BARNESIN A, DERIVATIVES AND USES THEREOF

Abstract

This invention relates to a compound according to general formula (IA), which acts as a selective cysteine protease inhibitor; to a pharmaceutical composition containing one or more of the compound(s) of the invention; to a combination preparation containing at least one compound of the invention and at least one further active pharmaceutical ingredient; and to uses of said compound(s), including the use as a medicament.

##STR00001##

Claims

1. A compound of the general formula (IA): ##STR00067## or a pharmacologically acceptable salt thereof, wherein R.sup.1A represents a hydrogen atom, —OR.sup.11, —NR.sup.11R.sup.12; or a (C.sub.1-C.sub.6)alkyl, (C.sub.2-C.sub.6)alkenyl, (C.sub.2-C.sub.6) alkinyl, or (C.sub.3-C.sub.6) cycloalkyl group, all of which groups may optionally be substituted; R.sup.11 and R.sup.12 each, independently of one another, represents a hydrogen atom, or a (C.sub.1-C.sub.6)alkyl, (C.sub.2-C.sub.6)alkenyl, (C.sub.2-C.sub.6) alkinyl or (C.sub.1-C.sub.6)heteroalkyl group, all of which groups may optionally be substituted, or R.sup.11 and R.sup.12 together with the nitrogen atom to which they are attached form a 5- to 8-membered heterocyclic or heteroaromatic ring that can be substituted with from 0 to 3 substituents which substituents are each independently selected from halogen atom, —OH, —NH.sub.2, —NHC.sub.1-6 alkyl, and —N(C.sub.1-6 alkyl).sub.2; R.sup.2A is a hydrogen atom, a group of formula —C(═NH)NH.sub.2, or a group of formula —C(═O)R.sup.21; R.sup.21 represents a (C.sub.1-C.sub.6)alkyl, (C.sub.2-C.sub.6)alkenyl, (C.sub.2-C.sub.6) alkinyl or (C.sub.1-C.sub.6)heteroalkyl group; all of which groups may optionally be substituted; R.sup.3A is an amino acid side chain; a hydrogen atom, a halogen atom, OH; or an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl or heteroaralkyl group; all of which groups may optionally be substituted; R.sup.4A is a hydrocarbon group containing 1 to 12 carbon atoms or a heteroaryl group containing from 5 to 10 ring atoms, and, optionally, 1 to 3 H atoms in the hydrocarbon and the heteroaryl group may, independently of each other, be replaced by a halogen atom, OH, NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, —NH(OC.sub.1-3 alkyl), unsubstituted C.sub.1-C.sub.3alkyl, (C.sub.1-C.sub.3)haloalkyl, (C.sub.1-C.sub.3)hydroxyalkyl, or (C.sub.1-C.sub.3)alkoxy group; R.sup.5A and R.sup.6A each, independently of one another, represents a hydrogen atom or a methyl group; and p is an integer of from 1 to 6.

2. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.1A represents —NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, —NH(OC.sub.1-3 alkyl), —NCH.sub.3(OC.sub.1-3 alkyl), —NH(C.sub.1-3 alkyl)CN; ##STR00068## wherein each R.sup.7A is independently selected from halogen atom, —OH, —NH.sub.2, and —NHC.sub.1-3 alkyl, and q is an integer of from 0 to 3; a C.sub.1-3 alkoxy group; or —OH.

3. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.2A is a hydrogen atom, a group of formula —C(═NH)NH.sub.2, a group of formula —C(═O)CH.sub.3, or a group of formula —C(═O)CH.sub.2CH.sub.2CH.sub.3.

4. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.3A is an optionally substituted amino acid side chain of a proteinogenic amino acid; or a group of formula (II): ##STR00069## wherein R.sup.31 represents a (C.sub.1-C.sub.6)alkyl, (C.sub.2-C.sub.6)alkenyl, or (C.sub.2-C.sub.6) alkinyl group, all of which groups may optionally be substituted.

5. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.3A represents the amino acid side chain of tyrosine, a group of formula (II); or an amino acid side chain of phenylalanine, leucine or isoleucine, wherein 1 to 3 H atoms in the respective side chain group may, independently of each other, be replaced by a halogen atom, OH, NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, —NH(OC.sub.1-3 alkyl), unsubstituted C.sub.1-C.sub.3alkyl, (C.sub.1-C.sub.3)haloalkyl, (C.sub.1-C.sub.3)hydroxyalkyl, or (C.sub.1-C.sub.3)alkoxy group.

6. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein R.sup.4A is a C.sub.1-7 alkyl, C.sub.2-7 alkenyl, (C.sub.2-C.sub.7) alkynyl, cyclohexyl, phenyl, benzyl or pyridyl group; wherein 1 to 3 H atoms in said groups may, independently of each other, be replaced by a halogen atom, OH, NH.sub.2, —NHCH.sub.3, —N(CH.sub.3).sub.2, —NH(OC.sub.1-3 alkyl), unsubstituted C.sub.1-C.sub.3alkyl, (C.sub.1-C.sub.3)haloalkyl, (C.sub.1-C.sub.3)hydroxyalkyl, or (C.sub.1-C.sub.3)alkoxy group.

7. The compound according to claim 1, or a pharmacologically acceptable salt thereof, wherein p is 3 or 4.

8. The compound according to claim 1, wherein R.sup.5A and R.sup.6A represent a hydrogen atom.

9. The compound according to claim 1, wherein the compound is: ##STR00070## ##STR00071## ##STR00072## ##STR00073## ##STR00074## ##STR00075## ##STR00076## ##STR00077## or a pharmacologically acceptable salt thereof.

10. A pharmaceutical composition comprising at least one compound according to claim 1 and, optionally, one or more carrier substance(s), excipient(s) and/or adjuvant(s).

11. A combination preparation containing at least one compound according to claim 1 and at least one further active pharmaceutical ingredient.

12. The compound according to claim 1 for use as a medicament.

13. The compound according to claim 1 claim 10 claim 11 for use in the prevention and/or treatment of a condition or disorder associated with a pathophysiological level of a proteasome or a cysteine protease.

14. The compound for use according to claim 13, wherein the condition or disorder associated with a pathophysiological level of a proteasome or a cysteine protease is a neurodegenerative disorder, a parasitic infection, an invasive cancer, or a metastatic cancer.

15. The compound according to claim 1, or the pharmaceutical salt thereof, for use as an inhibitor of a proteasome or a cysteine protease.

16. A synthetic nucleic acid comprising a sequence encoding a nonribosomal peptide-synthetase (NRPS)-polyketide synthase (PKS) gene cluster capable of synthesizing compound (1) of claim 9, wherein the sequence has a sequence identity to the full-length sequence of SEQ ID NO. 1 from at least 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% to 100%.

17. A method for the preparation of compound (1) of claim 9, the method comprising the steps of: (a) fermenting Sulfurospirillum barnesii (DSM 10660); and (b) separating and retaining the compound according to general formula (I) from the culture broth.

18. A method of treating a subject who is suffering from or susceptible to a condition or disorder associated with a pathophysiological level of a proteasome or a cysteine protease, comprising administering to a patient in need thereof an effective amount of a compound of claim 1.

19. A method of treating a subject who is suffering from or susceptible to a neurodegenerative disorder, a parasitic infection, an invasive cancer, or a metastatic cancer, comprising administering to a patient in need thereof an effective amount of a compound of claim 1.

Description

EXAMPLES

General Experimental Procedures

[0107] NMR measurements were performed on a Bruker AVANCE II 300 MHz, Bruker AVANCE III 500 MHz and a Bruker AVANCE III 600 MHz spectrometer, equipped with a Bruker Cryoplatform. The chemical shifts are reported in parts per million (ppm) relative to the solvent residual peak of DMSO-d.sub.6 (.sup.1H: 2.50 ppm, quintet; .sup.13C: 39.52 ppm, heptet), CDCl.sub.3 (.sup.1H: 7.26 ppm, singlet; .sup.13C: 77.16 ppm, triplet) and MeOD-d.sub.4 (.sup.1H: 3.31 ppm, quintet; .sup.13C: 49.00 ppm, heptet). LC-ESI-HRMS measurements were carried out on an Accela UPLC system (Thermo Scientific) coupled with a Accucore C18 column (100×2.1 mm, particle size 2.6 μm) combined with a Q-Exactive mass spectrometer (Thermo Scientific) equipped with an elecrospray ion (ESI) source. UHPLC-MS measurements were performed on a Shimadzu LCMS-2020 system equipped with single quadrupole mass spectrometer using a Phenomenex Kinetex C18 column (50×2.1 mm, particle size 1.7 μm, pore diameter 100 Å). Column oven was set to 40° C.; scan range of MS was set to m/z 150 to 2,000 with a scan speed of 10,000 u/s and event time of 0.25 s under positive and negative mode. DL temperature was set to 250° C. with an interface temperature of 350° C. and a heat block of 400° C. The nebulizing gas flow was set to 1.5 L/min and dry gas flow to 15 L/min. Semi-preparative HPLC was performed on a Shimadzu HPLC system using a Phenomenex Luna C18(2) and phenyl-hexyl 250×10 mm column (particle size 5 μm, pore diameter 100 Å), or a Biotage Isolera Prime. IR spectra were recorded on an FT/IR-4100 ATR spectrometer (JASCO). Optical rotations were recorded in MeOH on a P-1020 polarimeter (JASCO). Solid phase extraction was carried out using Chromabond C18ec cartridges filled with 1 g and 10 g of octadecyl-modified silica gel (Macherey-Nagel, Germany). PCR was performed on a Peqstar 2× Gradient cycler.

[0108] Chemicals: Methanol (VWR, Germany); water for analytical and preparative HPLC (Millipore, Germany), formic acid (Carl Roth, Germany); acetonitrile (VWR as LC-MS grade), media ingredients (Carl Roth, Germany). All reagents and solvents for synthesis were purchased from Acros Organics, Alfa Aesar, Carbolution Chemicals, Carl Roth, Sigma Aldrich, TCI, Th. Geyer and VWR and used without further purification.

Example 1 Biosynthesis and Biophysical Analysis of a Compound According to General Formula (I) of the Present Invention—Barnesin A (1)

[0109] Cultivation of Sulfurospirillum barnesii strain SES-3 (DSM 10660): Sulfurospirillum spp. were generally cultivated at 28° C. using the following conditions: For solid phase cultivation, S. barnesii was grown microoxically on R2A agar plates incubated in an anaerobic jar with approximately 0.2% oxygen in the gas phase for at least one week. When grown anaerobically, a defined mineral growth medium as described for S. multivorans was used with the following modifications: vitamin B12 (cyanocobalamin) and resazurine were omitted and pyruvate (40 mM) was used as electron donor and fumarate (40 mM) as electron acceptor. Small scale cultivations (100 mL) were performed in 200 mL rubber-stoppered serum bottles, large-scale cultivations in 2 L rubber-stoppered Schott bottles containing 1 L medium. Glass bottles used for cultivation were capped with Teflon-coated butyl rubber septa. Growth was monitored photometrically by measuring the optical density at 578 nm. Microaerobic cultivation with the aforementioned medium was performed using 2 L Schott bottles with an initial addition of 2% sterile air into the gas phase into the medium without fumarate as electron acceptor. Inoculation of the medium was performed with 10% of a preculture cultivated until the exponential phase.

[0110] Isolation and characterization: Cultures were centrifuged at 4000 rpm for 20 min and RT. The cell pellet was harvested and extracted using MeOH (4° C., overnight), cell debris was filtered off and methanolic extracts added to metabolites at a later step. The culture supernatant was mixed with activated HP20/XAD4 (1:1) resin and stirring overnight (4° C., 300 rpm). Then, the resin was filtered off, washed with 20% MeOH (if not mentioned otherwise, mixtures refers to MeOH in ddH.sub.2O) and metabolites eluted with 50% MeOH and 100% MeOH. Then methanolic cell pellet extracts were added to the 100% MeOH resin eluate. Combined extracts were concentrated under reduced pressure and redissolved in 10% MeOH. The crude extract was loaded on an activated and equilibrated SPE-C18 cartridge and fractionated using 50% and 100% MeOH. The concentrated 100% MeOH fraction was purified first by a semi-preparative HPLC over Luna C18(2) column to obtain subfractions (Fr. 1-34) using the following gradient: 0-3 min, 50% B, 3-30 min 50-100% B, 30-35 min 100% B, 35-41 min 100-50% B with a flow rate of 2.0 mL/min (A: ddH.sub.2O with 0.1% formic acid; B: MeOH). Concentrated fractions 10-12 were further separated by a second semi-preparative HPLC run over Phenyl-Hexyl column to yield pure barnesin A (1, 1.0 mg, t.sub.R=25.6 min) with the following gradient: 0-5 min 10% D, 5-38 min 10-75% D, 38-40 min 75-100% D, 40-45 min 100% D, 45-50 min 100-10% D (C: ACN; D: NH.sub.4OAc 20 mM, pH 7.0) with a flow rate of 2.0 mL/min. The samples were analyzed using analytical HPLC over Phenyl-Hexyl column (250×4.6 mm) or Luna C18(2) with the following gradient: 0-3 min 50% B, 3-30 min 50-100% B, 30-35 min 100% B, 37-40 min 100-50% B, 35-41 min 50% B (A: ddH.sub.2O with 0.1% formic acid; B: MeOH) (1, t.sub.R=14.38 min).

[0111] Marfey's reaction: Barnesin A (1, 0.2 mg) was hydrolyzed by 6 N HCl (1.0 mL) at 110° C. overnight (15 h). HCl was removed using SpeedVac (42° C.) and FDAA (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, 20 μL, 10 mg/mL in acetone) and NaHCO.sub.3 (100 μL, 1 M) were added. The reaction was performed under 80° C. for 10 min. The reaction was quenched by adding of HCl (50 μL, 2 N) and D-tyrosine reference were converted accordingly under the same condition. After centrifugation for 15 min at 13,000 rpm the supernatant was analyzed by LC-MS. 1 μL of the reaction was injected and analyzed using the following gradient: 0-1 min 10% D, 1-7 min 70% D, 7-10 100% D, 10-13.5 min 10% D (B: ddH.sub.2O with 0.1% formic acid; D: MeCN with 0.1% formic acid) at a flow rate of 0.7 mL/min.

[0112] Barnesin A (1): colourless solid; [α].sub.D.sup.25−5.76 (c 0.0910, MeOH); UV (MeOH): λ.sub.max 196; 214 nm; IR (ATR) v.sub.max: 1240, 1385, 1455, 1545, 1655, 2365, 2860, 2930, 3070, 3260 cm.sup.−1; ESI-HRMS: m/z: 488.2864 [M+H].sup.+; calcd. 488.2867.

[0113] Structure assignment: The molecular formula of barnesin A (1) was assigned as C.sub.25H.sub.37O.sub.5N.sub.5 based on ESI-HRMS analysis (m/z 488.2864 [M+H].sup.+, calcd. 488.2867, Δ=−0.69 ppm) and confirmed by the observation of 25 carbon signals from .sup.13C-NMR spectrum, including 11 olefinic or aromatic carbons, between δ.sub.C 114.8 ppm and δ.sub.C 157.4 ppm, three carbonyl carbons at δ.sub.C 164.7 ppm, 171.1 ppm and 171.4 ppm, three nitrogen-bearing carbons (δ.sub.C 39.9 ppm, 48.6 ppm, and 54.6 ppm), and eight aliphatic carbons between δ.sub.C 13.9 ppm and 37.4 ppm. Detailed analysis of the obtained 2D NMR spectra (DMSO-d.sub.6) revealed the structure of a modified di-peptides consisting of a tyrosine moiety, a modified arginine moiety and a fatty acid tail. The di-peptide was identified based on the presence of two α-carbons (δ.sub.H 4.47 ppm/δ.sub.C 54.6 ppm and δ.sub.H 4.34 ppm/δ.sub.C 48.6 ppm). The tyrosine moiety was assigned based on COSY correlations of NH(3) δ.sub.H 8.02 ppm) to H-10 δ.sub.H 4.47 ppm) to H-11 δ.sub.H 2.87/2.56 ppm, δ.sub.C 37.4 ppm), and correlations of the para-substituted aromatic protons H-13 δ.sub.H 6.99 ppm/δ.sub.C 130.0 ppm) to H-14 δ.sub.H 6.60 ppm/δ.sub.C 114.8 ppm). This assignment was confirmed by the HMBC correlations of H-10 to C-9/C-11/C-12, H-11 to C-9/C-10/C-12/C-13, H-13 to C-11/C-14/C-15 and H-14 to C-12/C-15. Furthermore, two unsaturated (trans, J=15.4 Hz and 15.7 Hz. respectively) spin systems were observed, one of which correlated to an α,β-unsaturated carbonyl group of a modified γ-amino acid connected to the tyrosine moiety (COSY correlation of NH(2) to H-4 and HMBC correlation of NH(2) to C-9). This was supported by COSY correlations from H-2 to H-3 to H-4 to H-5 to H-6 to H-7 and NH(1), and corresponding HMBC correlations of H-2/H-3 to C-1 (δ.sub.C 171.4 ppm). The presence of guanidine moiety was deduced based on the chemical shift of quaternary carbon at C-8 (δ.sub.C 157.4 ppm) together with the requirement of molecular formula and unsaturation degree, as well as HMBC correlation of H-7 to C-8. The second unsaturated spin system belonged to an unsaturated fatty acid moiety (COSY correlations from olefinic proton H-17 OH 5.89 ppm/δ.sub.C 124.3 ppm) to H-18 δ.sub.H 6.49 ppm/δ.sub.C 142.6 ppm) and to methylene protons H-19, H-20, H-21, H-22, and the presence of a methyl group δ.sub.H 0.86 ppm/δ.sub.C 13.9 ppm). HMBC correlations of H-17/H-18/NH(3) to C-16 revealed the connection to the N-terminus of assigned tyrosine moiety. The structure assignment was further confirmed by ESI-HR-MS/MS fragmentation, which revealed the fragment ion pair of m/z at 125.0964 and 364.1966 corresponding to the amine bond cleavage on the N-terminal of tyrosine moiety. The ion pair of m/z at 288.1583 and 201.1339 indicated the amine bond cleavage on the C-terminal of tyrosine moiety.

##STR00018##

[0114] In summary, on the basis of the obtained NMR data, the compound was named according to its producing organism: barnesin A (1).

TABLE-US-00001 TABLE 1 NMR data of barnesin A (1) (DMSO-d.sub.6, at 300K).sup.a. Position δ.sub.C, type.sup.b δ.sub.H, mult. (J in Hz) COSY HMBC NOESY  1 171.4, qC  2 127.8, CH 5.73, d (15.7) 3 1, 4 3, 4, 10, 11b, NH(2)  3 141.2, CH 6.42, dd 2, 4 1, 2, 4 2, 4, 5b, 6, NH(2) (15.7, 5.0)  4 48.6, CH 4.34, br s 3, 5a, 5b, NH(2) 2, 3, 5b, 6, 7a, 7b, NH(2)  5a 30.7, CH2 1.36, m 4, 5b 4, 7 3, 4, 5b, 7a, NH(2)  5b 1.68, m 4, 5a, 6 3, 4, 6, 7a, NH(2)  6 24.9, CH.sub.2 1.46, m 5b, 7a, 7b 4, 5, 7 3, 4, 5b, 7a, NH(2)  7a 39.9, CH.sub.2 3.05, m 6 8 4, 6, 5b  7b 3.09, m 6 4, 6, 5b  8 157.4, qC  9 171.1, qC 10 54.6, CH 4.47, t (8.4) 11a, 11b, NH(3) 9, 11, 12 6, 10, 11a, 11b, 13, NH(2), NH(3) 11a 37.4, CH.sub.2 2.56, t (12.0) 10, 11b 9, 10, 12, 13 10, 11b, 13, NH(2), NH(3) 11b 2.87, d (12.0) 10, 11a 9, 10, 12, 13 2, 10, 11a, 13, NH(2), NH(3) 12 128.1, qC 13 130.0, CH 6.99, d (8.0) 14 11, 14, 15 2, 10, 11a, 11b, 14, NH(1), NH(3) 14 114.8, CH 6.60, d (8.0) 13 12, 15 13 15 155.7, qC 16 164.7, qC 17 124.3, CH 5.89, d (15.4) 18 16, 19 18, 19, NH(3) 18 142.6, CH 6.49, dd 17, 19 16, 17, 19, 20 17, 19 (15.4, 6.7) 19 31.1, CH.sub.2 2.07, m 18, 20 17, 18, 20, 21 17, 18 20 27.5, CH.sub.2 1.36, m 19, 21 18, 19, 22 21 30.8, CH.sub.2 1.24, m 20, 22 19, 20, 22, 23 22 21.9, CH.sub.2 1.27, m 21, 23 20, 21, 23 23 13.9, CH.sub.3 0.86, t (8.6) 22 21, 22 NH(1) 10.09, br s 7a, 7b NH2) 8.50, d (8.1) 4 9 2, 3, 4, 5b, 6, 7a, 10, 11a, 11b, 13, NH(3) NH(3) 8.02, d (8.4) 10 10, 11, 16 10, 11a, 11b, 13, 17, NH(3) .sup.a600 MHz for .sup.1H NMR, COSY, HSQC and HMBC; 150 MHz for .sup.13C NMR; 500 MHz for NOESY; .sup.bnumbers of attached protons were determined by analysis of 2D spectra

Example 2 Synthesis According to Reaction Scheme 1

[0115] Specific examples for the preparation of compounds of formula (I) are provided below. Unless otherwise specified all starting materials and reagents are of standard commercial grade, and are used without further purification, or are readily prepared from such materials by routine methods. Those skilled in the art of organic synthesis will recognize that starting materials and reaction conditions may be varied including additional steps employed to produce compounds encompassed by the present invention.

##STR00019##

[0116] a) HBTU, DIPEA, DCM, r.t., o.n., 78%; b) 10%.sub.−aq KOH, MeOH, r.t., 3 h, quant.; c) 1. CuCO.sub.3.Math.Cu(OH).sub.2, H.sub.2O, r.t., 15 min, quant; 2. bis-Boc-pyrazolocarboxamidine, DIPEA, formamide, dioxane, r.t., o.n., 3. EDTA.Math.2Na.Math.2H.sub.2O, NaHCO.sub.3, H.sub.2O then Fmoc-OSu, acetone, r.t., o.n. 70% (three steps); d) N,O-dimethylhydroxylamin hydrochlorid, HBTU, DIPEA, DCM, r.t., o.n. 71%; e) 1. LiAlH.sub.4, THF, 0° C., 30 min, 2. triethyl phosphonoacetate, NaH, THF, 0° C., 45 min, 32% over two steps; f) 20% piperidine in DMF, r.t., 4 h, 86%; g) COMU, DIPEA, DMF, 0° C., 6 h, 27%; h) TFA, DCM, 0° C. to r.t., o.n.; i) porcine liver esterase, DMSO, H.sub.2O, Tris/HCl buffer, 37° C., 5 d, 47% over two steps.

2.1. Methyl (E)-oct-2-enoyl-tyrosinate (A)

[0117] ##STR00020##

[0118] To a solution of trans-2-octenoic acid (500 mg, 3.52 mmol) in DCM (3.5 mL) was added subsequently DIPEA (1.5 mL, 1.14 g, 8.79 mmol) and HBTU (1.60 g, 4.22 mmol). The mixture was stirred for 5 min at room temperature (r.t.) followed by the addition of H-Tyr-OMe HCl (896 mg, 3.87 mmol). The reaction was stirred at r.t. overnight. During this time the colourless suspension became a yellowish solution. The reaction was quenched by adding 10% aq. citric acid solution to the reaction mixture. The suspension was extracted three times with DCM. The combined organic layers were washed with sat. NaHCO.sub.3 and brine, dried over MgSO.sub.4, filtered and evaporated. The crude product was purified by flash chromatography over a silica gel column (mobile phase: cyclohexane/EtOAc 1:0 to 1:1). The appropriate fractions were collected and evaporated to afford ester A (878 mg, 2.75 mmol, 78% yield) as yellowish oil.

[0119] .sup.1H-NMR (600 MHz, CDCl.sub.3): δ=6.94-6.93 (m, 2H, 5-H), 6.85 (dt, J=15.3, 6.9 Hz, 1H, 10-H), 6.74-6.72 (m, 2H, 6-H), 5.96 (d, J=7.9 Hz, 1H, NH), 5.77 (d*, J=15.3 Hz, 1H, 9-H, *fine splitting), 4.93 (dt, J=7.9, 5.7 Hz, 1H, 2-H), 3.73 (s, 3H, OMe), 3.17 (dd, J=14.0, 5.7 Hz, 1H, 3-H), 3.04 (dd, J=14.0, 5.7 Hz, 1H, 3-H), 2.16 (q*, J=6.9 Hz, 2H, 11-H, *fine splitting), 1.43 (quint, J=7.3 Hz, 2H, 12-H), 1.32-1.25 (m, 4H, 13-H, 14-H), 0.88 (t, J=7.0 Hz, 3H, 15-H) ppm.

[0120] .sup.13C-NMR (150 MHz, CDCl.sub.3): δ=172.3, 165.7, 155.2 (3s, C-1, C-8, C-7), 146.4 (d, C-10), 130.3 (d, 2×C-5), 127.3 (s, C-4), 122.7 (d, C-9), 115.5 (d, 2×C-6), 53.3 (d, C-2), 52.4 (q, OMe), 37.2, 32.0, 31.3, 27.8, 22.4 (5t, C-3, C-11, C-13, C-12, C-14), 14.0 (q, C-15) ppm.

[0121] HRMS (ESI-TOF): calculated for C.sub.18H.sub.26NO.sub.4 [M+H].sup.+: 320.1856; found 320.1859.

2.2. (E)-Oct-2-enoyl-tyrosine (B)

[0122] ##STR00021##

[0123] added 10% aq. KOH (250 μl) and the mixture was stirred for 3 h at r.t. The reaction was acidified by adding 1M HCl and then extracted with EtOAc twice. The combined organic layers were washed with water and brine, dried over MgSO.sub.4, filtered and evaporated to afford acid B (79 mg, crude product) as clear oil. The crude product was used in the next step without further purification.

[0124] .sup.1H-NMR (600 MHz, MeOD-d.sub.4): δ=7.04-7.02 (m, 2H, 5-H), 6.74 (dt, J=15.4, 7.1 Hz, 1H, 10-H), 6.69-6.68 (m, 2H, 6-H), 5.95 (d*, J=15.4 Hz, 1H, 9-H, *fine splitting), 4.65 (dd, J=8.7, 5.2 Hz, 1H, 2-H), 3.11 (dd, J=14.0, 5.2 Hz, 1H, 3-H), 2.89 (dd, J=14.0, 8.7 Hz, 1H, 3-H), 2.18 (q*, J=7.0 Hz, 2H, 11-H, *fine splitting), 1.45 (quint, J=7.3 Hz, 2H, 12-H), 1.36-1.29 (m, 4H, 13-H, 14-H), 0.91 (t, J=7.0 Hz, 3H, 15-H) ppm.

[0125] .sup.13C-NMR (150 MHz, MeOD-d.sub.6): δ=174.9, 168.5, 157.3 (3s, C-1, C-8, C-7), 146.4 (d, C-10), 131.2 (d, 2×C-5), 129.1 (s, C-4), 124.2 (d, C-9), 116.2 (d, 2×C-6), 55.4 (d, C-2), 37.7, 33.0 (t, C-11), 32.5, 29.1, 23.5 (5t, C-3, C-11, C-13, C-12, C-14), 14.3 (q, C-15) ppm.

[0126] HRMS (ESI-TOF): calculated for C.sub.17H.sub.24NO.sub.4 [M+H].sup.+: 306.1700; found 306.1702.

2.3. Fmoc-Arg-(Boc).SUB.2.-OH (C)

[0127] ##STR00022##

[0128] mmol) and basic copper carbonate CuCO.sub.3 (656 mg, 2.97 mmol) dissolved in dest. H.sub.2O (8 mL) was stirred under reflux for 15 min. The blue reaction mixture was filtered, evaporated and dried under vacuum to afford a blue solid (1.22 g, quant).

[0129] Part of the blue solid (600 mg) was suspended in formamide (6 mL) and DIPEA (1.2 mL, 880 mg, 6.81 mmol) was added. To the blue solution was added drop wise bis-boc-pyrazolocarboxamidine (939 mg, 3.02 mmol) in dioxane (3 mL). The blue solution was stirred at r.t. overnight, and then quenched with H.sub.2O (20 mL) to yield a light blue solid precipitate. The suspension was extracted twice with EtOAc. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and evaporated to yield blue foam (1.48 g). The crude product was suspended in H.sub.2O (6 mL) and EDTA.Math.2Na.Math.2H.sub.2O (676 mg, 1.82 mmol) and sat. aq. NaHCO.sub.3 (597 mg, 7.11 mmol) were added, then 9-fluorenylmethyl-succinimidyl carbonate (1.22 g, 3.63 mmol), dissolved in acetone (13.5 mL), was added drop wise to the reaction mixture at r.t. The blue suspension turned light blue and the reaction mixture was stirred at r.t. overnight. Subsequently, the solvent was evaporated under vacuum, and H.sub.2O (25 mL) was added resulting in a slight basic light blue turbid mixture (pH 8). The mixture was acidified by adding 10% aq. citric acid solution (pH 2-3). The resulting solution was extracted twice with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO.sub.4, filtered and evaporated. The crude product was purified by chromatography over a silica gel column (mobile phase: cyclohexane/EtOAc+1% AcOH 10:1 to 1:1). The appropriate fractions were collected and evaporated to afford triprotected arginine C (1.22 g, 2.05 mmol, 70% yield over two steps) as colorless foam.

[0130] .sup.1H-NMR (600 MHz, CDCl.sub.3): δ=11.37 (br. s, 1H, NHCO.sub.2tBu), 8.50, 8.42* (s, 1H, NHCO.sub.2tBu), 7.75-7.74 (m, 2H, Ar), 7.62-7.59, 7.56* (m, 2H, Ar), 7.39-7.37 (m, 2H, Ar), 7.31-7.28 (m, 2H, Ar), 5.89*, 5.83 (d, J=8.1 Hz, NH (1)), 4.62*, 4.52*, 4.42-4.37 (m, 3H, 2-H, 8-H), 4.21 (t, J=7.0 Hz, 1H, 9-H), 3.46-3.39, 3.32* (m, 2H, 5-H), 1.98-1.94 (m, 1H, 3-H), 1.76-1.66 (m, 3H, 3-H, 4-H), 1.48 (s, 9H, NHCO.sub.2tBu), 1.47 (s, 9H, NHCO.sub.2tBu) ppm *minor rotamer.

[0131] .sup.13C-NMR (150 MHz, CDCl.sub.3): δ=174.9, 172.5* (s, C-1), 163.3*, 162.8 (s, C-6), 156.6*, 156.4 (s, C-7), 156.4 (s, NHCO.sub.2tBu), 153.3, 152.9* (s, NHCO.sub.2tBu), 144.0*, 143.8 (2s, Ar), 141.4 (s, 2Ar), 127.8 (d, 2Ar), 127.2 (d, 2Ar), 125.3, 124.9* (d, Ar), 120.1 (d, 2Ar), 84.3*, 83.7, 83.4* (2s, NHCO.sub.2tBu), 80.5*, 80.1, 79.7* (2s, NHCO.sub.2tBu), 67.3*, 67.1 (t, C-8), 54.1*, 53.7 (d, C-2), 47.3 (d, C-9), 40.6, 40.4* (t, C-5), 29.8*, 29.7 (t, C-3), 28.4*, 28.3 (q, NHCO.sub.2tBu), 28.2*, 28.2 (q, NHCO.sub.2tBu), 25.5, 25.3* (t, C-4) ppm *minor rotamer.

[0132] HRMS (ESI-TOF): calculated for C.sub.31H.sub.41N.sub.4O.sub.8 [M+H].sup.+ 597.2919; found 597.2924.

2.4. N1-Fmoc-N3,N4-Di-Boc-Arginine Weinreb Amide (D)

[0133] ##STR00023##

[0134] added subsequently DIPEA (263 μl, 195 mg, 1.51 mmol) and HBTU (229 mg, 0.603 mmol). The mixture was stirred at r.t. for 5 min followed by the addition of N,O-dimethylhydroxylamine hydrochlorid (98 mg, 1.01 mmol). The reaction was stirred at r.t. overnight. To the reaction mixture was added H.sub.2O and the resulting suspension was extracted twice with EtOAc. The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered and evaporated. The crude product was purified by flash chromatography over a silica column (mobile phase: cyclohexane/EtOAc 1:0 to 1:1). The appropriate fractions were collected and evaporated to afford weinreb amide D (228 mg, 0.356 mmol, 71% yield) as colorless foam.

[0135] .sup.1H-NMR (500 MHz, CDCl.sub.3): δ=11.50 (s, 1H, NHCO.sub.2tBu), 8.40, 8.28* (s, 1H, NHCO.sub.2tBu), 7.77-7.75 (m, 2H, Ar), 7.62-7.60, 7.57* (m, 2H, Ar), 7.41-7.38 (m, 2H, Ar), 7.33-7.30 (m, 2H, Ar), 5.61, 5.15* (d, J=8.9 Hz, 1H, NH(1)), 4.78-4.70, 4.60* (m, 1H, 2-H), 4.39 (dd, J=10.5, 7.5 Hz, 1H, 8-H), 4.35 (dd, J=10.5, 7.0 Hz, 1H, 8-H), 4.22 (t, J=7.0 Hz, 1H, 9-H), 3.77 (s, 3H, N(CH.sub.3)OCH.sub.3), 3.47-3.46, 3.36* (m, 2H, 5-H), 3.22, 3.11* (s, 3H, N(CH.sub.3OCH.sub.3), 1.84-1.80 (m, 1H, 3-H), 1.69-1.62 (m, 3H, 3-H, 4-H), 1.50 (s, 9H, NHCO.sub.2tBu), 1.49 (s, 9H, NHCO.sub.2tBu) ppm *minor rotamer.

[0136] .sup.13C-NMR (125 MHz, CDCl.sub.3): δ=172.2, 163.1, 156.1 (3s, C-1, C-6, C-7), 156.0, 153.2 (2s, NHCO.sub.2tBu), 143.9*, 143.8 (s, 2×Ar), 141.3*, 141.3 (s, 2Ar), 127.6 (d, 2×Ar), 127.0 (d, 2×Ar), 125.2*, 125.1 (d, 2×Ar), 119.9*, 119.9 (d, 2×Ar), 83.3, 79.5 (2s, NHCO.sub.2tBu), 67.0 (t, C-8), 61.6 (q, N(CH.sub.3)OCH.sub.3), 50.7, 47.2 (2d, C-2, C-9), 40.5 (t, C-5), 32.1 (q, N(CH.sub.3)OCH.sub.3), 29.9 (t, C-3), 28.3, 28.0 (2q, NHCO.sub.2tBu), 25.1 (t, C-4) ppm *minor rotamer.

[0137] HRMS (ESI-TOF): calculated for C.sub.33H.sub.46N.sub.5O.sub.8 [M+H].sup.+ 640.3341; found 640.3347.

2.5. N1-Fmoc-N3,N4-di-Boc-vinylogous arginine ethyl ester (E)

[0138] ##STR00024##

[0139] A solution of weinrebamide D (221 mg, 0.345 mmol) in dry THF (3.5 mL) was cooled to 0° C. followed by slow addition of LiAlH.sub.4 (66 mg, 1.73 mmol). The mixture was stirred for 30 min at 0° C. The reaction was quenched with 10% aq. citric acid solution and extracted twice with EtOAc. The combined organic layers were washed with water and brine, dried over MgSO.sub.4, filtered. The solvent was removed under reduced pressure to afford the aldehyde (146 mg) as colorless foam which was used in the next step without further purification.

[0140] To an ice-cooled solution of triethyl phosphonoacetate (60 μl, 68 mg, 0.302 mmol) in dry THF (0.5 mL) was added NaH (15 mg, 0.377 mmol, 60% in mineral oil). After 30 min of stirring at 0° C., the crude aldehyde (146 mg) dissolved in dry THF (2 mL) was added drop wise at 0° C. to the reaction mixture. Upon completed addition, the reaction was stirred for 45 min at 0° C. The mixture was poured into 10% aq. citric acid solution and the suspension was extracted twice with EtOAc. The combined organic layers were washed with brine, dried over MgSO.sub.4, filtered and evaporated. The crude product was purified by chromatography over a silica gel column (mobile phase: cyclohexane/EtOAc 5:1). The appropriate fractions were collected and evaporated to afford protected vinyl arginine E (71 mg, 0.109 mmol, 32% yield over two steps) as colorless oil.

[0141] .sup.1H-NMR (300 MHz, CDCl.sub.3): δ=11.52 (s, 1H, NHCO.sub.2tBu), 8.39 (m, 1H, NHCO.sub.2tBu), 7.76-7.73 (m, 2H, Ar), 7.66-7.59 (m, 2H, Ar), 7.40-7.35 (m, 2H, Ar), 7.32-7.25 (m, 2H, Ar), 6.84 (dd, J=15.6 Hz, 5.0 Hz, 1H, 3-H), 6.05 (d, J=8.9 Hz, 1H, NH(1)), 5.98 (d, J=15.6 Hz, 1H, 2-H), 4.44-4.41 (m, 3H, 4-H, 10-H), 4.22-4.14 (q, J=7.1 Hz, 3H, OCH.sub.2CH.sub.3, 11-H), 3.58-3.53 (m, 1H, 7-H), 3.32-3.26 (m, 1H, 7-H), 1.66-1.58 (m, 4H, 5-H, 6-H), 1.49 (s, 18H, 2 NHCO.sub.2tBu), 1.28 (t, J=7.1 Hz, 3H, OCH.sub.2CH.sub.3) ppm.

[0142] .sup.13C-NMR (75 MHz, CDCl.sub.3): δ=166.3, 163.5, 156.6 (3s, C-1, C-8, C-9), 156.0, 153.4 (2s, NHCO.sub.2tBu), 148.0 (d, C-3), 144.1, 143.8, 141.4, 141.3 (4s, Ar), 127.7 (d, 2×Ar), 127.1 (d, 2×Ar), 125.3, 125.1 (2d, Ar), 121.1 (d, C-2), 120.0 (d, 2×Ar), 83.3, 79.5 (2s, NHCO.sub.2tBu), 66.6 (t, C-10), 60.5 (t, OCH.sub.2CH.sub.3), 52.3, 47.4 (2d, C-4, C-11), 40.3 (t, C-7), 30.2, 29.7* (t, C-5), 28.3, 28.1 (2q, NHCO.sub.2tBu), 27.0*, 26.2 (t, C-6), 14.3 (q, OCH.sub.2CH.sub.3) ppm *minor rotamer.

[0143] HRMS (ESI-TOF): calculated for C.sub.35H.sub.47N.sub.4O.sub.8 [M+H].sup.+ 651.3388; found 651.3400.

2.6. N3,N4-Di-Boc-vinylogous arginine ethyl ester (F)

[0144] ##STR00025##

[0145] Compound E (71 mg, 0.109 mmol) was dissolved in DMF (1 mL) and piperidine (200 μl) was added. The solution was stirred at r.t. for 4 h. The reaction was evaporated and dried under vacuum overnight. The crude product was purified by chromatography over a silica gel column (mobile phase gradient: from cyclohexane/EtOAc 1:1 to 0:1, switched to DCM/MeOH 9:1). The appropriate fractions were collected and evaporated to afford amine F (40 mg, 0.093 mmol, 86% yield) as colorless oil.

[0146] .sup.1H-NMR (300 MHz, MeOD-d.sub.4): δ=6.88 (dd, J=15.7 Hz, 6.7 Hz, 1H, 3-H), 5.98 (dd, J=15.7 Hz, 1.2 Hz, 1H, 2-H), 4.18 (q, J=7.1 Hz, 2H, OCH.sub.2CH.sub.3), 3.53-3.47 (m, 1H, 4-H), 3.40-3.35 (m, 2H, 7-H), 1.68-1.57 (m, 4H, 5-H, 6-H), 1.52 (s, 9H, NHCO.sub.2tBu), 1.47 (s, 9H, NHCO.sub.2tBu), 1.28 (t, J=7.1 Hz, 3H, OCH.sub.2CH.sub.3) ppm.

[0147] .sup.13C-NMR (75 MHz, MeOD-d.sub.4): δ=168.1, 164.5 (2s, C-1, C-8), 157.6, 154.1 (2s, NCO.sub.2tBu), 152.7, 121.5 (2d, C-3, C-2), 84.4, 80.3 (2s, NHCO.sub.2tBu), 61.5 (t, OCH.sub.2CH.sub.3), 53.2 (d, C-4), 41.4, 34.5 (2t, C-7, C-5), 28.6, 28.2 (2q, NHCO.sub.2tBu), 26.5 (t, C-6), 14.5 (q, OCH.sub.2CH.sub.3) ppm.

[0148] HRMS (ESI-TOF): calculated for C.sub.20H.sub.37N.sub.4O.sub.6 [M+H].sup.+: 429.2708; found 429.2713.

2.7. (E)-Oct-2-enoyl-tyrosine-N3,N4-di-Boc-vinylogous arginine ethyl ester (G)

[0149] ##STR00026##

[0150] To a solution of amine F (40 mg, 0.093 mmol) and octenoic acid (43 mg, 0.140 mmol) in DMF (1.0 mL) was added DIPEA (81 μl, 60 mg, 0.467 mmol) (pH>7). The mixture was cooled to 0° C. followed by the addition of COMU (64 mg, 0.149 mmol). The solution was stirred for 6 h at this temperature. The reaction was poured into 10% aq. citric acid solution and the suspension was extracted with EtOAc twice and three times with a DCM/IPA (4:1) mixture. The combined organic layers were washed with H.sub.2O and brine, dried over Na.sub.2SO.sub.4, filtered and evaporated. The crude product was purified by HPLC using a semipreparative phenyl hexyl column (mobile phase: A: 20 mM aq. NH.sub.4OAc, pH 7, B: MeOH gradient: 80% B 5 min, from 80% to 97% B in 30 min, 97% B 15 min). The appropriate fractions were collected and evaporated to afford compound G (18 mg, 0.025 mmol, 27% yield) as yellowish oil.

[0151] .sup.1H-NMR (300 MHz, MeOD-d.sub.4): δ=7.05-7.02 (m, 2H, 13-H), 6.82-6.66 (m, 4H, 3-H, 14-H, 18-H), 5.97 (dt, J=15.4 Hz, 1.5 Hz, 1H, 17-H), 5.64 (dd, J=15.7 Hz, 1.5 Hz, 1H, 2-H), 4.60-4.55 (m, 1H, 10-H), 4.52-4.47 (m, 1H, 4-H), 4.18 (q, J=7.0 Hz, 2H, OCH.sub.2CH.sub.3), 3.39-3.33 (m, 2H, 7-H), 2.97 (dd, J=13.5 Hz, 8.0 Hz, 1H, 11-H), 2.84 (dd, J=13.5 Hz, 7.3 Hz, 1H, 11-H), 2.18 (q*, J=7.1 Hz, 2H, 19-H, *fine splitting), 1.67-1.53 (m, 4H, 5-H, 6-H), 1.52 (s, 9H, NHCO.sub.2tBu), 1.46 (s, 9H, NHCO.sub.2tBu), 1.45-1.41 (m, 2H, 20-H), 1.36-1.25 (m, 4H, 21-H & 22-H), 1.29 (t, J=7.0 Hz, 3H, OCH.sub.2CH.sub.3), 0.91 (t, J=7.0 Hz, 3H, 23-H) ppm.

[0152] .sup.13C-NMR (75 MHz, MeOD-d.sub.4): δ=173.4, 168.3, 167.9, 164.6 (4s, C-9, C-16, C-1, C-8), 157.6 (s, NHCO.sub.2tBu), 157.4 (s, C-15), 154.1 (s, NHCO.sub.2tBu), 148.8, 146.4 (2d, C-3, C-18), 131.3 (d, 2×C-13), 128.6 (s, C-12), 124.3, 122.0 (2d, C-17, C-2), 116.3 (d, 2×C-14), 84.4, 80.3 (2s, NHCO2tBu), 61.6 (t, OCH.sub.2CH.sub.3), 56.7, 51.2 (2d, C-10, C-4), 41.3, 38.5, 33.0, 32.5, 31.9, 29.1 (6t, C-7, C-11, C-19, C-21, C-5, C-20), 28.6, 28.3 (q, NHCO.sub.2tBu), 26.7 (t, C-6), 23.5 (2q, NHCO2tBu), 14.6 (q, OCH.sub.2CH.sub.3), 14.4 (q, C-23) ppm.

2.8. (E)-Oct-2-enoyl-tyrosine-vinylogous arginine ethyl ester (3)

[0153] ##STR00027##

[0154] A solution of ester G (18 mg, 25.1 μmol) in DCM (950 μl) was treated with TFA (50 μl) for 1 hour at 0° C. followed by stirring the reaction for 1.5 h at r.t. Additional TFA (50 μl) was added and the reaction was stirred at r.t. overnight. The reaction mixture was evaporated and dried under vacuum to afford the ester 3 as yellowish oil. The crude product was used in the next step without further purification.

[0155] .sup.1H-NMR (500 MHz, MeOD-d4): δ=7.05-7.03 (m, 2H, 13-H), 6.76 (dt, J=15.4 Hz, 7.0 Hz, 1H, 18-H), 6.70-6.68 (m, 2H, 14-H), 6.66 (dd, J=15.8 Hz, 5.6 Hz, 1H, 3-H), 5.98 (d*, J=15.4 Hz, 1H, 17-H, *fine splitting), 5.59 (dd, J=15.8 Hz, 1.5 Hz, 1H, 2-H), 4.49 (m, 2H, 4-H, 10-H), 4.19 (dq, J=7.1 Hz, 2H, OCH.sub.2CH.sub.3), 3.21-3.11 (m, 2H, 7-H), 2.95 (dd, J=13.6 Hz, 8.3 Hz, 1H, 11-H), 2.87 (dd, J=13.6 Hz, 7.1 Hz, 1H, 11-H), 2.19 (q*, J=7.1 Hz, 2H, 19-H, *fine splitting), 1.70-1.58 (m, 3H, 5-H, 6-H), 1.57-1.52 (m, 1H, 5-H), 1.49-1.44 (m, 2H, 20-H), 1.37-1.31 (m, 4H, 21-H, 22-H), 1.29 (t, J=7.1 Hz, 3H, OCH.sub.2CH.sub.3), 0.91 (t, J=6.9 Hz, 3H, 23-H) ppm.

[0156] .sup.13C-NMR (125 MHz, MeOD-d4): δ=173.7, 168.5, 167.9, 158.7, 157.5 (5s, C-9, C-16, C-1, C-8, C15), 148.2, 146.4 (2d, C-3, C-18), 131.3 (d, 2×C-13), 128.5 (s, C-12), 124.3, 122.2 (2d, C17, C-2), 116.4 (d, 2×C-14), 61.7 (t, OCH.sub.2CH.sub.3), 57.1, 50.7 (2d, C-10, C-4), 42.0, 38.3, 33.0, 32.5, 32.0, 29.1, 26.1, 23.5 (8t, C-7, C-11, C-19, C-21, C-5, C-20, C-6, C-22), 14.5 (q, OCH.sub.2CH.sub.3), 14.3 (q, C-23) ppm.

[0157] HRMS (ESI-TOF): calculated for C.sub.27H.sub.41N.sub.5O.sub.5 [M+H]+: 516.3180; found 516.3183.

2.9. Barnesin A (1)

[0158] ##STR00028##

[0159] Compound 3 was dissolved in DMSO (36 μl) and H.sub.2O (144 μl) followed by the addition of porcine liver esterase (36 mg) in Tris/HCl buffer (1 mL, 50 mM, pH 6.8). The mixture was shaken for 5 days at 37° C. The reaction was filtered twice over a SPE cartridge. The methanolic and aqueous fractions were combined and evaporated. The crude product was purified by HPLC using a semipreparative phenyl hexyl column (mobile phase: A: 20 mM aq. NH.sub.4OAc, pH 7, B: MeOH gradient: 30% B 5 min, from 30% to 80% B in 35 min, 80% B 10 min, from 80% to 95% B in 0.1 min, 95% B 4.9 min). The appropriate fractions were collected and evaporated to afford barnesin A (1) (7.0 mg, 14.4 μmol, 47% yield over two steps, colourless solid).

[0160] HRMS (ESI-TOF): calculated for C.sub.25H.sub.38N.sub.5O.sub.5 [M+H].sup.+: 488.2867; found 488.2876.

[0161] [α].sub.D.sup.25=−5.09° (0.0910; MeOH)

TABLE-US-00002 TABLE 1A NMR data of barnesin A (1) (DMSO-d.sub.6, at 300K).sup.a. position δ.sub.H, mult. (J in Hz) δ.sub.C, type.sup.b COSY HMBC  1 169.7, qC  2 5.73, d (15.4) 124.7, CH 3 1, 4  3 6.46, dd (15.7, 4.8) 144.3, CH 2, 4 1, 2, 4  4 4.34, m 48.9, CH 3, 5, NH(2)  5a 1.46.sup.d, m 30.7, CH.sub.2 4, 5b, 6  5b 1.69, m 4, 5a 6  6 1.45, m 24.9, CH.sub.2 5a, 5b, 7 4, 5, 7  7 3.06, m 40.4.sup.c, CH.sub.2 6 6, 8  8 157.4, qC  9 171.1, qC 10 4.47, m 54.6, CH 11a, 11b, NH(1) 9, 11, 12, 16 11a 2.87, m 37.3, CH.sub.2 10, 11b 10, 12 11b 2.59, m 10, 11a 10, 12 12 128.0, qC 13 7.00, d (8.3) 129.9, CH 14 11, 13, 14, 15 14 6.60, d (8.2) 114.8, CH 13 13, 14, 15 15 155.7, qC 16 164.7, qC 17 5.90, d (15.3) 124.3, CH 18, 19 16 18 6.50, dt (15.4, 6.8) 142.6, CH 17, 19 16, 17, 19 19 2.08, m 31.1, CH.sub.2 18, 20 17, 18, 20, 21 20 1.35, m 27.5, CH.sub.2 19 18, 20, 21 21 1.27, m 30.8, CH.sub.2 20, 22, 23 22 1.23, m 21.9, CH.sub.2 23 20, 21, 23 23 0.85, t (6.7) 13.9, CH.sub.3 22 21, 22 NH(3) 9.80, br. S 7 NH(2) 8.46, d (7.9) 4 9 NH(1) 8.05, d (8.5) 10 10, 11, 16 .sup.a600 MHz for .sup.1H NMR, COSY, HSQC and HMBC; 150 MHz for .sup.13C NMR; .sup.bnumbers of attached protons were determined by analysis of 2D spectra; .sup.cto be seen in HSQC; .sup.dto be seen in COSY.

Example 3 Synthesis According to Reaction Scheme 2

3.1. Preparation of Aldehyde

[0162] ##STR00029##

[0163] Weinreb amide route (GPC): Weinreb amide was solubilized in dry THF (1.0 mmol/mL) and cooled to 0° C. LiAlH.sub.4 (5.0 eq) was added in portions over 30 min and the suspension was stirred at 0° C. for additional 2 h. Reaction control was performed using TLC. In case reduction was incomplete after 2 h, the reaction mixture was warmed up to r.t., LiAlH.sub.4 (5.0-7.0 eq) was added and the reaction mixture was stirred for further 1-2 h depending on the reaction process. The reaction mixture was quenched using 10% aq. citric acid and extracted twice with EtOAc. The combined organic layers were washed twice with dest. H.sub.2O, twice with brine, dried over MgSO.sub.4, filtered and concentrated to obtain a yellow foamy oil. The crude product was used without further purification in the Horner-Wasdsworth-Emmons (HWE) reaction (see below).

[0164] Methyl ester route (GPD): Amino acid methyl ester was solubilized in dry DCM (50 μmol/mL) and cooled to −80° C. DIBAL-(H) (1.2 M in toluene, 2.0 eq) was added dropwise over 1 h and the reaction mixture stirred at −80° C. for one additional hour. Reaction control was performed using TLC. In case reduction was incomplete after one hour, additional 0.5 eq DIBAL-(H) was added every 30 min until full conversion of the methyl ester to the aldehyde. The solution was then quenched with a sat. solution of Rochelle salt until the production of gas ceased. The reaction mixture was extracted with DCM and warmed up at r.t. DCM and dest. H.sub.2O were added in order to get a clear suspension, which was strongly stirred for an additional hour. The aq. phase was at last extracted with DCM, and the combined org. layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated to obtain a yellowish oil. The crude product was used without further purification in the HWE-step (see below).

3.1.1 Products

[0165] ##STR00030##

[0166] Fmoc-Arg(ω,ω′-Boc) aldehyde (H): According to GPC, Fmoc-Arg(ω,ω′-Boc) Weinreb amide (110 mg, 0.17 mmol, 1.0 eq) was converted to aldehyde (H, 56 mg) as yellowish oil. According to GPD, Fmoc-L-Arg(ω,ω′-Boc)-OMe (720 mg, 1.18 mmol, 1.0 eq) was converted to aldehyde (H, 830 mg).

[0167] Fmoc-L-Orn(δ-Boc) aldehyde (I): According to GPD, Fmoc-Orn(δ-Boc)-OMe (340 mg, 0.73 mmol, 1.0 eq) was converted to aldehyde (I, 350 mg) as yellowish oil.

[0168] Fmoc-L-Lys(c-Boc) aldehyde (J): According to GPC, Fmoc-Lys(c-Boc) Weinreb amide (186 mg, 0.36 mmol, 1.0 eq) was converted to aldehyde (J. 126 mg) as yellowish oil. According to GPD, Fmoc-Lys(c-Boc)-OMe (340 mg, 0.70 mmol, 1.0 eq) was converted to aldehyde (J, 296 mg) as yellowish oil.

3.2. Solide Phase Peptide Synthesis (SPPS)

3.2.1 Loading of Resin

[0169] ##STR00031##

[0170] Wang Resin: Wang resin (275 mg, 1.40 mmol/g, 0.39 mmol, 1.0 eq) was activated by shaking in DMF (4 mL) for 1 h. The resin was filtrated, cooled to 0° C. and a solution of coupling reagents and diethylphosphonoacetic acid (377 mg, 1.92 mmol, 5.1 eq), dissolved in DMF (2 mL), was added at 0° C. The coupling reagents were either HBTU (719 mg, 1.93 mmol, 5.0 equ)/HOBt (265 mg, 1.94 mmol, 5.0 eq), or PyBOP (927 mg, 1.92 mmol, 5.1 eq). DIPEA (667 μL, 3.81 mmol, 10.0 eq) was added at the end. The resin was first shaken at 0° C. for 5 min, then at r.t. for additional 24 h. The reaction mixture turned red overtime. Lastly, the resin was filtrated and washed 3 times, alternatively, with DMF (2 mL), and IPA (2 mL). The last wash was with DMF. A capping step was added by shaking the resin a 20% acetanhydrid solution in DMF (2 mL) during 30 min. The resin was then again washed as previously described.

[0171] CTT Resin: CTT resin (300 mg, 0.875 mmol/g, 0.26 mmol, 1.0 eq) was swelled 1 h, while shaken, in dry DCM (4 mL). A solution of diethylposponoacetic acid (87 mg, 0.44 mmol, 2.5 eq), dissolved in dry DCM (2 mL), was poured on the resin with a first addition of DIPEA (61.1 μL, 0.35 mmol, 2.0 eq). After 5 minutes of shaking, at room temperature, DIPEA (91.7 μL, 0.53 mmol, 3.0 eq) was added a second time. The resin was them shaken at room temperature, overnight. The reaction mixture got yellow overtime. Lastly, the resin was capped by adding MeOH (300 μL), and shaken for further 30 minutes. The resin was filtrated and subsequently washed with DCM (2 mL), DMF (2 mL), DCM (2 mL) and, at the end with MeOH (2 mL).

3.2.2. Horner-Wasdsworth-Emmons Reaction on Solid Phase (GPE)

[0172] ##STR00032##

[0173] The amount of reagents was calculated estimating a resin loading of 0.50 mmol/g. The resin (300 mg, 0.50 mmol*g.sup.−1, 0.15 mmol, 1.0 eq) was activated by shaking in dry THF (4 mL) for 1 h. A solution of LiBr (26.1 mg, 0.30 mmol, 2.0 eq) and DIPEA (41.1 μL, 0.23 mmol, 1.5 eq), solubilized in dry THF (1 mL), was poured on the resin, followed by the addition of the aldehyde (Table 2), also solubilized in dry THF (1 mL). The resin was shaken for 24 h at r.t.

TABLE-US-00003 TABLE 2 Amount of α-amino aldehydes. Aldehyde Weight [mg] n [mmole] eq Fmoc-L-Arg (ω,ω′-Boc)-H (H) 261.3 0.45 3.0 Fmoc-L-Orn (δ-Boc)-H (I) 197.3 0.45 3.0 Fmoc-L-Lys (ε-Boc)-H (J) 206.8 0.46 3.1

[0174] Upon completed reaction, the resin was alternately washed three times with THF (2 mL) and IPA (2 mL). To determine the conversion during HWE reaction, the N-terminus was deprotected by shaking the resin three times with a 20% Piperidine DMF solution (1 mL) for 3 min. Piperidine was removed from the resin by washing alternately the resin three times with DMF (2 mL) and IPA (2 mL), and lastly with DMF (2 mL). An analytical amount of resin was then taken and analyzed using Kaiser test. In case of a positive Kaiser test, the peptide coupling step was performed (see GPF).

3.2.3. General Procedure for Peptide Coupling (GPF)

[0175] The general procedure F for 300 mg resin, with an estimated loading of 0.50 mmol/g, is summed up in

[0176] Table 3. All steps were carried out at room temperature. The coupling step was monitored using the Kaiser test (an analytical amount of the resin was taken, washed with DMF and analysed).

TABLE-US-00004 TABLE 3 Procedure for solid phase peptide synthesis. Volume Repeats × Step Operation Reagents/Solvent [mL] time [min] 1 Coupling Fmoc-Tyr(Bu)-OH, or fatty 3 1 × 30 acid (0.45 mmol, 3.0 eq), HOBt (50.7 mg, 0.38 mmol, 2.5 eq), HBTU (142.2 mg, 0.38 mmol, 2.5 eq), DIPEA (137.0 μL, 0.75 mmol, 5.0 eq) in DMF 2 Washing Alternating, DMF and IPA 2 3 × 1 3 Deprotection 20% piperidine in DMF 1 3 × 3 4.sup.b Washing Alternating, DMF and IPA 2 3 × 1

3.2.4. Cleavage of Protected Product from Resin

[0177] ##STR00033##

[0178] Wang Resin: The resin was shaken in 95% TFA, 2.5% TES and 2.5% H.sub.2O (1 mL pro 100 mg resin) for 24 h at r.t. in order to get the unprotected peptide.

[0179] CTT Resin: The resin was shaken in HFIP:DCM (1:4) (1 mL pro 100 mg resin) for 15 min at r.t. in order to get the protected peptide.

[0180] Depending on the quantity of crude product, the peptide was either purified by semi-preparative HPLC (see methods above) or by reverse-phase flash chromatography.

3.2.5. Products

[0181] ##STR00034##

[0182] Protected barnesin (K): HRMS (ESI-TOF): calculated for C.sub.39H.sub.62O.sub.9N.sub.5 [M+H].sup.+ 744.4542; found 744.4537.

[0183] Protected 17,18-Dihydrobarnesin (L): HRMS (ESI-TOF): calculated for C.sub.39H.sub.64O.sub.9N.sub.5 [M+H].sup.+ 746.4699; found 744.4701.

[0184] Protected Lysin-Barnesin (M): HRMS (ESI-TOF): calculated for C.sub.34H.sub.54O.sub.7N3 [M+H]+616.3956; found 616.3955.

3.3 Modification of Protected Lipodipeptides

3.3.1 Synthesis of Protected Barnesin Weinreb Amide (T)

[0185] ##STR00035##

[0186] Protected barnesin (K, 4.5 mg, 6.0 μmol, 1.0 eq) was solubilized in DCM (2 mL) and cooled to 0° C. and then treated with HBTU (1.2 eq) and DiEPA (2.1 eq). Then hydrochloride salt of N,O-dimethylhydroxylamine (2.0 eq.) was added. The progress of the reaction was monitored by TLC. The reaction was quenched by aqueous work-up (brine, sodium carbonate solution) and the organic layer was dried over Na.sub.2SO.sub.4 and evaporated under reduced pressure. The pure Boc-Weinreb amide was isolated after column chromatography as colourless oil.

[0187] Protected barnesin weinreb amide (T): HRMS (ESI-TOF): calculated for C.sub.41H.sub.67N.sub.6O.sub.9 [M+H].sup.+ 787.4970; found 787.4969.

3.3.2 Modifications of C-terminus (GPG)

[0188] The protected purified peptide was solubilized in dry MeOH (2 mL) and cooled to 0° C. TMS-diazomethane (5.0 eq) was added dropwise. A production of gas (N2) was observed and the solution turned yellow. The reaction mixture was stirred at r.t. The reaction was monitored by UHPLC-MS and in case of uncomplete conversion additional TMS-diazomethane (5.0 eq) was added every hour until full conversion of the peptide into the methyl ester. The reaction mixture was concentrated to obtain a yellowish oil, which was purified by semi preparative HPLC.

##STR00036##

[0189] Protected barnesin methyl ester (O): According to GPG, protected peptide (K, 4.5 mg, 6.0 μmol 1.0 eq) was converted to ester (O, 2.5 mg after purification, 3.3 μmol 53% yield, colourless oil). HRMS (ESI-TOF): calculated for C.sub.40H.sub.64O.sub.9N.sub.5 [M+H].sup.+ 758.4699; found 758.4694.

[0190] Protected 17,18-dehydrobarnesin methyl ester (P): According to GPG, protected peptide (L, 5.6 mg, 7.5 μmol 1.0 eq) was converted to ester (P) (2.7 mg after purification, 3.6 μmol 47% yield, colourless oil). HRMS (ESI-TOF): calculated for C.sub.40H.sub.66O.sub.9N5 [M+I-1]+760.4855; found 758.4860.

[0191] Protected lysin-barnesin methyl ester (Q): According to GPG, protected peptide (M, 6.7 mg, 10.5 μmol 1.0 eq) was converted to ester (Q) (2.6 mg after purification, 4.1 μmol 38% yield, colourless oil). HRMS (ESI-TOF): calculated for C.sub.35H.sub.56O.sub.7N.sub.3 [M+H].sup.+ 630.4113; found 630.4114.

3.3.3 Deprotection (GPH)

[0192] The protected purified peptide was cooled to 0° C. A solution of 95% TFA in DCM (1 mL) was poured on the peptide and the mixture was stirred and allowed to warm to r.t. over 24 h. Upon completed reaction, the mixture was concentrated to obtain the deprotected peptide as yellowish oil. It was purified on semi-preparative HPLC.

##STR00037##

[0193] Barnesin A (1): According to GPH, protected barnesin (0, 4.4 mg, 5.9 μmol, 1.0 eq) was converted to barnesin (1) (1.5 mg, 3.1 μmol, 52% yield, colourless oil). HRMS (ESI-TOF): calculated for C.sub.25H.sub.38O.sub.5N.sub.5 [M+H].sup.+ 488.2867; found 488.2871.

[0194] 17,18-Dihydrobarnesin (4): According to GPH, protected purified 17,18-dihydrobarnesin (P, 5.6 mg, 7.5 μmol, 1.0 eq) was converted to 17,18-dihydrobarnesin (4, 3.5 mg, 7.1 μmol, 95% yield, colourless oil). HRMS (ESI-TOF): calculated for C.sub.25H.sub.40O.sub.5N.sub.5 [M+H].sup.+ 490.3024; found 490.3020. IR (ATR) v.sub.max: 3273, 2956, 2927, 2856, 1632, 1541, 1516, 1457, 1393. [α].sub.D.sup.25: −9.0° (c 1.0; MeOH). Weinreb barnesin (6): According to GPH, protected purified weinreb barnesin (T, 6 mg, 7.6 μmol, 1.0 eq.) was converted to Weinreb barnesin (6, 3 mg, 5.6 μmol, 73%, colourless oil). HRMS (ESI-TOF): calculated for C.sub.27H.sub.44N.sub.6O.sub.5 [M+H].sup.+ 533.3451; found 533.3453.

[0195] Lysin-barnesin (20): According to GPH, protected purified lysin-barnesin (Q, 4.5 mg, 7.3 μmol, 1.0 eq) was converted to lysin-barnesin (20, 2.5 mg, 5.4 μmol, 74% yield, colourless oil). HRMS (ESI-TOF): calculated for C.sub.25H.sub.38O.sub.5N.sub.3 [M+H].sup.+ 460.2806; found 460.2801.

3.3.4 Stereoisomers

[0196] Stereoisomers were synthesized according to the procedures outlined above using appropriate isomeric building blocks as starting materials. Final purification of the crude products was carried out on a Shimadzu HPLC system using a Phenomenex Luna C18 (2) 250×10.

[0197] a) Barnesin-D-Arginine-L-Tyrosine (1A)

##STR00038##

TABLE-US-00005 TABLE 1B NMR data of Barnesin-D-Arginine-L-Tyrosine (1A) (DMSO-d.sub.6, at 300K).sup.a. position δ.sub.H, mult. (J in Hz) δ.sub.C, type.sup.b COSY HMBC  1  2 5.70, d (15.5) 128.7, CH 2 1, 4  3 6.37, dd (15.9, 5.3) 141.2, CH 2, 4 1, 2, 4  4 4.29, m 48.7, CH NH(2)  5a 1.58.sup.d, m 30.9, CH.sub.2 5b, 6  5b 1.45, m 5a, 6 6  6 1.29, m 22.1, CH.sub.2 5a, 5b, 7 4, 5, 7  7 2.97, m 40.4.sup.c, CH.sub.2 6 6, 8  8  9 170.8, qC 10 4.52, m 54.6, CH 11a, 11b, NH(1) 9, 11, 12, 16 11a 2.82, m 37.5, CH.sub.2 10, 11b 10, 12 11b 2.66, m 10, 11a 10, 12 12 128.0, qC 13 7.00, d (8.4) 130.0, CH 14 11, 13, 14, 15 14 6.60, d (8.4) 114.9, CH 13 13, 14, 15 15 155.9, qC 16 164.6, qC 17 5.93, d (15.5) 124.4, CH 18, 19 16 18 6.50, dt (15.1, 7.1) 142.7, CH 17, 19 16, 17, 19 19 2.07, m 31.2, CH.sub.2 18, 20 17, 18, 20, 21 20 1.35, m 27.5, CH.sub.2 19 18, 20, 21 21 1.26, m 30.8, CH.sub.2 20, 22, 23 22 1.23, m 21.9, CH.sub.2 23 20, 21, 23 23 0.85, t (6.9) 13.9, CH.sub.3 22 21, 22 NH(3) 10.02, br. S 7 NH(2) 8.21, d (8.3) 4 9 NH(1) 8.15, d (8.3) 10 10, 11, 16 .sup.a600 MHz for .sup.1H NMR, COSY, HSQC and HMBC; 150 MHz for .sup.13C NMR; .sup.bnumbers of attached protons were determined by analysis of 2D spectra; .sup.cto be seen in HSQC; .sup.dto be seen in COSY.

[0198] b) Barnesin-L-Arginine-D-Tyrosine (1B)

##STR00039##

TABLE-US-00006 TABLE 1C NMR data of Barnesin-L-Arginine-D-Tyrosine (1B) (DMSO-d.sub.6, at 300K).sup.a. position δ.sub.H, mult. (J in Hz) δ.sub.C, type.sup.b COSY HMBC  1  2 5.70, d (15.8) 129.2, CH 3 4  3 6.36, dd (15.8, 5.2) 140.8, CH 2, 4 1, 4  4 4.28, m 49.2, CH 3, 6, NH(2)  5a 1.58, m 29.1, CH.sub.2 5b, 6  5b 1.45, m 5a, 6  6 1.29, m 22.5, CH.sub.2 5a, 5b, 7 5, 7  7 2.97, m 40.4.sup.c, CH.sub.2 6  8  9 171.2, qC 10 4.52, m 55.0, CH 11a, 11b, NH(1) 11a 2.82, m 38.0, CH.sub.2 10, 11b 12, 13 11b 2.66, m 10, 11a 12, 13 12 128.3, qC 13 6.99, d (8.3) 130.5, CH 14 11, 13, 15 14 6.60, d (8.5) 115.4, CH 13 12, 14, 15 15 156.3, qC 16 165.0, qC 17 5.94, d (15.8) 124.8, CH 18, 19 16, 19 18 6.53, dt (15.0, 7.2) 143.1, CH 17, 19 16, 19, 20 19 2.07, m 31.6, CH.sub.2 18, 20 17, 18, 20, 21 20 1.35, m 27.9, CH.sub.2 19 18, 21, 22 21 1.24, m 31.3, CH.sub.2 20, 22, 23 22 1.23, m 22.3, CH.sub.2 23 20, 21, 23 23 0.85, t (6.7) 14.3, CH.sub.3 22 21, 22 NH(3) 9.99, br. S NH(2) 8.21, d (8.1) 4 9 NH(1) 8.15, d (8.1) 10 16 .sup.a600 MHz for .sup.1H NMR, COSY, HSQC and HMBC; 150 MHz for .sup.13C NMR; .sup.bnumbers ofattached protons were determined by analysis of 2D spectra; .sup.cto be seein in HSQC; .sup.dto be seen in COSY.

[0199] c) Barnesin-D-Lysin (20A)

##STR00040##

TABLE-US-00007 TABLE 1D NMR data of Barnesin-D-Lysin (20A) (DMSO-d.sub.6, at 300K).sup.a. position δ.sub.H, mult. (J in Hz) δ.sub.C, type.sup.b COSY HMBC  1 167.1, qC  2 5.73, dd (15.7, 1.5) 121.2, CH 3 1, 4  3 6.70, dd (15.7, 5.5) 147.9, CH 2, 4 1, 2, 4, 5  4 4.39, m.sup.c 49.1, CH 5a, 5b, 2, 3, 5 NH(2)  5a 1.57, m.sup.c 32.8, CH.sub.2 4 4, 6  5b 1.44, m.sup.c 4 4, 6  6 1.27, m.sup.c 22.2, CH.sub.2  7 1.51, m 36.6, CH.sub.2 8 6, 8  8 2.75, t (7.57) 38.7, CH.sub.2 7 6, 7  9 171.1, qC 10 4.45, m 54.6, CH 11a, 11b, 8, 9, 11 NH(1) 11a 2.85, m.sup.c 37.1, CH.sub.2 10, 11b 9, 10, 12, 13 11b 2.67, m.sup.c 10, 11a 9, 10, 12, 13 12 127.8, qC 13 7.03, d (8.3).sup.c 130.0, CH 14 11, 13, 14, 15 14 6.62, d (8.3).sup.c 114.9, CH 13 12, , 13, 14, 15 15 155.8, qC 16 164.8, qC 17 5.95, d (15.4).sup.c 124.2, CH 18 16, 19 18 6.54, dt (15.5, 7.1).sup.c 142.9, CH 19, 17 16, 17, 19, 20 19 2.09, q (6.8, 31.1, CH.sub.2 18, 20 17, 18, 20, 21 20 1.37, m.sup.c 27.4, CH.sub.2 19, 21 18, 19, 22 21 1.25, m.sup.d 30.8, CH.sub.2 20 22 22 1.27, m.sup.c 21.9, CH.sub.2 23 20, 23 23 0.86, t (7.0) 13.9, CH.sub.3 22 21, 22 NH(1) 8.09, d (8.3) 10 10, 16 NH(2) 8.18, d (8.3) 4 4, 9 NH2 (3) 9.17, br, s .sup.a600 MHz for .sup.1H NMR, .sup.13C NMR, COSY, HSQC, HMBC; .sup.bnumbers of attached protons were determined by analysis of 2D spectra; cdetermined through COSY; .sup.ddetermined through HSQC.

[0200] d) Barnesin-L-Phenylalanine (10)

##STR00041##

TABLE-US-00008 TABLE 1E NMR data of Barnesin-L-Phenylalanine (10) (DMSO-d.sub.6, at 300K).sup.a. position δ.sub.H, mult. (J in Hz) δ.sub.C, type.sup.b COSY HMBC  1 170.6, qC  2 5.72, d (15.4) 126.2, CH 3 1, 4  3 6.49, m 142.3, CH 2, 4 1, 2, 4  4 4.36, m 48.8, CH 3, 5, NH(2) 5, 6, 9  5a 1.39, m 30.7, CH.sub.2 4, 5b, 6 4, 6, 7  5b 1.67, m 4, 5a 4, 6, 7  6 1.46, m 24.9, CH.sub.2 5a, 5b, 7 4, 5, 7  7 3.07, m 40.1.sup.c, CH.sub.2 6 5, 6, 8  8 157.3, qC  9 170.9, qC 10 4.56, m 54.3, CH 11a, 11b, NH(1) 9, 11, 12, 16 11a 2.98, m 38.2, CH.sub.2 10, 11b 9, 10, 12, 13, 14 11b 2.71, m 10, 11a 9, 10, 12, 13, 14 12 138.0, qC 13 7.23, m 129.1, CH 11, 13, 14, 15 14 7.23, m 128.0, CH 15 13, 14, 15 15 7.16, m 129.7, qC 14 13, 14 16 164.7, qC 17 5.89, d (15.5) 124.3, CH 18 16, 19 18 6.49, m 142.8, CH 17, 19 16, 17, 19 19 2.06, q (7.3, 14.2) 31.2, CH.sub.2 18, 20 17, 18, 20, 21 20 1.35, m 27.5, CH.sub.2 19 18, 20, 21 21 1.27, m 30.8, CH.sub.2 20, 22, 23 22 1.23, m 21.9, CH.sub.2 23 20, 21, 23 23 0.85, t (7.0) 13.9, CH.sub.3 22 21, 22 NH(3) 9.79, br. S 7 NH(2) 8.47, d (8.3) 4 4, 9 NH(1) 8.13, d (8.5) 10 10, 11, 16 .sup.a600 MHz for .sup.1H NMR, COSY, HSQC and HMBC; 150 MHz for .sup.13C NMR; .sup.bnumbers of attached protons were determined by analysis of 2D spectra; .sup.cto be seen in HSQC; .sup.d to be seen in COSY.

3.4 Preparation of Comparative Compounds R and S

3.4.1 Synthesis of protected 2,3-17,18-Tetrahydrobarnesin (N)

[0201] ##STR00042##

[0202] Peptide (L) (16 mg, 21.4 μmol 1.0 eq) was solubilized in methanol and put under argon atmosphere in a dried round-bottom flask. A spatula tip of Pd on carbon (10% weight) was added to the solution. The suspension was put under H.sub.2 atmosphere, and stirred at r.t. for 5 h. An analysis by UPLC-MS confirmed the completion of the reaction. The reaction mixture is filtered over Celite and washed with methanol. The solution was then dried under vacuum to obtain hydrogenated peptide (N) (13.6 mg, 18 μmol 85% yield) as yellowish oil. HRMS (ESI-TOF): calculated for C.sub.39H.sub.66O.sub.9N.sub.5 [M+H].sup.+ 747.4855; found 748.4847.

3.4.2 Preparation of Comparative Compound R

[0203] 2,3,17,18-Tetrahydrobarnesin A (R): According to GPH (see section 3.3.3 above) protected purified 2,3-17,18-tetrahydrobarnesin (N, 12 mg, 16.0 μmol 1.0 eq) was converted to 2,3-17,18-tetrahydrobarnesin (R, 3.8 mg, 7.7 μma 48% yield, colourless oil).

##STR00043##

[0204] HRMS (ESI-TOF): calculated for C.sub.25H.sub.42O.sub.5N.sub.5 [M+H].sup.+ 492.3180; found 492.3177. IR (ATR) v.sub.max: 3288, 2949, 2838, 1644, 1557, 1518, 1449, 1402. [α].sub.D.sup.25: +8.3° (c 1.0; MeOH).

3.4.3 Preparation of Comparative Compound S

[0205] 2,3-17,18-Tetrahydrobarnesin A methyl ester (S): Treatment of R with acidic methanol afforded methyl ester (S, 2.9 mg, 5.7 μmol colourless oil).

##STR00044##

[0206] HRMS (ESI-TOF): calculated for C.sub.26H.sub.44O.sub.5N.sub.5 [M+H].sup.+ 506.3337; found 506.3336. IR (ATR) v.sub.max: 3280, 2929, 2856, 1652, 1540, 1517, 1438. [α].sub.D.sup.25: +10.22° (c 1.0; MeOH).

Example 4 Assessment of Protease Inhibition

[0207] Protease Assays with azocasein: Protease inhibition assays against the proteases papain, ficin, trypsin, pepsin and thermolysin were performed according to the protocol of Garcia-Carreño (loc. cit.) in a reaction buffer containing 25 mM TRIS-HCl, 0.15 M NaCl, pH 7.2 (or pH 3.5 for aspartic protease). The total assay volume was 47 μL buffer with 2 μL of the protease as well as 1 μL of suitable inhibitor or corresponding inhibitor solvent (100% MeoH or water). Final concentration of proteases was as follows: 2 mg/mL for papain (Sigma-Aldrich, P4762), 600 μg/mL for ficin (Sigma-Aldrich, F6008), 80 μg/mL for trypsin (Thermo Fisher, 23266), 60 mg/mL for pepsin (Sigma-Aldrich P6887)) and 2 μg/mL for thermolysin (Sigma-Aldrich, P1512).

[0208] Inhibitors were dissolved in 100% MeOH or water and added to the assay (1 μL); final concentration: 2% MeOH. Samples of control inhibitors were prepared as follows: (1) serine protease (trypsin): PMFS (2 mM) and soybean trypsin inhibitor (240 μM) (Sigma-Aldrich, T1021), (2) cysteine proteases (papain and ficin): iodacetamide (2 mM), (3) asparagine protease (pepsin): pepstatin A (1 μg/mL) (Sigma-Aldrich, P4265), (4) metalloprotease (thermolysin): EDTA (10 mM). In all cases the given concentration inhibited enzyme activity >95%. Negative controls were performed with protease alone (without inhibitor) and MeOH at a final concentration of 2% (reference for activity of 100%). Extract absorbance controls were performed using inhibitor (1 μL in MeOH) without protease. Blanks were performed using 49 μL buffer and 1 μL MeOH.

[0209] Samples were incubated with buffer and suitable inhibitor (or without for negative controls) for 1 h at r.t. and the reaction was started afterward by adding 50 μl of 1 azocasein substrate and incubated for 1 h at 37° C. After stopping the reaction with trichloracetic acid (TCA), the separation of the precipitate was accomplished by centrifugation at 6500 g for 5 min. The hydrolyzed substrate supernatant was incubated in an equal volume of 0.5 N NaOH for 5 min and the absorbance was measured at 440 nm. OD values of the protease control without inhibitor were used as reference for a 100% activity.

[0210] The log IC.sub.50 values were determined using the standard curves analyzing tool with four parameter logistic equation of SigmaPlot12 with technical triplicates. Propagation of error was calculated using the standard error and log IC.sub.50 values where the equation of error propagation is defined as Δy=0.43Δx/x.

[0211] Cathepsin B inhibition assay. Cathepsin B inhibition assay was determined according to Hiwasa et al. (loc. cit.) with minor changes. Cathepsin B was purchased from Sigma Aldrich (C0150) and stored in 50 mM sodium acetate, 1 mM EDTA, pH 5 (adjusted with acetic acid). Cathepsin B was activated by preincubation at 40° C. for 10 min in assay buffer (0.1 M sodium acetate 1.3 mM EDTA, pH 6.0 adjusted with acetic acid, 2 μM DTT, 2.6 mM cysteine and 0.05% Triton X100). The total assay volume was 47 μL buffer with 2 μL cathepsin B as well as 1 μL of suitable inhibitor or corresponding inhibitor solvent (100% MeOH or water). Final concentration of cathepsin B was 0.1 mg/ml. Samples were incubated with buffer and suitable inhibitor (or without for negative controls) for 20 min at 4° C. and the reaction was started afterwards by adding 1 μL of 10 mM Z-Arg-Arg-AMC (Peptanova, 3123-v) and incubated for 20 min at 40° C. After stopping the reaction with 85 μL buffer containing 100 mM sodium monochlor acetate, 30 mM sodium acetate, 70 mM acetic acid, the hydrolyzed substrate was detected at an excitation wavelength of 380 nm and a fluorescence wavelength of 450 nm, using a fluorophotometer. 1 μL of 1.5 mM leupeptin was used as enzyme inhibition control.

[0212] The results of the inhibition assays are summarized in Tables 4A and B below.

TABLE-US-00009 TABLE 4A Evaluation of protease inhibition activities Protease class (inhibitory activity as IC50 value (μM)) Metallo Serine Aspartic (3.4.24) Cysteine (3.4.22) (3.4.21) (3.4.23) Thermo- Compound Papain Ficin Cathepsin B Trypsin Pepsin lysin Barnesin A (1) 15.96 μM (±5.8) 3.43 μM (±0.15) 91.72 nM (±5.8) n.i. n.i. n.i. Barnesin A  2.89 μM (±0.13) 3.43 μM (±0.38)  23.99 nM (±0.13) n.i. n.i. n.i. ethyl ester (3) 17,18-  4.78 μM (±2.9) 1.16 μM (±0.07) 87.56 nM (±2.9) n.i. n.i. n.i. Dihydro- barnesin (4) Comparative n.i. n.i. n.i. n.i. n.i. n.i. Compound (R) Comparative n.i. n.i. n.i. n.i. n.i. n.i. Compound (S)

TABLE-US-00010 TABLE 4B Reported protease inhibition activities of known compounds Protease class (inhibitory activity as IC50 value (μM)) Metallo Cysteine (3.4.22) Serine Aspartic (3.4.24) Cath- (3.4.21) (3.4.23) Thermo- Compound Papain Ficin epsin B Trypsin Pepsin lysin Leupeptin  0.86 μM n.r.  21.5 nM 2.2 μM n.r. n.r. Cyclo- 0.0054 μM n.r.  0.71 μM n.r. n.r. n.r. propenone 1’S Cyclo-    22 μM n.r. 0.044 μM n.r. n.r. n.r. propenone 1’R Cystatin  0.029 μM n.r. n.r. 3.5 μM n.r. n.r. Miraziri- n.r. n.r.  2.05 μM  60 μM n.r. n.r. dine A Tokar- n.r. n.r.  62.4 nM n.r. n.r. n.r. amide A YM 51084   2.2 μM n.r.  12.0 nM n.r. n.r. n.r. a) n.r. = not reported in literature; n.i. = no inhibition

[0213] Activity Tests Against Cathepsin B, Cathepsin L and Rhodesain.

[0214] Rhodesain is a central cysteine protease from Trypanosoma brucei rhodesiense and a potential drug target against human African trypanosomiasis (sleeping sickness).

[0215] The determination of the activity of the inhibitors against hCatL and RD was performed in fluorescence-based assays in accordance with the assays described in Giroud et al., ChemMedChem 12 (2017), 257-270 and Schirmeister, Bioorganic & Medicinal Chemistry Letters 27 (2017) 45-50. The biological activities against hCatL were determined using Cbz-Phe-Arg-AMC as substrate, which releases AMC (7-amino-4-methylcoumarin) after amide bond cleavage by the enzyme. The proteolytic activity of the enzyme can be monitored spectrophotometrically by the increase of fluorescence intensity by release of AMC (emission at 460 nm) upon hydrolysis. An initial screen at an inhibitor concentration of 20 mM was performed to identify ligands with an inhibition of hCatL and RD higher than 80%.

TABLE-US-00011 TABLE 5 Evaluation of protease inhibition activities Cathepsin B Cathepsin L Rhodesain Compound (%) (%) (%) [00045] 50 95 99 [00046] 73 96  9 [00047] 41 90 99 [00048] 15 20 60 [00049]  5  7 ni [00050] 30 80 99

Example 5 Determination of Metabolic Stability

[0216] Glutathione (GSH) Assay:

[0217] Glutathione (GSH), a thiol-containing tripeptide (γ-glutamyl-cysteinyl-glycine), is a key antioxidant in many species. It has been highly implicated in the detoxification/elimination of antibiotics and xenobiotics (naturally occurring harmful compounds such as free radicals, hydroperoxides etc.) and in the maintenance of the oxidation state of protein sulfhydryl groups. In addition, GSH plays a pivotal role in the pathogenesis of numerous human diseases including cancer and cardio-vascular diseases. Glutathione is present in cells in both reduced (GSH) and oxidized (GSSG) forms—GSH being, the predominant species under normal physiological conditions inside cells. Furthermore, electrophiles that cause the depletion of the cellular GSH pool can cause cytotoxicity.

[0218] Procedure: A standard GSH assay includes a phosphate buffer (720 μL), a GSH solution (40 μL, 100 mM) and the test solution of the test compound (20 μM in 10% DMSO in phosphate buffer (100 mM, pH 7.4)). As negative control (NC) serves the test item (20 μM in 10% DMSO in phosphate buffer (100 mM, pH 7.4) in phosphate buffer (760 μL).

[0219] Samples are prepared as follows: phosphate buffer (720 μL) and GSH solution (40 μL, 100 mM) were preincubated at 37° C., and 40 μL of the test item solution (or internal standard) added. Then 100 aliquots of the assay mixture were mixed with 100 μL ACN were at the following time points: t=0, 30 and 90 min; in case of the test item, t=0 and 90 min in case of PC or NC. The samples were mixed for 2 min at 150 rpm, centrifuged at 16.1 (krcf). 5 μL of each samples were injected to a UPLC/HRMS system. The peak areas of the extracted ion chromatograms were determined using the standard software.

[0220] The results of the assay are shown in Table 6 below. As will be recognized, the compounds of the invention, i.e. barnesin and derivatives thereof, are remarkably unreactive towards general thiol nucleophiles such as GSH. In other words, the compounds of the invention have a high stability towards soft nucleophiles. More specifically, an intracellular detoxification mechanism, unwanted side reactions and unregulated depletion of the GSH pool by simple, unselective 1,4-addition of GSH to the vinylogous double bond of the claimed compounds can be excluded.

[0221] Thus, the experimental data demonstrate that the compounds of the invention have an advantageous pharmacokinetic property, namely high stability towards soft nucleophiles.

TABLE-US-00012 TABLE 6 Metabolic stability in GSH Assay Time Area Area rel. Test item Sample Type [min] Area rel. [%] [00051] FM049_1 FM049_7 FM049_10 FM049_4 FM049_13 Test item   NC  0 30 90  0 90 367407693 342890085 345965508 363205474 333706916 1.000 0.933 0.942 1.000 0.919 100.0  93.3  94.2 100.0  91.9 [00052] FM049_2 FM049_8 FM049_11 FM049_5 FM049_14 Test item   NC  0 30 90  0 90 519869606 542406698 509683755 510530074 530330916 1.000 1.043 0.980 1.000 1.039 100.0 104.3  98.0 100.0 103.9 [00053] FM049_3 FM049_9 FM049_12 FM049_6 FM049_15 Test item   NC  0 30 90  0 90 680377355 698665622 722442552 705274108 717968682 1.000 1.027 1.062 1.000 1.018 100.0 102.7 106.2 100.0 101.8 [00054] FM062_29 FM062_35 Test item  0 90 202656073 201316491 1.000 0.993 100.0  99.3 [00055] FM062_30 FM062_36 FM062_40 FM062_42 Test item NC  0 90  0 90 115525226 113959407 117560798 108084764 1.000 0.986 1.000 0.919 100.0  98.6 100.0  91.9 [00056] FM062_31 FM062_37 Test item  0 90 489372587 507468070 1.000 1.037 100.0 103.7 [00057] FM062_32 FM062_38 Test item  0 90 501743817 516317782 1.000 1.029 100.0 102.9 [00058] FM062_33 FM062_39 FM062_41 FM062_43 Test sample NC  0 90  0 90 40293650.6 45445179.6 37458620.2 40800514.3 1.000 1.128 1.000 1.089 100.0 112.8 100.0 108.9 IR76 FM062_28 PC  0 29338493 1.000 100.0 (internal standard) FM062_34 90 17109.4453 0.001  0.1

[0222] Microsome Stability Assay

[0223] The liver is the main organ of drug metabolism in the body. Subcellular fractions such as liver microsomes are useful in vitro models of hepatic clearance as they contain many of the drug metabolising enzymes found in the liver. Liver microsomes are subcellular fractions which contain membrane bound drug metabolising enzymes.

[0224] The microsomes are incubated with the test compound at 37° C. in the presence of the co-factors, which initiates the reaction. The reaction is terminated by the addition of organic solvents containing internal standard. Following centrifugation, the supernatant is analysed on the LC-MS/MS. The disappearance of test compound is monitored over certain time period.

[0225] Here, a microsome stability assay was used to investigate the metabolism of the compounds; using this assay it is possible to measure in vitro the intrinsic clearance or to identify metabolites formed.

[0226] For a standard assay the following solutions were used: 360 μL Microsomal solution (1.1 mg/mL in phosphate buffer), 360 μL NADP-regeneration mix (containing NADP (10 mM), MgCl.sub.2 (50 mM), glucose-6-phosphate (50 mM), glucose-6-phosphate dehydrogenase (50 U/ml)) and 40 μL phosphate buffer (100 mM, pH 7.4). Diclofenac (10 μL, 200 μM) was used as positive control (360 μL Microsomal solution, 360 μL NADP-regeneration mix and 40 μL Phosphate buffer) and phosphate buffer (360 μL Microsomal solution, 360 μL NADP-regeneration mix) served as negative control. The test substance was dissolved to 20 μM in 10% DMSO (phosphate buffer (100 mM, pH 7.4)) and used as working solution.

[0227] The following procedure was applied: 40 μL of the working solution was added to a pre-incubated solution containing the microsomal solution and the NADP-regeneration mix at 37° C./1500 rpm. Samples were prepared as follows: Proteine precipitation was induced by addition of 100 μL ACN to 100 μL aliquots of the assay mixture at t=0, 30 and 90 min in case of the test item, t=0 and 30 min in case of PC and t=0 and 90 min in case of NC. Samples were mixed at 1500 rpm for 2 min, centrifuged for 2 min at 16.1 krcf. 5 uL of each sample was injected to a UPLC/HRMS system and the peak areas of the extracted ion chromatograms were determined

TABLE-US-00013 TABLE 7 Metabolic stability in liver microsomes Time Area Area rel. Sample Type [min] Area rel. [%] [00059] FM052_13 FM052_19 FM052_22 FM052_16 FM052_25 Sub- stance   NC  0 30 90  0 90 235792454 208205685 196717888 211564777 195341003 1.000 0.883 0.834 1.000 0.923 100.0  88.3  83.4 100.0  92.3 [00060] FM052_14 FM052_20 FM052_23 FM052_17 FM052_26 Sub- stance   NC  0 30 90  0 90 361788562 298891411 288411590 336394843 322058861 1.000 0.826 0.797 1.000 0.957 100.0  82.6  79.7 100.0  95.7 [00061] FM052_15 FM052_21 FM052_24 FM052_18 FM052_27 Sub- stance   NC  0 30 90  0 90 330765136 281767935 262648398 280077767 249809907 1.000 0.852 0.794 1.000 0.892 100.0  85.2  79.4 100.0  89.2 Diclo FM052_10 PC  0 4.18E+08 1.000 100.0 FM052_11 10 2.05E+08 0.490  49.0 FM052_12 35 1.60E+07 0.038  3.8 [00062] FM062_1 FM062_7 FM062_13 FM062_18 FM062_23 Sub- stance   NC  0 30 90  0 90 179467850 142403215 121177629 143469802 147452392 1.000 0.793 0.675 1.000 1.028 100.0  79.3  67.5 100.0 102.8 [00063] FM062_2 FM062_8 FM062_14 FM062_19 FM062_24 Sub- stance   NC  0 30 90  0 90 95241639.3 85162953 82923793.2 84799508 89736887.8 1.000 0.894 0.871 1.000 1.058 100.0  89.4  87.1 100.0 105.8 [00064] FM062_3 FM062_9 FM062_15 FM062_20 FM062_25 Sub- stance   NC  0 30 90  0 90 328347157 304396247 302016018 332191329 346639421 1.000 0.927 0.920 1.000 1.043 100.0  92.7  92.0 100.0 104.3 [00065] FM062_4 FM062_10 FM062_16 FM062_21 FM062_26 Sub- stance   NC  0 30 90  0 90 396622297 379045795 389518824 363662768 361941845 1.000 0.956 0.982 1.000 0.995 100.0  95.6  98.2 100.0  99.5 [00066] FM062_5 FM062_11 FM062_17 FM062_22 FM062_27 Sub- stance   NC  0 30 90  0 90 38412530.4 33877398.1 38180235.5 25518415.7 28546900.9 1.000 0.882 0.994 1.000 1.119 100.0  88.2  99.4 100.0 111.9

[0228] As can be taken from Table 7, all compounds have a good metabolic stability compared to the positive control. In essence, the compounds of the invention are able to reach the cellular target and are not metabolist instantaneously. Accordingly, this data shows that the compounds of the invention have good pharmacokinetic properties which makes them suitable as active agents in various applications, including oral drugs.

[0229] The above results confirm that the compounds according to the invention act as selective cysteine protease inhibitors in the low molecular range.

[0230] The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention.