30-8).

[00183] To a solution of 4’-hydroxy-2,4-dimethoxybenzophenone 14 g, 0.054 mol) in acetone (60 mL) at room temperature benzyl-bromoacetate was added (13.5 g, 0.059 mol). The mixture was stirred at rt for 16 h.

[00184] Hydrolysis:

[00186] To a solution of the acid (0.98 g, 3.09 mmol) in i-PrOH (20 mL) were added TEA (0.313 g, 3.09 mol) and NaBH ₄ (0.584 g, 15.43 mmol). The mixture was refluxed for 2 h. After evaporation of the solvent, water was added to the residue. The resulting mixture was acidified with 3% HC1 (pH 4.0) and then extracted with AcOEt.

[00187] The organic layer was washed with brine, dried over anhydrous MgS0 ₄ , and evaporated to give the crude alcohol.

[00188] The acid was attached to the amino solid support using 5eq of the acid and 5eq of the DIC in DMF for 5 hours.

[00189] The alcohol attached to the resin was chlorinated using the same method previously described. The protected amino alcohol was added using the same methodology previously used. The loadings obtained using the procedure are around 0.2 mmol/g.

[00190] Example 12: Optical purity

[00191] The optical purity was investigated during the synthesis of LARGY-Phe-ol. The loss of configuration during the attachment of the Fmoc-amino alcohol to the solid support was investigated. Using 5eq of Fmoc-phenylalaninol and 10 eq of diisopropylethylamine (DIEA). After the synthesis of the previous mentioned peptide alcohol it was observed a loss of configuration of approximately 70% determined by HPLC integration (see, FIG. 7).

[00192] After several experiments it was found the loss of configuration was totally suppressed using 5.5 eq of 2,6 lutidine as base during the attachment of the Fmoc-amino alcohol (e g Fmoc-Phenylalaninol). The result is shown in FIG. 8.

[00193] This provides a robust procedure of preloading Fmoc or BOC amino alcohols to a matrix (PS or PEG) having Rink, Ramage and Sieber as linkers, as shown in the list below.

00194] Example 13: Synthesis of Octreotide

[00195] Using this resin with ethyl cyanohydroxyiminoacetate/DIC in a microwave peptide synthesizer, 165mg (~70% yield) of the reduced form of Octreotide was obtained in a good quality in 70 min. The successful synthesis result is depicted in FIG. 9A, showing the HPLC trace, and in FIG. 9B, showing the desired LCMS peak. This demonstrates that the resins are versatile.

[00196] Example 14: Synthesis of Trichogin

[00197] Using this resin, Trichogin was obtained. The successful synthesis result is depicted in FIG. 10, showing the desired LCMS peak.

[00198] Example 15: Synthesis of Alamethacin

Using this resin, Alamethacin was obtained. It was synthesized in the same way, using ethyl cyanohydroxyiminoacetate/DIC on a microwave synthesizer. An LCMS is depicted in FIG. 11A. Albeit this MS not looking very clean, two majors masses, 775.76 and 1204.12 are presented along with the alamethicin molecular weight. This peptide was cleaved from the resin using 95% TFA, TIS and water 2.5 % each for two hours. Trying to find the origin of those additional peptides (775.76 and 1204.12) it was found in the literature that in order to avoid the hydrolysis of Aib-Pro amide bond (as shown in FIG. 11B), the cleavage cocktail used for cleaving alamethicin is 50% TFA in DCM. If the only problem with the synthesis is the cleavage, the two additional masses observed in the MS should correspond to the peptides formed after the hydrolysis of the Aib-Pro linkage. In order to add more evidence to the fact that Aib-Pro bond is susceptible towards hydrolysis and the fact that the combination of the resin in a microwave-assisted synthesis with ethyl cyanohydroxyiminoacetate /DIC is very effective for difficult couplings, a segment of Alamethicin A30 was synthesized. The LCMS of the fragment is shown in FIG. 11C and the finding regarding the cleavage was verified. [00199] Summarizing Examples 13 to 15, a microwave-assisted solid phase synthesis of the following peptide alcohols using the resin in combination with ethyl cyanohydroxyiminoacetate and DIC was successfully shown:

• Octreotide

• Alamethicin (an important antibiotic petaibol)

• Trichogin GA IV (Tinidazole) fragment (without the octyl side chain at the N- terminus); and

• Alamethicin fragment K.

The synthesis of all those molecules took between 60min to 90min depending of the length of the peptide.

[00200] Example 16: Fmoc amino protected alcohols attached to the resin can be used for the synthesis of fully protected peptides alcohols

[00201] As an example, RESKY-Phe-ol fully protected was synthesized, denoted as follows: H-Arg(Pbf)-Glu(OtBu)-Ser(tBu)-Lys(BOC)-Tyr(tBu)-Phe-ol and shown below.

[00202] In this example, the most common protecting groups used during the Fmoc/tBu chemistry were incorporated, e. g. 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for Arginine, tertbutyloxycarbonyl (BOC) for Lysine and tert-Butyl for Tyrosine and Glutamic acid. After the synthesis, the fully protected peptide alcohol was cleaved from the resin using a solution 20% Hexafluoroisopropanol (HFIP) in dicholoromethane (DCM) for two hours. The solution was concentrated and the peptide was precipitated using diethyl ether.

[00203] The peptide crude was analysed by MS, shown as FIG. 19A (M+l= 1336 cal= 1335.71), and HPLC, shown as FIG. 19B, with a purity around 91%. [00204] Example 17: Fmoc amino protected alcohols attached to the resin can be used for the synthesis of fully protected peptides amides when the serine is the first residue in the synthesis. The fully protected peptide amide using the resin Fmoc-Ser(PS-Rink)-NH ₂ was synthesized:

[00205] As an example, RESKY-Ser-NH ₂ fully protected was synthesized using the following Fmoc-Ser(PS-Rink)-NH ₂ :

H-Arg(Pbf)-Glu(OtBu)-Ser(tBu)-Lys(BOC)-Tyr(tBu)-Ser-NH ₂ All protecting groups previously described were used for the synthesis of the peptide alcohol mentioned above. After the synthesis, the fully protected peptide amide was cleaved from the resin using a solution of 20% Hexafluoroisopropanol (HFIP) in dicholoromethane (DCM) for two hours. The solution was concentrated and the peptide was precipitated using diethyl ether. The peptide crude was analysed by MS, as shown in FIG. 20A (M+1 =1290, cal=l288.6l), and HPLC as shown in FIG. 20B, with a purity around 91%.

[00206] Example 18: Fmoc amino protected alcohols attached to the resin can be used for the synthesis of peptides esters when tyrosine is the first residue in the synthesis.

[00207] The following peptide methyl ester using the resin Fmoc-Tyr(PS-Rink)-OMe was synthesized:

The Fmoc-Tyr(PS-Rink)-OMe has the following structure:

[00208] The Fmoc-Tyr(PS-Rink)-OMe was used to synthesize the following stapled peptide name VIP65-OMe. After standard Fmoc/tBu chemistry, the peptide was cleaved from the resin using a cocktail of TFA, TIS, water 95:2.5:2.5% for two hours. The peptide was isolated by precipitation using ether. The peptide methyl ester was analysed by MS, as shown in FIG. 21A (M+l =1885, cal 1884.16), and HPLC, as shown in FIG. 21B, with a purity around 90%.

[00209] Herein, it was verified that a number of hydroxy amino acids and amino alcohols can be loaded on the linker system. The loadings are about 0.7 mmol/g. The peptides or peptide alcohols can be subsequently synthesized in one pot manually or by using a peptide synthesizer. The solid-supported hydroxy amino acids and solid-supported amino alcohols are stable at 4 °C. It was also shown that any solid support may be used for the synthesis of the solid- supported hydroxy amino acids and solid- supported amino alcohols. Further Examples illustrate the wide applicability of the technology.

[00210] Example 19: Synthesis of Stapled Peptide Alcohols and Their Activity as Mdm2 Inhibitor

[00211] The use and testing of peptide alcohols (PAs) in SAR is limited because of the lack of commercially available resins that enable their reliable and fast synthesis. It is well known that peptide C-terminus can have a strong influence on the activity. The solid-supported amino alcohol provides a great opportunity to evaluate the effect of changing the amide or carboxylic group to their reduced form (alcohol). Here there is presented a solid phase synthesis of stapled PAs and a comparison with their peptide amide counterparts. The change of the C- terminal amide to alcohol increases the hydrophobicity and doubles the p53 activation over their C-terminus amide counterparts. Even though the amide and PAs have similar K _d , an increase in hydrophobicity can enhance the activation of p53 by making the PAs more cell permeable.

[00212] Stapled peptides are peptides wherein the side chains are stapled together by a bridge. All-hydrocarbon staples peptides have been extensively used to stabilize a-helical conformations, whereby the all-hydrocarbon bridge enhances the bioactivity, creating a new generation of functional probes and potential therapeutics. The well-known stapled peptides are formed using the i,i+3 ; i,i+4 and i,i+7 positions of a peptide. Those positions correspond to approximately 3.6 residues (one turn of the a -helix) for the i,i+3 and i,i+4 and two turns for the i,i+7. In all those cases, the C-terminus are acids or amides. No stapled peptide alcohol is reported in the literature in which the C-terminus is an alcohol. The effect of having a C- terminus alcohol is unknown.

[00213] The use of peptide alcohols (PAs) in Structure-Activity Relationship (SAR) studies is not wide spread, one of the reasons is the lack of easy and reliable Fmoc/tBu methodology. One of the methods already reported involved the loading of Chloro-trityl resin with a Fmoc amino alcohol. However the method has not been generalized for all commercially available Fmoc amino alcohols. The inconsistency of the loading, and the time consuming process related to it, tends to limit the synthesis and use of PAs.

[00214] Using the hydroxy alcohol resins, it was possible to synthesize several i,i+7 stapled peptide alcohol (SPALs) analogs of the most active amide counterparts. For the synthesized stapled peptide alcohols, the following terminology was utilized accompanying the following results:

[00215] L- VIP65-ol (also known as VIP65 SPAL) with a sequence Ac-

TSF(R8)EYWALL(S5)-ENF-ol has a better EC50 than the amidated version (see FIG. 14B)

[00216] II.- VIP82-ol (also known as VIP82 SPAL) with a sequence Ac-KK-Ahx- TSF(R8)EYWALL(S5)-ENF-ol has a better EC50 than the amidated version (see FIG. 14B).

[00217] III.- VIPI I6-oI (also known as VIP116 SPAL) with a sequence Ac-K-Ahx- TSF(R8)EYWALL(S5)-ENF-ol has a better binding to Mdm2 and Mdm4 than the amidated version (see FIG. 13B).

[00218] IV.- VIP1 l6-Pro-ol (also known as VIP116 SPAL) with a sequence Ac-K-Ahx- TSF(R8)EYWALL(S5)-ENF-CH ₂ -CH ₂ -CH ₂ -OH has a better EC50 than the amidated version

(see FIG. 14B).

[00219] V.- VIPl9-ol (also known as VIP 19 SPAL) with a sequence Ac-

TSF(R8)EYWALL(S5)-E-ol has a better EC50 than the amidated version (see FIG. 14B).

[00220] VI.- ATSP704l-ol (also known as ATSP7041 SPAL) with a sequence Ac- LTF(R8)EYWAQ(Cba)(S5)-SAA-ol has a better binding to Mdm2 and Mdm4 than the amidated version (see FIG. 13D) (Cba= Cyclobutyl-alanine). Further examples may be found in FIG. 13E.

[00221] In all the cases it was found that the Stapled Peptide Alcohol (SPAL) is more hydrophobic than the Stapled peptide amide analog (FIG. 12).

[00222] All SPALs were synthesized using the hydroxy amino acid resins and amino alcohol resins. The Fmoc-tryptophan was not Boc protected. Automated peptide synthesis was performed on CEM Liberty Blue microwave peptide synthesizer using Fmoc-protected amino acids (5 equiv.) Diisopropyl carbodiimide (DIC) as a coupling reagent and ethyl cyanohydroxyiminoacetate as an additive. The peptides were synthesized using standard protocols on the CEM instrument and then cleaved from the resin using a cocktail of 95:2.5:2.5, trifluoroacetic acid:triisopropyl silane:water for 2 h. The resin was removed by filtration and the resulting peptide solution was precipitated using ethyl ether. Ring-closing metathesis (RCM) of resin-bound, N-acetylated peptides was performed manually using a 5 mg/mL solution of Grubbs I catalyst (25 mol%) x3. It was found that all SPALs can be synthesized using the hydroxy amino acid resins and amino alcohol resins with a high purity and recovery. With this tool available for research, it is possible to extend the scope of any peptide research by adding peptide alcohols to the SAR. This technology allows to study the effect of having a C-terminus alcohol vs amides or acids in a very straightforward manner. Hence, a set of SPALs was synthesized based on the best stapled peptide amides (SPAMs). A comparison of the structures is shown in FIG. 18.

[00223] Even though almost all SPALs have less binding affinity to Mdm2 (see the K _d in FIG. 13A, FIG. 13B, FIG. 13C and FIG. 13D) those peptides show better EC50s in almost all the cases than the SPAMs (FIG. 14A and FIG. 14B). This finding suggests that the increase of hydrophobicity of the SPALs could help to have a better internalization and bioavailability.

[00224] SPALs and SPAMs activate P53 in a similar fashion on Acute Myeloid Leukemia cells (AML2). As a consequence of p53 activation, the downstream activation of the cyclin-dependent kinase inhibitor p2l indicates cell-cycle arrest on those cells (FIG. 17).

[00225] Activation of p53 cannot be related to cell membrane damage. According to the lactose dehydrogenase assay, all the SPAMs and SPALs do not cause a significant LDH release (FIG. 15A and FIG. 15B).

[00226] The p53 tumor suppressor is well known for its ability to trigger cell death by apoptosis, cell-cycle arrest or induce cellular senescence. The cleavage of Poly(ADP-ribose) polymerase (PARP1, H6kDa) by several caspases produces a fragment of 89kDa. The presence of this fragment is an indication of the cells going into apoptosis. The preliminary findings indicate due to the presence of P21 and the 89kDa fragment on the western blot some SPALs can trigger both, cell-cycle arrest and apoptosis (FIG. 17).

[00227] Example 20: Stability of the resins

[00228] Real Time Monitoring MT, 3000 cells per well in 384 well format, 3 replicates per point:

• 24 Jul 2018: peptide freshly purified and dissolved

• 17 Dec 2018: peptide used after 7 months of storage at -20°C

• 8 Jan 2019: peptide freshly dissolved after 8 months of storage in the powder form at - 20°C

• Peptide stocks: lOmM in 100% DMSO in all cases

[00229] Storage conditions seem to affect the activity of the peptide alcohols therefore some optimization may be needed

[00230] Caspase 3/7 Glo, 20,000 cells per well in 96 well format, 2 replicates per point: • 17 Jan 2019: peptide freshly purified and dissolved

• 24 Jan 2019: peptide used after 7 months of storage at -20°C

• 8 Feb 2019: peptide freshly dissolved after 8 months of storage in the powder form at - 20°C

• Peptide stocks: lOmM in 100% DMSO in all cases

[00231] Summarizing the stability experiments, it may be concluded, that keeping the resins at -20°C is the best. They may be used within 6 months.

[00232] There was developed a robust and facile synthesis of an all-hydrocarbon stapled peptide alcohols (i,i+7), using the solid- supported hydroxy amino acids and solid-supported amino alcohols. It was demonstrated with preliminary data that a small change in the peptide structure e.g. at the C-terminus from amide to alcohol, can drive a change in the biological activity. These solid- supported hydroxy amino acids and solid- supported amino alcohols facilitate and increase the scope of SAR in peptide research adding peptide alcohols as a routinely peptide synthesis in SAR.

[00233] Using the solid- supported hydroxy amino acids and solid- supported amino alcohols it was possible to see the difference of having a stapled peptide amide (SPAMs) or the Stapled peptide alcohols (SPALs) in which the only modification is the C-terminus of the peptide. In all the cases there was an increase in hydrophobicity compared to the SPAMs, which was noticed by the increase on the retention time in the HPLC. Higher hydrophobicity had an impact in the biological properties of the peptides (only modification is the C-terminus from amide to alcohol, see FIG. 18).

[00234] This tool for SAR in peptide research makes the evaluation of peptides alcohols possible in a very straightforward manner. Easy synthesis, high yield and purity of the crude peptide alcohols makes the peptide alcohols a common modification in any SAR of peptides.

[00235] Advantages of the present disclosure

I. This technology provides a robust procedure to obtain preloaded Rink-PS resins with Fmoc amino alcohols.

II. This technology provides a robust procedure to obtain preloaded Ramage-PS resins with Fmoc amino alcohols.

III. This technology provides a robust procedure to obtain preloaded Sieber-PS resins with Fmoc amino alcohols.

IV. This technology allows the commercialization of all the pre-loaded Fmoc amino alcohol resins previously described for research purpose. V. This technology provides a robust procedure to obtain preloaded Polyethylene glycol resins with Fmoc amino alcohols with all the previous linkers

VI. The PA synthesis can be done in one pot as a conventional solid phase peptide synthesis using all the above linkers and solid supports (matrixes).

VII. This technology provides a robust procedure to obtain preloaded benzhydryl-PS resins with Fmoc amino alcohols for BOC chemistry.

VIII. The resins having a Rink, Ramage and Sieber linkers, preloaded with Fmoc-amino alcohols can be released from the resin using mild conditions allowing the synthesis of peptaibols.

IX. This method facilitates the loading of phthalimide in order to be in the synthesis ofthalidomes peptides.

X. Well-known PAs like octreotide can be synthesized by this method opening the path for afast synthesis and SAR.

XI. This technology can be used for the synthesis of stapled peptide alcohols, providing an additional tool in the search of more potent Mdm2/p53 agonists.

XII This technology can be used for the synthesis of fully protected peptide alcohols, providing an additional tool in the synthesis long peptides by segment condensation.

XIII: This technology can be used for the synthesis of peptide C-teminus di-ols (glycols) providing an additional tool for the synthesis of fully-protected and non-protected stapled and non-stapled peptides aldehydes.