Protease inhibitors

Abstract

The present invention relates to a new class of compounds based on alkylated oligopeptides featuring distinct head and tail modifications at their termini. The compounds are useful as inhibitors of viral proteases, particularly of flaviviral proteases, and can therefore be used for treatment of viral infections.

Claims

1. A compound of general formula (0) or a salt thereof: ##STR00321## wherein m is 3 or 2; link is C(O)or is absent; tail is H or C1-24alkyl; head is NH.sub.2, NH-C1-24alkyl, C(O)NH.sub.2, C(O)NH-C1-24alkyl, or5-6-membered (hetero)aryl; AA.sup.1 is an amino acid residue that is connected to its neighbouring AA.sup.n via an amide bond to which it contributes a donor carboxylic acid, and that via its amine is connected to link; AA.sup.n is for each instance independently an amino acid residue.

2. The compound according to claim 1, wherein m is 3.

3. The compound according to claim 1, wherein m is 2.

4. The compound according to claim 1, wherein tail is CH.sub.3(CH.sub.2).sub.14;

5. The compound according to claim 1, wherein link is C(O)- and tail is C1-24 alkyl.

6. The compound according to claim 1, wherein link is C(O).

7. The compound according to claim 4, wherein link is C(O).

8. The compound according to claim 1, wherein link is absent and tail is H.

9. The compound according to claim 1, wherein head is NH.sub.2, NH(CH.sub.2).sub.15CH.sub.3, -phenyl, CH.sub.3, or C(O)NH.sub.2.

10. The compound according to claim 1, wherein head is NH.sub.2, -phenyl, CH.sub.3, or C(O)NH.sub.2.

11. The compound according to claim 4, wherein head is NH.sub.2, -phenyl, CH.sub.3, or C(O)NH.sub.2.

12. The compound according to claim 8, wherein head is NHC1-24alkyl or C(O)NH-C1-24alkyl.

13. The compounds according to claim 1, wherein AA.sup.1 is lysine, arginine, or alanine.

14. The compounds according to claim 1, wherein each AA.sup.n is individually selected from alanine, arginine, glutamine, lysine, phenylalanine, histidine, proline, (4-OBn-phenyl)glycine, tryptophan, and homophenylalanine.

15. The compounds according to claim 1, wherein at least one instance of AA.sup.n that is not adjacent to head is alanine, glutamine, or histidine.

16. The compounds according to claim 1, wherein the instance of AA.sup.n that is adjacent to head is lysine, phenylalanine, homophenylalanine, histidine, tryptophan, or (4-OBn-phenyl)glycine.

17. The compounds according to claim 1, wherein at least one instance of AA.sup.n that is not adjacent to head is glutamine.

18. The compounds according to claim 1, wherein the instance of AA.sup.n that is adjacent to head is phenylalanine.

19. The compounds according to claim 1, wherein AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAAK, ARQK, KAF, RQ-homoPhe, KAK, RAF, RQF, KAH, KAAH, KPA-(4-OBn-phenylGly), KPAK, KAAW, KPH-homoPhe, KPAF, KPA-homoPhe, or KPAW.

20. The compounds according to claim 1, wherein AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAAK, (D-K)AAK, K (D-A) AK, KA (D-A) K, KAA (D-K), ARQK, KAF, RQ-homoPhe, KAK, RAF, RQF, (D-R) QF, KAH, KAAH, KAA (D-H), KPA-(4-OBn-phenylGly), KPAK, KAAW, KPH-homoPhe, KPAF, KPA (D-F), KPA-homoPhe, KPA-(D-homoPhe), KPAW, (D-K) PAW, or KPA (D-W).

21. The compound according to claim 1, wherein i. tail is H, link is absent; AA.sup.1 and each instance of AA.sup.n together form a peptide represented by ARQK; head is NH(CH.sub.2).sub.15CH.sub.3; ii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAAK; head is -phenyl; iii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAF; head is NH.sub.2; iv. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by RQ-homoPhe; head is NH.sub.2; v. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KA (D-A) K; head is NH.sub.2; vi. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by ARQK; head is NH.sub.2; vii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAK; head is -phenyl; viii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by RAF; head is NH.sub.2; ix. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAA (D-K); head is NH.sub.2; x. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by (D-R) QF; head is NH.sub.2; xi. tail is H, link is absent; AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAK; head is NH(CH.sub.2).sub.15CH.sub.3; xii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAAK; head is NH.sub.2; xiii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAH; head is NH.sub.2; xiv. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAAK; head is C(O)NH.sub.2; xv. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by RQF; head is NH.sub.2; xvi. tail is H, link is absent; AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAAK; head is NH(CH.sub.2).sub.15CH.sub.3; xvii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAK; head is C(O)NH.sub.2; xviii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by (D-K)AAK; head is NH.sub.2; xix. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAAH; head is NH.sub.2; xx. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAA (D-K); head is C(O)NHCH.sub.2-phenyl; xxi. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KPA-(4-OBn-phenylGly); head is NH.sub.2; xxii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAK; head is CH.sub.3; xxiii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KPAK; head is NH.sub.2; xxiv. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by K (D-A) AK; head is NH.sub.2; xxv. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAA (D-H); head is NH.sub.2; xxvi. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAAW; head is NH.sub.2; xxvii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KPH-homoPhe; head is NH.sub.2; xxviii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KPAF; head is NH.sub.2; xxix. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KPA-homoPhe; head is NH.sub.2; xxx. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KPA (D-homoPhe); head is NH.sub.2; xxxi. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KPA (D-F); head is NH.sub.2; xxxii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by (D-K) PAW; head is NH.sub.2; or xxxiii. tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KPA (D-W); head is NH.sub.2.

22. The compound according to claim 1, wherein it is of formula (KAAK-4): ##STR00322##

23. The compound according to claim 1, wherein it is of formula (RQF-1): ##STR00323##

24. Method of treating, preventing, or delaying a viral infection or a condition related to a viral infection in a subject in need thereof, the method comprising the step of administering to the subject an effective amount of a compound as defined in claim 1.

25. The method according to claim 24, wherein the viral infection is a dengue viral infection.

26. The method according to claim 24, wherein the compound is of general formula (0) or a salt thereof wherein tail is CH.sub.3(CH.sub.2).sub.14, link is C(O); AA.sup.1 and each instance of AA.sup.n together form a peptide represented by KAA (D-K); head is NH.sub.2.

27. A compound of general formula (1) or a salt thereof: ##STR00324## wherein tail is C1-24alkyl, OC1-24alkyl, 5-20-membered (hetero)aryl, or 3-20-membered (hetero)cycloalkyl, wherein tail is optionally unsaturated, wherein tail is optionally substituted with halogen, C1-3 (halo)alkyl, or C1-3 (halo)alkoxyl; head is H, -h1, O-h1, C(O)-h1, C(O)N(H)h1, N(h2) h1, or head is -C1-24alkyl, (NH).sub.0-1-5-20-membered (hetero)aryl, or (NH).sub.0-1-3-20-membered (hetero)cycloalkyl, wherein head is optionally unsaturated, wherein head is optionally substituted with halogen, C1-3 (halo)alkyl, or C1-3 (halo)alkoxyl; h1 is H, OH, S(O).sub.0-2OH, C1-8 (halo)alkyl, C1-8 (halo)alkoxyl, S(O).sub.0-2-C1-8 (halo)alkyl, 3-8-membered (hetero)cycloalkyl, S(O).sub.0-2-[3-8-membered (hetero)cycloalkyl], -C1-4alkyl-[3-8-membered (hetero)cycloalkyl], 5-6-membered (hetero)aryl, S(O).sub.0-2-[5-6-membered (hetero)aryl], or C1-4alkyl[5-6-membered (hetero)aryl], wherein h1 is optionally substituted with halogen, C1-3 (halo)alkyl, or C1-3 (halo)alkoxyl; h2 is H, OH, S(O).sub.0-2OH, C1-8 (halo)alkyl, C1-8 (halo)alkoxyl, S(O).sub.0-2-C1-8 (halo)alkyl, 3-8-membered (hetero)cycloalkyl, S(O).sub.0-2-[3-8-membered (hetero)cycloalkyl], -C1-4alkyl-[3-8-membered (hetero)cycloalkyl], 5-6-membered (hetero)aryl, S(O).sub.0-2-[5-6-membered (hetero)aryl], or C1-4alkyl[5-6-membered (hetero)aryl], wherein h2 is optionally substituted with halogen, C1-3 (halo)alkyl, or C1-3 (halo)alkoxyl; AA.sup.1 is an amino acid residue that is connected to its neighbouring AA.sup.n via an amide bond to which it contributes a donor carboxylic acid, and that is connected to tail via an amide bond of a secondary amide to which it contributes a donor amine; AA.sup.n is for each instance independently an amino acid residue, wherein the carbonyl moiety in the residue adjacent to head can instead together with head be replaced by B(OH).sub.2 or a C1-6alkyl ester thereof, P(O)(OH).sub.2 or a C1-6alkyl ester thereof, S(O).sub.2-halogen, or 5-12 membered (hetero)aryl that is optionally substituted with halogen, C1-3 (halo)alkyl, or C1-3 (halo)alkoxy; m is 1, 2, 3, 4, or 5.

Description

DESCRIPTION OF DRAWINGS

[0206] FIG. 1-Body weights of experimental groups of mice following treatment. Error bars are MeanSEM)

[0207] FIG. 2-Spleen weights in experimental groups of mice following treatment. Error bars are MeanSD. * p<0.05 significantly different from vehicle control (G1).

[0208] FIG. 3-viral load (plaque assay) in plasma of experimental groups of mice following treatment. Error bars are MeanSEM. * p<0.05 significantly different from vehicle control (G1).

[0209] FIG. 4-plasma concentrations of TNF-alpha, IL-6, and IL-12 in plasma of experimental groups of mice at the end of treatment. Error bars are meanSEM. * p<0.05 significantly different from vehicle control (G1).

[0210] FIG. 5A-multidose PK-profiles of compound 113 in mice after intravenous (IV), intraperitoneal (IP) and subcutaneous (SC) administration to mice (dosed twice per day for 5 days, mean of 3 mice per group).

[0211] FIG. 5B-viral load (plaque assay) in plasma of mice following treatment with vehicle or with compound 113 (20 mg/kg, b.i.d. IP and SC starting at 5h post-infection). Error bars are MeanSEM.

[0212] FIG. 5C-tissue distribution after intravenous injection in rats, measured by ratio concentration tissue over plasma.

[0213] FIG. 5D-multidose PK-profiles of compound 43 in mice after intravenous (IV), intraperitoneal (IP) and subcutaneous (SC) administration to mice (dosed twice per day for 5 days, mean of 3 mice per group).

EXAMPLES

Abbreviations

[0214] AA Amino Acid [0215] Ac Acetyl [0216] ACOH Acetic acid [0217] Ala Alanine [0218] AM Amino mehtyl [0219] BB Building Block [0220] Bn Benzyl [0221] Boc Tert-butyloxycarbonyl [0222] Bs Brosyl [0223] calcd calculated [0224] Cbz Carboxybenzylcarbonyl [0225] CDI Carbonyl Diimidazole [0226] CTC-Cl 3-Chlorotrityl chloride [0227] Cyp Cyclopropyl [0228] DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene [0229] DCM Dichloromethane [0230] DEAD Diethyl azodicarboxylate [0231] DIC Diisopropylcarbodiimide [0232] DIPEA Diisopropylethylamine [0233] DMAP 4-(N,N-dimethyl)aminopyridine [0234] DMF Dimethylformamide [0235] DMP Dess-Martin Periodinane [0236] ESI Electron Spray Ionization [0237] Fmoc Fluorenylmethoxycarbonyl [0238] HATU HexafluorophosphateAzabenzotriazole Tetramethyl Uronium [0239] hHis Homo-Histidine [0240] HMDS bis(trimethylsilyl)amide [0241] HOBt Hydroxybenzotriazole [0242] Hpg Hydroxyphenylglycine [0243] HRMS High-resolution mass spectrometry [0244] Hyp Hydroxyproline [0245] IPA Isopropan -2-ol [0246] KHMDS Potassium bis(trimethylsilyl)amide [0247] LDA Lithium diisopropylamide [0248] LRMS Liquid-phase Peptide synthesis [0249] LPPS Low resolution mass spectrometry [0250] MBHA 4-Methylbenzhydrylamine [0251] Mmt 4-Methyltrityl [0252] MS Mass Spectrometry [0253] Oxd Oxadiazole [0254] Oxyma Ethyl cyano (hydroxyimino)acetate [0255] PDA Poly diode array [0256] Ph Phenyl [0257] PHTI Phthalimide [0258] Pin (1S,2S,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptane-2,3-diol [0259] Pip. Plperidine [0260] PPh3 Triphenylphosphine [0261] Pro Proline [0262] quant. Quantitative [0263] rac Racemate [0264] R.sub.f Retardation factor [0265] RP-HPLC Reversed Phase-HPLC [0266] RT Room temperature [0267] SFC Supercritical Fluid Chromatography [0268] SPPS Solid-phase peptide synthesis [0269] SOC Synthetic Organic Chemistry [0270] Su Succinimidyl [0271] TEA Triethylamine [0272] TFA Trifluoroacetic acid [0273] TFE Trifluoroethanol [0274] THF Tetrahydrofuran [0275] TIC Total Ion Chromatogram [0276] TIPS Triisopropylsilane [0277] TLC Thin-Layer Chromatography [0278] TMS Tetramethylsilane [0279] TsCl Tosylchloride [0280] V Volumes [0281] wt weight

##STR00290## ##STR00291## ##STR00292##

[0282] The scheme on the previous pages shows compound examples covering compound classes according to synthetic procedures. It describes the compounds of the invention classified by their synthetic accessibility: compounds can be grouped as a group with 1, a group with 2, 3, 4, a group with 5, 6, 7, 8, a group with 9, 10, 11, and a group with 12. Different examples in the same group required same synthetic strategy but different building blocks. Similar compounds can be synthetized accordingly. Building blocks and intermediates can be either purchased commercially or obtained following well established chemistry. All compounds were obtained after global deprotection and purification via preparative HPLC.

[0283] As shown in Scheme 1 (next page), peptides primary amides as Example 1 were prepared via SPPS, using Rink amide MBHA resin and commercially available Fmoc-AAs (1-3) Compounds according to examples 2, 3, and 4 were obtained via late stage derivatizations of the same peptide precursor Intermediate 1, which was synthesized side-chain protected via SPPS, using CTC-Cl resin (Barlos' Resin)(4).

[0284] Compounds according to Examples 5-8 were obtained by joining via amide coupling side-chain protected peptide intermediate 2 with the appropriate building block 1-4. Intermediate 2 was synthesized side-chain protected via SPPS, using CTC-Cl resin (Barlos' Resin)(4). Building Blocks 1 and 2 are prepared from the appropriately protected Lys precursor, adapting literature procedures reported for other amino acids (ketone (5, 6) in Scheme 9, hydroxyamide (7) in Scheme 11) or commonly found in the synthesis of pharmaceuticals (oxadiazole (6-10) in Scheme 10). Building block 4 was obtained via Matteson homologation (11), adapting known literature procedures (Scheme 12). (12, 13) To obtain compounds according to examples 7 (Scheme 11) and 8 (Scheme 12), prior to global deprotection, the appropriate functional group interconversion was performed, respectively to oxidize the hydroxyamide to alpha-ketoamide, (14) and convert the bromide to an amino group. (12)

##STR00293##

[0285] Example compounds 9-11 can be obtained via SPPS using Rink-amide resin, however their synthesis required the ad-hoc preparation of Fmoc-AAs as Building Blocks 5-7, whose synthesis is detailed in Schemes 13-15. BB5 was prepared adapting the procedure described by Behnam and coworkers (15, 16) as detailed in Scheme 13. BB6 was prepared adapting the procedure of Bloemhoff and coworkers (17) as detailed in Scheme 14. BB7 was prepared adapting the procedure described by Elliot and coworkers (18) as detailed in Scheme 15.

##STR00294##

[0286] Compounds according to Example 12 (Scheme 16) were synthesized via LPPS (19) joining pre-assembled intermediate 3 (i3), and intermediate 4 (i4). i4 was prepared via LPPS, from BB8 and BB9. Boc-protected AA BB8 was obtained via Mitsunobu displacement (20) from commercially available amino- and carboxyl-protected commercially available Hyp (BB8a)(21). BB9 was prepared via LPPS from commercially available AAs.

##STR00295##

General Experimental

[0287] All peptides were purified via RP-HPLC(unless stated otherwise) using a Shimadzu LC-20A Prominence system (Shimadzu, 's Hertogenbosch, The Netherlands) equipped with a C18 Gemini-NX column, 15021.20 mm, particle size 10 m (Phenomenex, Utrecht, The Netherlands), a pre-column guard and UV detection at 215 and 254 nm. RP-HPLC elution was performed with 0.1% TFA in a MeCN/milliQ H2O solution (isocratic 10% MeCN in H2O over 5 min, gradient from 10 to 100% MeCN in H2O over 20 min, with a solvent flow rate of 10.0 mL/min, unless stated otherwise). Chemicals were purchased from Fluorochem, VWR, Fischer Scientific or Sigma-Aldrich and used as received, unless stated otherwise. Dry DCM and THF as were obtained extra-pure from commercial vendors and used as ultra-pure after purification with MBraun Solvent Purification System (MB SPS). Reactions were magnetically stirred and carried out under inert atmosphere of dry nitrogen or argon, unless stated otherwise. Standard syringe techniques were applied for the transfer of dry solvents and air- or moisture-sensitive reagents. Reactions were followed either via analytical LCMS or TLC. R.sub.f values are obtained using thin layer chromatography (TLC) on silica gel-coated plates (Merck 60 F254) with the indicated solvent mixture. Detection was performed with UV-light, and/or by charring at 150 C. after dipping into a solution of either KMnO.sub.4 (7.5 g/L), K.sub.2CO.sub.3 (50 g/L) and 10% NaOH (6.25 ML/L) in H2O, or (NH.sub.4).sub.6MO.sub.7O.sub.24. 4H.sub.2O (25 g/L) and (NH.sub.4).sub.4Ce(SO.sub.4).sub.4 .Math.2H.sub.2O (10 g/L) in 10% H.sub.2SO.sub.4. Alternatively, iodine staining was performed burying the plate in 12 adsorbed on SiO.sub.2 for 30 seconds. LCMS spectrograms were recorded on a Thermo Finnigan LCQ-Fleet ion trap mass spectrometer (ESI-IT-MS) coupled to a Shimadzu analytical HPLC [LC-20AD (pump) and SPD-M30A (photodiode array detector)], equipped with a Gemini C18 110A column, 50 mm2 mm, particle size 3 m (Phenomenex, Utrecht, The Netherlands), eluting with 0.1% formic acid in a MeOH/milliQ H.sub.2O solution (isocratic 5% MeOH in H.sub.2O over 5 min, gradient from to 95% MeOH in H.sub.2O over 20 min, with a solvent flow rate of 1.0 mL/min). NMR spectra were recorded using either a Bruker Avance 400 (400 MHZ) or a Bruker Avance III (500 MHZ) spectrometer, in MeOH (d4), CDCl.sub.3, or D.sub.2O solutions, unless stated otherwise. Chemical shifts are given in ppm with respect to residual non-deuterated solvents or TMS as internal standard for CDCl.sub.3. Coupling constants are reported as J-values in Hz. The following abbreviations are used to explain multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, dd=doublet of doublets, dp=doublet of quintets, ddd=doublet of doublet of doublets, dtd=doublet of triplet of doublets, td=triplet of doublets, m=multiplet. Flash column chromatography was carried out using ACROS silica gel (0.035-0.070 mm, and ca 6 nm pore diameter). High resolution mass spectra (HRMS) were recorded on a JEOL AccuToF CS JMS-T100CS (ESI-HRMS). Regular MS (ESI-MS) measurements were recorded on Thermo Finnigan LCQ-Q Advantage Max.

General Information SPPS

[0288] Fmoc deprotection. The resin was swollen with DCM (10 mL/gram of resin, 1 min) and DMF (210 mL/gram of resin, 1 min) and treated with 20% pip in DMF (10 mL/gram of resin) and left to shake for 20 minutes. The suspension was filtered and the resin was washed with DMF (210 mL/gram of resin, 1 min) and treated with a second portion of 20% pip in DMF (10 mL/gram of resin) and left to shake for 10 minutes. The suspension was then filtered and the resin was washed with DMF (310 mL/gram of resin, 1 min), DCM (310 mL/gram of resin, 1 min) and MeOH (310 mL/gram of resin, 1 min). Deprotection efficiency was determined by means of Kaiser or chloranil tests (for Pro deprotection).

[0289] Loading of the first amino acid (Rink Amide MBHA/AM resin). The resin was swollen with DCM (1 min) and DMF (21 min). Fmoc-amino acid (3 equiv) and HOBt (3 equiv) were dissolved in DMF (10 mL/gram of resin) and the resulting solution was added to the resin. Next DIPCDI (3 equiv) was added and the reactor was left to shake for 16 h. The suspension was then filtered and the resin was washed with DMF (31 min), DCM (31 min), before treating with a capping solution of acetic anhydride/pyridine (3:2, 10 mL/gram of resin) for 20 minutes. The suspension was then filtered and the resin was washed with DMF (31 min), DCM (31 min) and MeOH (31 min).

Coupling Efficiency was Determined by Means of a Kaiser Test

[0290] Loading of the first amino acid (CTC-Cl Barlos' resin). The Fmoc-amino acid (3 equiv) was dissolved in dry DCM (10 mL/gram of resin) and collidine (3 equiv), and the resulting solution was added to resin. The reactor was left shaking overnight, and the suspension was filtered before treating with a capping solution of 5% DIPEA in MeOH (10 mL/gram of resin) for 15 minutes. The suspension was then filtered and the resin was washed with DMF (31 min), DCM (31 min) and MeOH (3 1 min).

[0291] Peptide coupling. The resin was swollen with DCM (1 min) and DMF (21 min). Fmoc-amino acid (3 equiv) and HOBt (3 equiv) were dissolved in DMF (10 mL/gram of resin) and the resulting solution was added to the resin. Next DIPCDI (3 equiv) was added and left to shake for 3 h. The suspension was then filtered and the resin was washed with DMF (31 min), DCM (31 min) and MeOH (3 1 min). Coupling efficiency was determined by means of a Kaiser or chloranil tests (for couplings on Pro).

[0292] Coupling to palmitic acid. The resin was swollen with DCM (1 min) and DMF (21 min). Palmitic acid (3 equiv) and HATU (2.9 equiv) were dissolved in DCM/DMF (1:1, 10 mL/gram of resin) and the resulting solution was added to the resin. Next DIPEA (3 equiv) was added and the reactor was left to shake for 3 h. The suspension was then filtered and the resin was washed with DMF (31 min), DCM (31 min) and MeOH (31 min). Coupling efficiency was determined by means of Kaiser or chloranil tests (for couplings on Pro).

[0293] Peptide cleavage (Rink Amide MBHA/AM resin). The peptidyl-resin was washed with DCM (31 min) and dried under nitrogen. The resin was treated with a cleavage solution (95% TFA, 2.5% TIPS, 2.5% H.sub.2O, 5 mL) and left to shake for 2 h (unless stated otherwise). The mixture was filtered and the resin was washed with DCM (31 min), filtrates were collected, combined and volatiles were removed in vacuo. The crude residue was triturated in dry Et.sub.2O and after centrifuge the precipitate was collected by decantation. Solvent leftovers were removed under high-vacuum.

[0294] Purification. The crude material was dissolved in minimal amount of MeOH (unless stated otherwise), filtered through a 0.20 m syringe filter, and purified using preparative RP-HPLC (isocratic 20% MeCN in H.sub.2O over 5 min, gradient from 20 to 80% MeCN in H.sub.2O over 15 min, with a solvent flow rate of 10.0 mL/min, at 30 C.). All fractions containing product were combined, concentrated to 5 mL in vacuo, prior to lyophilization (unless stated otherwise) to afford the pure materials.

[0295] Peptide cleavage (CTC-Cl Barlos' resin). The peptidyl-resin was washed with DCM (31 min) and dried under nitrogen before being suspended in a cleavage solution of AcOH/TFE/DCM (1:1:8, 10 mL/g of resin) and shaken for 1 h, then the resin was washed with DCM (31 min), collecting all the filtrates. The cleavage process was repeated three time in total, then the filtrates were combined and reduced to 10 mL in vacuo before lyophilization. The resulting white powder was suspended in dry Et.sub.2O and after centrifuge the precipitate was collected by decantation. Solvent leftovers were removed under high-vacuum.

Example 1

##STR00296##

[0296] N(S)-6-amino-1-(((S)-1-(((S)-1-(((R)-1,6-diamino-1-oxohexan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxohexan -2-yl) palmitamide bis-TFA [Palmitoyl-Lys-Ala-Ala-D-Lys-NH.sub.2 bis-TFA salt (Example 1)]. was prepared according the general procedure for SPPS using 700 mg Rink amide resin (0.26 mmol). The crude peptide was purified by preparative HPLC, all fractions containing the product were combined and concentrated in vacuo to 5 mL, prior to lyophilization, to afford peptide Example 1 (140 mg) as a white powder.

HRMS (ESI-MS m/z): mass calcd for C.sub.34H.sub.67N.sub.7O.sub.5 [M+H].sup.+, 654.5276, found 654.5268.

[0297] Compounds made following the synthesis of example 1:11, 12, 13, 14, 15, 16, 17, 33, 35, 37, 40, 43, 44, 47, 49, 50, 51, 52, 53, 54, 57, 58, 63, 64, 70, 72, 73, 75, 77, 80, 82, 84, 86, 88, 90, 91, 92, 93, 95, 97, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 131, 132, 133, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 167, 168, 169, 170, 171, 190, 191, 192, 193, 194, 195, 196

Example 2

##STR00297##

[0298] N.sup.6-(tert-butoxycarbonyl)-N.sup.2-N.sup.6-(tert-butoxycarbonyl)-N.sup.2-palmitoyl-L-lysyl-L-alanyl-L-alanyl-L-lysine[Palmitoyl-Lys(Boc)-Ala-Ala-Lys(Boc)-OH(Intermediate 1)]. was prepared according the general procedure for SPPS using 2.5 g CTC-Cl resin (3.3 mmol), to recover Intermediate 1 (1.7 g, 60%) as a white powder. The crude peptide was used as such in the next synthetic step.

[0299] LCMS (ESI-MS m/z): mass calcd for C.sub.44H.sub.83N.sub.6O.sub.10 [M+H].sup.+, 855.62; found 854.96.

[0300] Palmitoyl-L-lysyl-L-alanyl-L-alanyl-L-lysine bis-TFA [Palmitoyl-Lys-Ala-Ala-Lys-OH bis-TFA salt (Example 2)]. To a DCM (2.0 mL) solution of Intermediate 1 (110 mg, 0.12 mmol, 1.0 equiv), TFA (2.0 mL) was added, and the resulting mixture was left stirring 2 h at RT. The reaction mixture was concentrated in vacuo and the crude residue was dissolved in H.sub.2O, filtered through a 0.20 m syringe filter, and purified using preparative RP-HPLC. All fractions containing product were combined, concentrated to 5 mL in vacuo, prior to lyophilization, to recover Example 2 (73 mg, 66%) as a white powder.

[0301] LCMS (ESI-MS m/z): mass calcd for C.sub.36H.sub.72N.sub.7O.sub.5 [M+H].sup.+, 655.51, found 655.28.

[0302] Compounds made following the synthesis of example 2:45, 46, 134, 188, 189.

Example 3

##STR00298##

[0303] Tert-butyl ((10S,13S,16S,19S)-19-(methoxy(methyl)carbamoyl)-2,2,13,16-tetramethyl-4,11,14,17-tetraoxo-10-palmitamido-3-oxa-5,12,15,18-tetraazatricosan -23-yl)carbamate[Palmitoyl-Lys(Boc)-Ala-Ala-Lys(Boc)-N(OMe)Me (Intermediate 1a)]. To a DMF solution (5.0 mL) of Intermediate 1 (300 mg, 0.35 mmol, 1.0 equiv) NH(OMe)Me hydrochloride was added at 0 C., (41 mg, 0.42 mmol, 1.2 equiv), followed by HATU (150 mg, 0.38 mmol, 1.1 equiv) and DIPEA (180 L, 1.0 mmol, 3.0 equiv). The resulting mixture was left warming up at RT over 16 h, 10 before it was quenched by pouring over 50 mL of ice and 10% aqueous citric acid. The so-formed solid was filtered, washed with water (220 mL) and dried in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 1:19 to 1:20). Fractions containing product were combined and concentrated in vacuo, the residue was dissolved in dioxane and lyophilized to recover intermediate 1a (290 mg, 93%) as a white powder. LCMS (ESI-MS m/z): mass calcd for C.sub.46H.sub.88N.sub.7O.sub.10 [M+H].sup.+, 898.66; found 898.12. N-((5S,8S,11S,14S)-18-amino-5-(4-aminobutyl)-3,8,11-trimethyl-4,7,10,13-tetraoxo-2-oxa-3,6,9,12-tetraazaoctadecane -14-yl) palmitamide bis-TFA [Palmitoyl-Lys-Ala-Ala-Lys-N(OMe)Me bis-TFA salt (Example 3)]. To a DCM (2.0 mL) solution of Intermediate 1a (107 mg, 0.12 mmol, 1.0 equiv), TFA (2.0 mL) was added, and the resulting mixture was left stirring 2 h at room temperature. The reaction mixture was concentrated in vacuo and the crude residue was dissolved in H.sub.2O, filtered through a 0.20 m syringe filter, and purified using preparative RP-HPLC(isocratic 20% MeCN in H.sub.2O over 5 min, gradient from 20 to 100% MeCN in H.sub.2O over 15 min). All fractions containing product were combined, concentrated to 5 mL in vacuo, prior to lyophilization, to recover Example 3 (83 mg, 75%) as a white powder.

[0304] LCMS (ESI-MS m/z): mass calcd for C.sub.36H.sub.72N.sub.7O.sub.5 [M+H].sup.+, 698.55, found 698.48.

[0305] Compounds made following the synthesis of example 3:18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 36, 38, 69, 71, 74, 76, 78, 79, 81, 83, 85, 87, 89, 94, 96, 98, 126, 177, 184, 185, 187,

Example 4

##STR00299##

[0306] Tert-butyl ((10S,13S,16S,19S)-10-formyl-2,2,13,16-tetramethyl-4,12,15,18-tetraoxo-19-palmitamido-3-oxa-5,11,14,17-tetraazatricosan -23-yl)carbamate[Palmitoyl-Lys(Boc)-Ala-Ala-Lys(Boc)-H (Intermediate 1b)]. To a THF solution (10 mL) of Intermediate 1a (206 mg, 0.23 mmol, 1.0 equiv), LiAlH.sub.4 (36.0 mg, 0.95 mmol, 4.1 equiv) was added at 0 C., and the resulting mixture was left stirring for 0.5 h. Next, the reaction mixture was quenched by the addition of saturated aqueous NH.sub.4Cl (20 mL) and diluted with EtOAc (40 mL). After phase separation the organic layer was diluted with DCM (3.0 mL), IPA (3.0 mL) and MeOH (3.0 mL), washed with saturated aqueous potassium tartrate (320 mL), brine (210 mL), dried over sodium sulfate, and concentrated in vacuo to recover a crude residue, which was purified via flash column chromatography (SiO.sub.2, MeOH/CHCl.sub.3, from 1:49 to 1:9). Fractions containing product were combined and concentrated in vacuo. The residue was dissolved in dioxane and lyophilized to recover intermediate 1b (50 mg, 26%) as a white powder.

[0307] LCMS (ESI-MS m/z): mass calcd for C.sub.36H.sub.72N.sub.7O.sub.5 [M+H].sup.+, 839.62, found 839.16.

[0308] N((S)-6-amino-1-(((S)-1-(((S)-1-(((S)-6-amino-1-oxohexan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxohexan -2-yl) palmitamide bis-TFA [Palmitoyl-Lys-Ala-Ala-Lys-H bis-TFA salt (Example 4)]. To a DCM (2.0 mL) solution of Intermediate 1b (110 mg, 0.12 mmol, 1.0 equiv), TFA (2.0 mL) was added, and the resulting mixture was left stirring 2 h at RT. The reaction mixture was concentrated in vacuo and the crude residue was dissolved in H.sub.2O, filtered through a 0.20 m syringe filter, and purified using preparative RP-HPLC. All fractions containing product were combined, concentrated to 5 mL in vacuo, prior to lyophilization, to recover Example 4 (41 mg, 33%) as a white powder.

[0309] LCMS (ESI-MS m/z): mass calcd for C.sub.34H.sub.65N.sub.6O.sub.4 [MH.sub.2O+H].sup.+, 621.51; found 621.48.

[0310] Compounds made following the synthesis of example 4:180, 181, 186.

Intermediate 2a and 2b

##STR00300##

[0311] N6-(tert-butoxycarbonyl)-N2-palmitoyl-L-lysyl-L-alanyl-L-alanine[Palmitoyl-Lys(Boc)-Ala-Ala-OH(Intermediate 2a)]. was prepared according the general procedure for SPPS using 0.50 g CTC-Cl resin (0.85 mmol), to recover Intermediate 2a (340 mg, 63%) as a white powder. The crude peptide was used as such in the next synthetic step.

[0312] MS (ESI-MS m/z): mass calcd for C.sub.33H.sub.63N.sub.4O.sub.7 [M+H].sup.+, calcd 627.5; found 627.1.

[0313] N6-(benzyloxy)carbonyl)-N2-palmitoyl-L-lysyl-L-alanyl-L-alanine[Palmitoyl-Lys (Cbz)-Ala-Ala-OH(Intermediate 2b)]. was prepared according the general procedure for SPPS using 0.50 g CTC-Cl resin (0.85 mmol), to recover intermediate 2b (320 mg, 60%) as a white powder. The crude peptide was used as such in the next synthetic step.

[0314] LCMS (ESI-MS m/z): mass calcd for C.sub.36H.sub.61N.sub.4O.sub.7 [M+H].sup.+, calcd 861.45; found 661.00.

Example 5

##STR00301##

[0315] Benzyl tart-butyl (6-(methoxy(methyl)amino)-6-oxohexane-1,6-diyl)(S)-dicarbamate[Boc-Lys (Cbz)-NOMe)Me (BB1a]. To a DMF solution (20.0 ml) of Boc-Lys (Cbz)-OH (2.0 g, 5.3 mmol, 1.0 equiv), NH(OMe)Me hydrochloride (760 mg, 7.9 mmol, 1.5 equiv) was added at 0 C., followed by HATU (3.0 g, 7.9 mmol, 1.1 equiv) and DIPEA (4.7 mL, 16 mmol, 3.0 equiv). The resulting mixture was left stirring over 10 minutes, before it was quenched by diluting with H.sub.2O (50 mL). The aqueous layer was extracted with EtOAc (3100 mL), and the combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, 1:4), to recover Weinreb amide BB1a (1.4 g, 64%) as a light yellow oil.

[0316] R.sub.f=0.7 (SiO.sub.2, EtOAc)

[0317] LCMS (ESI-MS m/z): mass calcd for C.sub.21H.sub.34N3O.sub.6 [M+H].sup.+, 424.24; found 424.42.

[0318] Benzyl tert-butyl (6-oxo-6-phenylhexane-1,5-diyl)(S)-dicarbamate[Boc-Lys (Cbz)-Ph (BB1b)]. To a THF (15 mL) solution of BB1a (1.5 g, 3.3 mmol, 1.0 equiv), 1 M PhMgBr in THF (10.6 mL, 10.6 mmol, 3.0 equiv) was added dropwise over 5 minutes at 0 C. The resulting mixture was warmed to RT and stirred for 2 h, before it was quenched by diluting with saturated aqueous NH.sub.4Cl (30 mL). The aqueous layer was extracted with EtOAc (3100 mL), and the combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, 1:5), to recover BB1b (0.35 g, 22%) as a light yellow oil.

[0319] R.sub.f=0.5 (SiO.sub.2, EtOAc/pet-ether, 1:1)

[0320] LCMS (ESI-MS m/z): mass calcd for C.sub.25H.sub.33N.sub.2O.sub.5 [M+H].sup.+, 441.24; found 441.49.

[0321] Benzyl(S)-(5-amino-6-oxo-6-phenylhexyl)carbamate TFA [H-Lys (Cbz)-Ph TFA salt (BB1)]. To a TFA/DCM solution (1:5, 7.0 mL), BB1a (0.35 g, 0.79 mmol, 1.0 equiv) was added at 0 C. The resulting mixture was warmed to RT and stirred for 1 h, before it was concentrated in vacuo, to recover amine hydrochloride BB1 (0.25 g, quant.) as crude material, which was used as such in the next synthetic step.

[0322] LCMS (ESI-MS m/z): mass calcd for C.sub.20H.sub.25N.sub.2O.sub.3 [M+H].sup.+, 341.19 found 341.37.

[0323] Tert-butyl ((9S, 12S,15S, 18S)-9-benzoyl-12,15-dimethyl-3,11,14,17-tetraoxo-18-palmitamido-1-phenyl-2-oxa-4,10,13,16-tetraazadocosan -22-yl)carbamate[Palmitoyl-Lys(Boc)-Ala-Ala-Lys (Cbz)-Ph (Intermediate 2c)]. To a DMF (20 mL) solution of Intermediate 2a (300 mg, 0.47 mmol, 1.0 equiv) was added CDI at 0 C., followed by imidazole (48 mg, 0.71 mmol, 1.5 equiv). The resulting mixture was warmed to RT and stirred for 10 minutes, before BB1 (97 mg, 0.28 mmol, 0.6 equiv) was added. The resulting mixture was stirred for 16 h, before it was quenched by diluting with H.sub.2O (50 mL). The aqueous layer was extracted with EtOAc (3100 mL), and the combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo, to recover Intermediate 2c (0.4 g, quant.) as crude material which was used as such in the next synthetic step.

[0324] (ESI-MS m/z): mass calcd for C.sub.53H.sub.85N.sub.6O.sub.9 [M+H].sup.+, 949.64 found 949.65.

[0325] N((S)-6-amino-1-(((S)-1-(((S)-1-(((S)-6-amino-1-oxo-1-phenylhexan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxohexan -2-yl) palmitamide bis-TFA [Palmitoyl-Lys-Ala-Ala-Lys-Ph bis-TFA salt (Example 5)]. To a TFA/TIPS/H.sub.2O (95:2.5:2.5, 8.0 mL) solution, Intermediate 2c (0.40 g, 0.42 mmol, 1.0 equiv) was added at 0 C. The reaction mixture was heated to 50 C. and stirred for 2 h, before it was concentrated in vacuo, and the crude residue was purified via RP-HPLC. All fractions containing product were combined, concentrated to 5 mL in vacuo, prior to lyophilization, to recover Example 5 (31 mg, 8%) as a white powder.

[0326] MS (ESI-MS m/z): mass calcd for C.sub.40H.sub.71N.sub.6O.sub.5 [M+H].sup.+, 715.5 found 715.5.

[0327] .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 8.27 (d, J=8.0 Hz, 1H), 7.98-7.91 (m, 5H), 7.68-7.64 (m, 7H), 7.53 (t, J=8.0 Hz, 2H), 5.30-5.21 (m, 1H), 4.29-4.19 (m, 3H), 2.74 (t, J=7.6 Hz, 4H), 2.51 (t, J=2.0 Hz, 4H), 2.10 (t, J=7.6 Hz, 2H), 1.83-1.70 (m, 1H), 1.65-1.39 (m, 9H), 1.41-1.14 (m, 34H), 0.85 (t, J=6.4 Hz, 3H).

[0328] Compounds made following the synthesis of example 5:42, 128, 136, 137, 174, 175

Example 6

##STR00302##

[0329] Benzyl ter-butyl (6-(2-acetylhydrazineyl)-6-oxohexane-1,5-diyl (S)-dicarbamate[Cbz-Lys(Boc)-NHNHAc (BB2a]. To a DMF solution (13 ml) of CBz-Lys(Boc)-OH (500 mg, 1.3 mmol, 1.0 equiv) Acetyl Hydrazide (130 mg, 1.7 mmol, 1.3 equiv) was added, followed by HATU (550 mg, 1.4 mmol, 1.1 equiv) and DIPEA (080 L, 1.7 mmol, 1.3 equiv). The resulting mixture was left stirring over 16 h, before it was quenched by diluting with H.sub.2O (200 mL). The aqueous layer was extracted with EtOAc (2100 mL), and the combined organic layers were washed with brine (2100 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 0:100 to 1:9) to recover BB2a (500 mg, 86%) as transparent crystals.

[0330] R.sub.f=0.35 (SiO.sub.2, MeOH/DCM, 1:9)

[0331] HRMS (ESI-MS m/z): mass calcd for C.sub.21H.sub.32N.sub.4NaO.sub.6 [M+Na].sup.+, 459.22195, found 459.22252. Benzyl tert-butyl (1-(5-methyl-1,3,4-oxadiazol-2-yl)pentane-1,5-diyl)(S)-dicarbamate[Cbz-Lys(Boc)-Oxd-Me (BB2b)]. To a DCM solution (18 mL) of BB2a (850 mg, 1.9 mmol, 1.0 equiv),

[0332] TsCl (742 mg, 3.9 mmol, 2.0 equiv) was added, followed by DIPEA (1.6 mL, 12 mmol, 6.3 equiv) and DIPEA (690 L, 1.7 mmol, 1.3 equiv). The resulting mixture was left stirring over 5 h, before it was quenched by diluting with saturated aqueous NH.sub.4Cl (20 mL). After layer separation, the aqueous layer was extracted with DCM (240 mL), and the combined organic layers were washed with brine (240 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 0:100 to 1:9) to recover BB2b (590 mg, 72%) as a yellow oil.

[0333] R.sub.f=0.57 (SiO.sub.2, MeOH/DCM, 1:9)

[0334] HRMS (ESI-MS m/z): mass calcd for C.sub.21H.sub.30N.sub.4NaO.sub.5 [M+Na].sup.+, 441.21139, found 441.21214. Tert-butyl(S)-(5-amino-5-(5-methyl-1,3,4-oxadiazol-2-yl)pentyl)carbamate[H-Lys(Boc)-Oxd-Me (BB2)]. To a MeOH (18 mL) solution of Compound BB2b (580 mg, 1.4 mmol, 1.0 equiv) was added 10% Pd on Carbon (150 mg, 0.14 mmol, 0.1 equiv) followed by Hydrogen gas by means of a balloon. The resulting mixture was left stirring for 2 h before hydrogen was purged and backfilled with Ar, before filtering over Celite, washing with MeOH (20 mL). The filtrate was concentrated in vacuo to recover amine BB2 (390 mg, 98%) as a yellow oil.

[0335] R.sub.f=0.32 (SiO.sub.2, MeOH/DCM, 1:9)

[0336] HRMS (ESI-MS m/z): mass calcd for C.sub.12H.sub.24N.sub.4NaO.sub.3 [M+Na].sup.+, 307.17461, found 307.17739. Tert-butyl ((10S, 13S, 16S, 19S)-2,2,13,16-tetramethyl-10-(5-methyl-1,3,4-oxadiazol-2-yl)-4,12,15,18-tetraoxo-19-palmitamido-3-oxa-5,11,14,17-tetraazatricosan -23-yl)carbamate[Palmitoyl-Lys(Boc)-Ala-Ala-Lys(Boc)-Oxd-Me (Intermediate 2d)]. To a DMF (5.0 mL) solution of amine BB2 (140 mg, 0.50 mmol, 1.0 equiv) acid intermediate 2a (350 mg, 0.55 mmol, 1.1 equiv) was added at 0 C., followed by HATU (210 mg, 0.55 mmol, 1.1 equiv) and DIPEA (0.33 mL, 1.9 mmol, 3.5 equiv). The resulting mixture was left stirring 2 h, before it was quenched by diluting with H.sub.2O (150 mL) at 0 C. The so-formed precipitate was filtered, washed with H.sub.2O (15 mL) and recovered dissolving in MeOH (30 mL). The methanolic solution was concentrated in vacuo, and the crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 0:100 to 1:9) to recover peptide intermediate 2d (110 mg, 24%) as a white amorphous solid.

[0337] R.sub.f=0.36 (SiO.sub.2, MeOH/DCM, 1:9)

[0338] HRMS (ESI-MS m/z): mass calcd for C.sub.46H.sub.84N.sub.8O.sub.9 [M+H].sup.+, 893.64395, found 893.84690.

[0339] N(S)-6-amino-1-(((S)-1-(((S)-1-(((S)-5-amino-1-(5-methyl-1,3,4-oxadiazol-2-yl)pentyl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxohexan -2-yl) palmitamide bis-TFA [Palmitoyl-Lys-Ala-Ala-Lys-Oxd-Me bis-TFA salt (Example 6)]. To a DCM (2.0 mL) solution of Intermediate 2d (108 mg, 0.12 mmol, 1.0 equiv), TFA (2.0 mL) was added, and the resulting mixture was left stirring 2 h at RT. The reaction mixture was concentrated in vacuo and the crude residue was dissolved in H.sub.2O, filtered through a 0.20 m syringe filter, and purified using preparative RP-HPLC. All fractions containing product were combined, concentrated to 5 mL in vacuo, prior to lyophilization, to recover Example 6 (13 mg, 12%) as a white powder.

[0340] HRMS (ESI-MS m/z): mass calcd for C.sub.36H.sub.68N.sub.8O.sub.5 [M+H].sup.+, 693.53909, found 693.53875.

[0341] Compounds made following the synthesis of example 6:1, 2, 3, 4.

Example 7

##STR00303##

[0342] Benzyl tert-butyl (6-oxohexane-1,5-diyl)(S)-dicarbamate[Cbz-Lys(Boc)-H (BB3a)]. To a dry THE solution (50 mL) of BB1a (2.2 g, 5.2 mmol, 1.0 equiv), LiAlH.sub.4 (390 mg, 10.4 mmol, 2.0 equiv) was added at 0 C., and the resulting mixture was left stirring for 2 h. Next, the reaction mixture was quenched by the addition of 1 M aqueous KHSO.sub.4 (20 mL), before THF was concentrated in vacuo. The aqueous residue was diluted with 1 M aqueous HCl (20 mL) and Et.sub.2O (50 mL). After phase separation, the aqueous layer was extracted further with Et.sub.2O (350 mL), and the 10 combined organic layers were washed with 1 M aqueous HCl (320 mL), saturated aqueous NaHCO.sub.3 (320 mL), brine (320 mL), dried over MgSO4 and concentrated in vacuo, to obtain crude aldehyde BB3a (1.7 g, 89%) as a yellow oil. The crude material was used as such in the next synthetic step. Spectral and analytical data are in accordance to those published before by Lindgren and coworkers (24)

[0343] Benzyl tert-butyl ((5S)-6-cyano-6-hydroxyhexane-1,5-diyl)dicarbamate (BB3b). To a DCM solution (5 mL) of aldehyde BB3a (240 mg, 0.66 mmol, 1.0 equiv), acetone cyanohydrine (0.30 mL, 3.3 mmol, 5.0 equiv) was added, followed by TEA (0.91 mL, 6.6 mmol, 10 equiv), and the resulting mixture was left stirring for 16 h, at RT. Next, the reaction mixture was concentrated in vacuo, and the residue was dissolved in EtOAc (20 mL), and the organic layer was washed with H.sub.2O (310 mL), saturated aqueous NaHCO.sub.3 (10 mL), brine (10 mL), dried over MgSO4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 0:100 to 1:9) to recover cyanohydrine BB3b (250 mg, 96%) as a yellow oil.

[0344] R.sub.f=0.51 (SiO.sub.2, MeOH/DCM, 1:9).

[0345] .sup.1H NMR (400 MHZ, CDCl.sub.3) 7.36 (s, 5H), 5.43 (s, 1H), 5.13 (s, 2H), 4.58 (bs, 1H), 4.57 (d, J=16.3 Hz, 1H), 3.92 (s, 1H), 3.82 (s, 1H), 3.12 (m, 2H), 1.50 (m, 15H).

[0346] Benzyl tert-butyl ((5S)-7-amino-6-hydroxy-7-oxoheptane-1,5-diyl)dicarbamate (BB3c). To a MeOH solution (30 mL) of cyanohydrine BB3b (1.8 g, 4.6 mmol, 1.0 equiv), LiOH (130 mg, 5.5 mmol, 1.2 equiv) was added at 0 C., followed by H.sub.2O.sub.2 (10 mL, 115 mmol, 25 equiv), and the resulting mixture was left stirring for 4 h. Next, the reaction mixture was was quenched by diluting with 5% aqueous Na.sub.2S.sub.2O.sub.3 (30 mL), and the aqueous layer was extracted with DCM (3100 mL). The combined organic layers were dried over MgSO4 and concentrated in vacuo, the crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 0:100 to 1:9) to recover hydroxyamide BB3c (710 mg, 44%) as a white solid.

[0347] R.sub.f=0.47 (SiO.sub.2, MeOH/DCM, 1:9).

[0348] .sup.1H NMR (400 MHZ, CDCl.sub.3) 7.33-7.13 (m, 5H), 6.75 (s, 1H), 5.56 (s, 1H), 5.04 (s, 1H), 4.96 (d, J=7.6 Hz, OH), 4.24 (s, 0.5H), 4.14 (s, 0.5H), 3.78 (s, 1H), 2.68 (m, 2H), 2.18-1,85 (m, 2H), 1.45 (s, 5H), 1.43 (s, 4H).

[0349] Tert-butyl ((5S)-5,7-diamino-6-hydroxy-7-oxoheptyl)carbamate (BB3). To a MeOH (22 mL) solution of Cbz-protected hydroxyamide BB3c (920 mg, 2.2 mmol, 1.0 equiv), 10% Pd/C(230 mg, 0.22 mmol, 0.1 equiv) was added, and the mixture was left to stir in H.sub.2 atmosphere (1.0 atm) over 1 h. Next, the reaction mixture was filtered over Celite, washing the pad with MeOH (20 mL), the combined filtrate was concentrated in vacuo, and the residue was dissolved in dioxane (10 mL), prior to lyophilization, to recover free amine BB3 (610 mg, 99%) as a white solid. The crude material was used as such in the next synthetic step.

[0350] .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 7.20-7.13 (2 br s, 2H), 6.75 (t, 1H), 5.37 (bs, 1H), 3.71 (d, J=4.1 Hz, 1H), 3.65 (d, J=3.2 Hz, 1H), 2.89 (m, 2H), 2.79 (m, 1H), 1.84-1.69 (2 bs, 2H), 1.37 (s, 13H), 1.27-1.09 (m, 2H).

[0351] Tert-butyl ((10S, 13S, 16S,19S)-19-(2-amino-1-hydroxy-2-oxoethyl)-2,2,13,16-tetramethyl-4,11,14,17-tetraoxo-10-palmitamido-3-oxa-5,12,15,18-tetraazatricosan -23-yl)carbamate[Palmitoyl-Lys(Boc)-Ala-Ala-Lys(Boc)-CHOHCONH.sub.2 (Intermediate 2e)]. To a DMF (5.0 mL) solution of amine BB3 (100 mg, 0.36 mmol, 1.0 equiv) acid intermediate 2a (250 mg, 0.40 mmol, 1.1 equiv) was added at 0 C., followed by HATU (140 mg, 0.40 mmol, 1.1 equiv) and DIPEA (0.22 mL, 1.3 mmol, 3.5 equiv). The resulting mixture was left stirring 4 h, before it was quenched by diluting with H.sub.2O (50 mL) at 0 C. The so-formed precipitate was filtered, washed with H.sub.2O (15 mL) and recovered dissolving in MeOH (30 mL). The methanolic solution was concentrated in vacuo, and the crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 0:100 to 1:9) to recover peptide intermediate 2e (83 mg, 26%) as a white amorphous solid.

[0352] R.sub.f=0.41 (SiO.sub.2, MeOH/DCM, 1:9)

[0353] HRMS (ESI-MS m/z): mass calcd for C.sub.45H.sub.85N.sub.7NaO.sub.10 [M+Na].sup.+, 906.62556; found, 906.62468. Tert-butyl ((10S,13S,16S, 19S)-19-(2-amino-2-oxoacetyl)-2,2,13,16-tetramethyl-4,11,14,17-tetraoxo-10-palmitamido-3-oxa-5,12,15,18-tetraazatricosan -23-yl)carbamate[Palmitoyl-Lys(Boc)-Ala-Ala-Lys(Boc)-CONH.sub.2 (Intermediate 3a)]. To a DCM (8 mL) solution of Intermediate 2e (83 mg, 0.094 mmol, 1.0 equiv), DMP (130 mg, 0.28 mmol, 3.0 equiv) was added and the resulting mixture was left stirring for 12 h. Next, the reaction mixture was was quenched by diluting with saturated aqueous NaHCO.sub.3 (6 mL) and 10% aqueous Na.sub.2S.sub.2O.sub.3 (6 mL), and the resulting mixture was stirred for 0.33 h, before diluting with DCM (20 mL) and stirring additional 0.33 h. After layer separation, the organic layer was collected, washed with H.sub.2O (240 mL), dried over MgSO4 and concentrated in vacuo, to recover Intermediate 3a (82 mg, 99%) as a white solid. The crude material was used as such in the next synthetic step.

[0354] HRMS (ESI-MS m/z): mass calcd for C.sub.45H.sub.83N.sub.7NaO.sub.10 [M+Na].sup.+, 904.60991; found, 904.61264. N((S)-6-amino-1-(((S)-1-(((S)-1-(((S)-1,7-diamino-1,2-dioxoheptan -3-yl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxohexan -2-yl) palmitamide bis-TFA [Palmitoyl-Lys-Ala-Ala-Lys-CONH.sub.2 bis-TFA salt (Example 7)]. To a DCM (2.0 mL) solution of Intermediate 2d (83 mg, 0.094 mmol, 1.0 equiv), TFA (2.0 mL) was added, and the resulting mixture was left stirring 2 h at RT. The reaction mixture was concentrated in vacuo and the crude residue was dissolved in H.sub.2O, filtered through a 0.20 m syringe filter, and purified using preparative RP-HPLC. All fractions containing product were combined, concentrated to 5 mL in vacuo, prior to lyophilization, to recover Example 7 (29 mg, 34%) as a white powder.

[0355] HRMS (ESI-MS m/z): mass calcd for C.sub.35H.sub.66N.sub.7O.sub.5 [MH.sub.2O+H].sup.+, 664.51199, found 664.51246. Compounds made following the synthesis of example 7:5, 6, 9, 10, 176.

Example 8

##STR00304## ##STR00305##

[0356] (3aS,4S,6S,7aR)-2-(4-bromobutyl)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborole (BB4a). Catechol Borane (2.3 mL, 21 mmol, 1.2 equiv) was added to 4-bromobut-1-ene (2.2 mL, 22 mmol, 1.3 equiv) and the resulting mixture was heated to 100 C. for 3 h. After cooling to RT, the reaction mixture was added dropwise to a dry THF (20 mL) solution of (1S,2S,3R,5S)-2,6,6-trimethylbicyclo[3.1.1]heptane-2,3-diol (3.0 g, 18 mmol, 1.0 equiv) at 0 C., and the resulting mixture was warmed to RT and stirred for 16 h. Next, the reaction mixture was concentrated in vacuo and the crude residue was purified via flash column chromatography (SiO.sub.2. Heptane/EtOAc, from 99:1 to 9:1) to recover boronic ester BB4 (4.5 g, 81%) as a transparent oil.

[0357] .sup.1H NMR (400 MHZ, CDCl.sub.3) 4.26 (dd, J=8.7, 2.0 Hz, 1H), 3.41 (t, J=6.9 Hz, 2H), 2.34 (ddt, J=14.5, 8.7, 2.4 Hz, 1H), 2.22 (dtd, J=10.8, 6.1, 2.2 Hz, 1H), 2.08-2.00 (m, 1H), 1.95-1.79 (m, 4H), 1.63-1.51 (m, 3H), 1.38 (s, 3H), 1.29 (s, 3H), 1.09 (d, J=10.9 Hz, 1H), 0.84 (s, 5H).

[0358] (3aS,4S,6S,7aR)-2-((S)-5-bromo-1-chloropentyl)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborole (BB4b). To a THF/cyclohexane (2:1, 30 mL) solution of boronic ester BB4 (2.7 g, 8.7 mmol, 1.0 equiv) DCM (0.73 mL, 11 mmol, 1.3 equiv) was added at 20 C. Next, a 1M THF solution of LDA (11 mL, 11 mmol, 1.3 equiv) was added dropwise over 0.5 h, followed by a cold (20 C.).sub.1M THF solution of ZnCl2 (14 mL, 14 mmol, 1.6 equiv). The reaction mixture was warmed to RT and stirred for 16 h, before it was concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2. Heptane/EtOAc, from 9:1 to 20:3) to recover chlorohomologation product BB4b (3.2 g, 58%) as a transparent oil.

[0359] .sup.1H NMR (400 MHZ, CDCl.sub.3) 4.37 (dd, J=8.8, 2.0 Hz, 1H), 3.51-3.38 (m, 3H), 2.42-2.31 (m, 1H), 2.30-2.19 (m, 1H), 2.10 (d, J=5.0 Hz, 1H), 1.97-1.79 (m, 5H), 1.55 (d, J=0.7 Hz, 3H), 1.42 (s, 3H), 1.29 (d, J=3.8 Hz, 3H), 1.17 (d, J=11.0 Hz, 1H), 0.85 (s, 3H).

[0360] (R)-5-bromo-1-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pentan -1-amine hydrochloride (BB4). To a dry THF (13 mL) solution of chlorohomologation product BB4b (1.9 g, 5.3 mmol, 1.0 equiv) a 0.5 M toluene solution of KHMDS (13 mL, 6.4 mmol, 1.2 equiv) was added at 40 C., and the resulting mixture was stirred overnight. Next, the reaction mixture was concentrated in vacuo and the crude residue was suspended in heptane (15 mL), before being filtered through a CELITE pad, washing with heptane (15 mL). The filtrates were combined and concentrated in vacuo, and the residue was re-dissolved in heptane (15 mL), before it was diluted by the dropwise addition of a 4 M 1,4-dioxane solution of HCl (3.0 mL, 12 mmol, 2.1 equiv) at 20 C. The resulting mixture was stored at 20 C. for 16 h, before it was concentrated in vacuo, and the crude residue was triturated in heptane (15 mL). The solids were separated via decantation, washed with cold heptane (15 mL) and dried in vacuo, to recover amine hydrochloride BB4 (750 mg, 37%) as a white solid.

[0361] .sup.1H NMR (400 MHZ, cdcl.sub.3) 8.28 (s, 3H), 4.46-4.35 (m, 1H), 3.42 (td, J=6.9, 1.8 Hz, 2H), 2.96 (d, J=5.8 Hz, 1H), 2.40-2.18 (m, 2H), 2.10-2.03 (m, 1H), 1.95-1.80 (m, 5H), 1.80-1.56 (m, 3H), 1.43 (s, 3H), 1.29 (s, 3H), 1.16 (d, J=11.1 Hz, 1H), 0.83 (s, 3H).

[0362] Benzyl ((S)-6-(((S)-1-(((S)-1-(((R)-5-bromo-1-(3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pentyl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-6-oxo-5-palmitamidohexyl)carbamate[Palmitoyl-Lys (Cbz)-Ala-Ala-5-bromo-1-BO.sub.2Pin-pentylamide (Intermediate 2f)].To a DMF (8 mL) solution of amine BB4 (170 mg, 0.44 mmol, 1.0 equiv) acid intermediate 2b (290 mg, 0.44 mmol, 1.0 equiv) was added at 0 C., followed by HATU (180 mg, 0.48 mmol, 1.1 equiv) and DIPEA (0.17 mL, 0.96 mmol, 2.2 equiv). The resulting mixture was left stirring 2 h, before it was quenched by diluting with H.sub.2O (80 mL) at 0 C. The so-formed precipitate was filtered, washed with H.sub.2O (15 mL) and recovered dissolving in MeOH (30 mL). The methanolic solution was concentrated in vacuo, the crude residue was purified via flash column chromatography (SiO.sub.2, CHCl.sub.3/MeCN, from 7:3 to 1:4) to recover peptide intermediate 2f (152 mg, 35%) as a white amorphous solid.

[0363] R.sub.f=0.3 (SiO.sub.2, CHCl.sub.3/MeCN, 1:4)

[0364] LCMS (ESI-MS m/z): mass calcd for C.sub.51H.sub.86BBrN.sub.5O.sub.8 [M+H].sup.+, 986.57; found, 986.88.

[0365] Benzyl ((S)-6-(((S)-1-(((S)-1-(((R)-5-azido-1-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pentyl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-6-oxo-5-palmitamidohexyl)carbamate[Palmitoyl-Lys (Cbz)-Ala-Ala-5-azido-1-BO.sub.2Pin-pentylamide (Intermediate 3b)]. To a DMF (1.5 mL) solution of intermediate2 f (150 mg, 0.15 mmol, 1.0 equiv), NaN.sub.3 (9.7 mg, 0.15 mmol, 1.0 equiv) was added, and the resulting mixture was heated to 100 C. and stirred for 1 h. The reaction mixture was quenched by diluting with H.sub.2O (20 mL) at 0 C., and the so-formed precipitate was filtered, washed with H.sub.2O (10 mL) and recovered dissolving in MeOH (15 mL). The methanolic solution was concentrated in vacuo, to recover azide intermediate 3b (130 mg, 91%) as a white amorphous solid. The crude material was used as such in the next synthetic step.

[0366] LCMS (ESI-MS m/z): mass calcd for C.sub.51H.sub.86BN.sub.8O.sub.8 [M+H].sup.+, 949.67; found, 949.32.

[0367] N((S)-6-amino-1-(((S)-1-(((S)-1-(((R)-5-amino-1-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahydro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)pentyl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxohexan -2-yl) palmitamide[Palmitoyl-Lys-Ala-Ala-5-amine-1-BO.sub.2Pin-pentylamide (Intermediate 4a)]. To a MeOH (10 mL) solution of intermediate 3b (130 mg, 0.13 mmol, 1.0 equiv) 10% wt Pd/C(29 mg, 0.027 mmol, 0.2 equiv) was added, followed by H.sub.2 (balloon). The resulting mixture was left stirring for 1.5 h, before a 4 N 1,4-dioxane solution of HCl (0.067 mL, 0.27 mmol, 2.0 equiv) was added, and the resulting mixture was stirred for 0.5h. Next, the reaction mixture was filtered through a CELITE pad, washing with MeOH (15 mL). The filtrates were combined and concentrated in vacuo to recover intermediate 4a (111 mg, 95%) as a white amorphous solid. The crude material was used as such in the next synthetic step.

[0368] LCMS (ESI-MS m/z): mass calcd for C.sub.43H.sub.82BN.sub.6O.sub.6 [M+H].sup.+, 789.64; found, 789.40.

[0369] ((5R,8S,11S,14S)-1-amino-14-(4-aminobutyl)-8,11-dimethyl-7,10,13,16-tetraoxo-6,9,12,15-tetraazahentriacontan -5-yl) boronic acid bis-TFA [Palmitoyl-Lys-Ala-Ala-5-amine-1-B(OH).sub.2-pentylamide (Example 8)]. To a Heptane/MeOH (1:1, 10 mL) mixture of intermediate 4a (100 mg, 0.12 mmol, 1.0 equiv) isobutyl boronic acid (71 mg, 0.70 mmol, 6.0 equiv) was added, followed by a 1 M aqueous HCl (0.46 mL, 0.46 mmol, 6.0 equiv). The resulting mixture was stirred for 16 h, before it was diluted with heptane (5 mL) and MeOH (5 mL). After layer separation, the heptane layer was further extracted with MeOH (5 mL), and the methanolic layers were combined, washed with heptane (210 mL), and concentrated in vacuo. The crude residue was dissolved in H.sub.2O, filtered through a 0.20 m syringe filter, and purified using preparative RP-HPLC(isocratic 30% MeCN in H.sub.2O over 5 min, gradient from 30 to 100% MeCN in H.sub.2O over 30 min). All fractions containing product were combined, concentrated to 5 mL in vacuo, prior to lyophilization, to recover Example 8 (16 mg, 15%) as a white powder.

[0370] HRMS (ESI-MS m/z): mass calcd for C.sub.33H.sub.67BN.sub.6NaO.sub.6 [M+Na].sup.+, 677.51074; found, 677.51419. Compounds made following the synthesis of example 8:39, 41, 48, 60, 61, 182, 183.

Example 9

##STR00306##

[0371] (R)-2-amino-2-(4-(benzyloxy)phenyl) acetic acid (BB5a). To a H.sub.2O (20 mL) solution of CuSO.sub.4 (1.8 g, 36 mmol, 3.0 equiv), a H.sub.2O (20 mL) solution of 1 N aqueous NaOH (24 mL, 24 mmol, 2.0 equiv) and H-(4-hydroxy) Phg-OH (2.0 g, 12 mmol, 1.0 equiv) was added at 50 C. The resulting mixture was stirred at that temperature for 0.5 h, before it was cooled to 0 C. and stirred further 0.25 h. The so-formed precipitate was filtered, washed with H.sub.2O (20 mL) and dried. Next, the solid was take up in MeOH (40 mL), and the resulting suspension was diluted with 1 N aqueous NaOH (12 mL, 12 mmol, 1.0 equiv), followed by the addition of BnBr (2.2 g, 12 mmol, 1.1 equiv). The resulting mixture was stirred for 16 h at RT, before the so-formed precipitate was filtered, washed with H.sub.2O (100 mL) and triturated in 1 N aqueous HCl (250 mL). The precipitated was filtered, washed with H.sub.2O (100 mL), Et.sub.2O (100 mL) and dried in vacuo, to recover AA BB5a (700 mg, 23%), as an off-white solid.

[0372] LCMS (ESI-MS m/z): mass calcd for C15H16NO3 [M+H].sup.+, 258.11; found, 258.34.

[0373] (R)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(4-(benzyloxy)phenyl) acetic acid [Fmoc-(OBn)-Hpg-OH(BB5)]. To a H.sub.2O (14 mL) solution of BB5a (0.70 g, 2.7 mmol, 1.0 equiv),

[0374] NaHCO.sub.3 (0.46 g, 5.4 mmol, 2.0 equiv) was added, followed by a acetone (14 mL) solution of Fmoc-OSu (1.0 g, 2.9 mmol, 1.1 equiv). The resulting mixture was stirred at RT for 16 h, before it was quenched by the addition of saturated aqueous KHSO.sub.4 and the aqueous layer was extracted with EtOAc (3100 mL). The combined organic layer were washed with brine (100 mL), dried over NA.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, DCM/MeOH, from 19:1 to 9:1), to recover Fmoc-(OBn)-Hpg-OH BB5 (500 mg, 39%) as an off-white solid.

[0375] R.sub.f=0.5 (SiO.sub.2, MeOH/DCM, 1:9)

[0376] LCMS (ESI-MS m/z): mass calcd for C30H.sub.26NO5 [M+H].sup.+, 480.18; found, 480.36.

[0377] N((S)-6-amino-1-(((S)-1-(((S)-1-(((S)-2-amino-1-(4-(benzyloxy)phenyl)-2-oxoethyl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxohexan -2-yl) palmitamide TFA [Palmitoyl-Lys-Ala-Ala-(OBn) Hpg-NH.sub.2 TFA salt (Example 9)]. was prepared according the general procedure for SPPS using 1.5 g Rink amide AM resin (0.90 mmol). The crude peptide was purified by preparative RP-HPLC, all fractions containing the product were combined and concentrated in vacuo to 5 mL, prior to lyophilization, to afford peptide Example 9 (19 mg) as a white powder.

[0378] HRMS (ESI-MS m/z): mass calcd for C.sub.43H.sub.69N.sub.6O.sub.6 [M+H].sup.+, 765.5273; found, 765.5255. Compounds made following the synthesis of example 9:129, 130, 138, 139.

Example 10

##STR00307##

[0379] 4-(2-chloroethyl)-1H-imidazole (BB6b). To a 4 M 1,4-dioxane solution of HCl (75 mL), 2-(1H-imidazol-4-yl)ethan -1-ol (BB6a)(15 g, 130 mmol, 1.0 equiv) was added, and the resulting mixture was stirred for 0.5 at RT. Next, the reaction mixture was concentrated in vacuo, and the residue was co-evaporated with toluene (250 mL), before it was dissolved in CCl.sub.4 (75 mL) and cooled to 0 C. Then, SOCl.sub.2 (75 mL) was added, and the resulting mixture was stirred at RT for 16 h, before being warmed to 90 C. and stirred at that temperature for 0.5 h. After cooling to RT, the reaction mixture was diluted with benzene (150 mL), and the so-formed precipitate was washed with Et.sub.2O (100 mL), to recover crude chloride BB6b (15 g, 67%), which was used as such in the next synthetic step.

[0380] LCMS (ESI-MS m/z): mass calcd for C.sub.6H.sub.8ClN.sub.2 [M+H].sup.+, 131.04; found, 131.05.

[0381] 2-Acetamido-4-(1H-imidazol-4-yl) butanoic acid [Ac-hHis-OH(BB6c)]. To a EtOH (200 mL) solution of chloride BB6b (20 g, 150 mmol, 1.0 equiv) was added a EtOH (175 mL) solution of Na (8.8 g, 380 mmol, 2.5 equiv) dropwise at 0 C., over the course of 10 minutes. To the resulting mixture, a EtOH (25 mL) solution of Diethyl acetamidomalonate (50 g, 230 mmol, 1.5 equiv) was added dropwise over 10 minutes, before the reaction mixture was heated to 85 C. and stirred for 5 h. After cooling to RT, the reaction mixture was quenched by the addition of 1 N aqueous HCl (200 mL), and concentrated in vacuo. The crude residue was washed with pet-ether (300 mL) to recover crude Ac-hHis-OH BB6c (30 g, quant.) as a brown gum, which was used as such in the next synthetic step.

[0382] LCMS (ESI-MS m/z): mass calcd for C.sub.9H.sub.13N.sub.3O.sub.3 [M+H].sup.+, 212.10; found, 212.25.

[0383] 2-Amino-4-(1H-imidazol-4-yl) butanoic acid [H-hHis-OH(BB6d)]. To 6 N aqueous HCl (300 mL), Ac-hHis-OH BB6c (30 g, 120 mmol, 1.0 equiv) was added, and the resulting mixture was heated to 110 C., stirring for 5 h. Next, the reaction mixture was quenched by the addition of 2 M aqueous NaOH (1.0 L), and concentrated in vacuo, and the crude residue was purified via preparative RP-HPLC, to recover H-hHis-OH BB6d (5.5 g, 27%) as an off-white solid. LCMS (ESI-MS m/z): mass calcd for C.sub.7H.sub.12N.sub.3O.sub.2 [M+H].sup.+, 170.09; found, 170.12.

[0384] 2-(1,3-dioxoisoindolin-2-yl)-4-(1H-imidazol-5-yl) butanoic acid [PHTI-hHis-OH(Not isolated)]. To a H.sub.2O (55 mL) solution of H-hHis-OH BB6d (4.5 g, 27 mmol, 1.0 equiv), Na.sub.2CO.sub.3 (2.8 g, 27 mmol, 1.0 equiv) was added, followed by N-carbethoxyphthalimide (5.8 g, 27 g, 1.0 equiv). The resulting mixture was stirred at RT for 3, before it was quenched by the addition of 1 N aqueous HCl (75 mL) and concentrated in vacuo. The crude residue was suspended in MeOH (100 mL), filtered, and the filtrate was concentrated in vacuo, to recover crude PTHI-hHis-OH (8.1 g, quant.) as a brown solid, which was used as such in the next synthetic step.

[0385] LCMS (ESI-MS m/z): mass calcd for C.sub.15H.sub.14N.sub.3O.sub.4 [M+H].sup.+, 300.10; found, 300.21.

[0386] 2-(1,3-dioxoisoindolin-2-yl)-4-(1-(diphenyl(p-tolyl)methyl)-1H-imidazol-5-yl) butanoic acid [PHTI-hHis (Mmt)-OH(BB6e)]. To a DMF/DCM (1:2, 90 mL) solution of crude PHTI-hHis-OH (8.1 g, 27 mmol, 1.0 equiv), TEA (15 mL, 110 mmol, 4.0 equiv) was added at 0 C., followed by Mmt-Cl (15 g, 53 mmol, 2.0 equiv). The resulting mixture was warmed to RT and stirred for 16 h, before it was quenched by the addition of saturated aqueous KHSO.sub.4 (200 mL). The aqueous layer was extracted with EtOAc (3200 mL), and the combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo, the crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 1:19 to 1:9) to recover PHTI-hHis (Mmt)-OH BB6e (2.4 g, 16%) as an off-white solid.

[0387] LCMS (ESI-MS m/z): mass calcd for C.sub.35H.sub.30N.sub.3O.sub.4 [M+H].sup.+, 556.22; found, 556.49.

[0388] 2-Amino-4-(1-(diphenyl(p-tolyl)methyl)-1H-imidazol-5-yl) butanoic acid [H-hHis (Mmt)-OH(BB6f)]. To a EtOH (40 mL) solution of PTHI-hHis (Mmt)-OH BB6e (2.0 g, 3.6 mmol, 1.0 equiv), hydrazine hydrate (0.34 mL, 7.2 mmol, 2.0 equiv) was added, and the resulting mixture was stirred for 3 h at RT. Next, the so-formed solids were filtered, and the filtrate was concentrated in vacuo, to recover crude H-hHis (Mmt)-OH BB6f (2.0 g, quant) as an off-white solid, which was used as such in the next synthetic step.

[0389] LCMS (ESI-MS m/z): mass calcd for C.sub.27H.sub.28N.sub.3O.sub.2 [M+H].sup.+, 426.22; found, 426.46 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(1-{diphenyl(p-tolyl)methyl)-1H-imidazol-5-yl) butanoic acid [Fmoc-(+/) hHis (Mmt)-OH (rac-BB6)]. To a H.sub.2O (20 mL) solution of H-hHis (Mmt)-OH BB6f (2.0 g, 4.7 mmol, 1.0 equiv), NaHCO.sub.3 (0.78 g, 9.3 mmol, 2.0 equiv) was added, before the reaction mixture was cooled to 0 C., and an acetone (20 mL) solution of Fmoc-OSu (2.3 g, 7.0 mmol, 1.5 equiv) was added drop-wise. The resulting mixture was warmed to RT and stirred for 16 h, before it was quenched by the addition of saturated aqueous KHSO.sub.4 The aqueous layer was extracted with EtOAc (3200 mL), the combined organic layers were dried over Na.sub.2SO.sub.4 and it was concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM+1% AcOH, from 1:9 to 3:15) to recover Fmoc-(+/) hHis (Mmt)-OH rac-BB6 (1.5 g, 57%) as a light-yellow gum.

[0390] LCMS (ESI-MS m/z): mass calcd for C.sub.42H.sub.38N.sub.3O.sub.4 [M+H].sup.+, 648.29; found, 648.47

[0391] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(1-(diphenyl(p-tolyl)methyl)-1H-imidazol-5-yl) butanoic acid [Fmoc-(S)-hHis (Mmt)-OH(L-BB6)]. Fmoc-(+/) hHis (Mmt)-OH (rac-BB6) was purified by chiral SFC, to recover two sets of fractions. The set of fractions eluting at first showed negative optical rotation value [ ].sub.D.sup.25=31.16, therefore the structure was assigned to be the D-enantiomer. Concentration in vacuo of the secondly eluting fraction allowed to recover Fmoc-deprotected L-BB6, which was re-submitted to Fmoc-protection as in the synthesis of rac-BB6, to recover Fmoc-(+) hHis (Mmt)-OH L-BB6 (400 mg, 27%) as a white powder.

[0392] LCMS (ESI-MS m/z): mass calcd for C.sub.42H.sub.38N.sub.3O.sub.4 [M+H].sup.+, 648.29; found, 648.47

[0393] N((S)-6-amino-1-(((S)-1-(((S)-1-(((S)-1-amino-4-(1H-imidazol-4-yl)-1-oxobutan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxohexan -2-yl) palmitamide bis-TFA [Palmitoyl-Lys-Ala-Ala-hHis-NH.sub.2 bis-TFA salt (Example 10)]. was prepared according the general procedure for SPPS using 0.4 g Rink amide AM resin (0.90 mmol). The crude peptide was purified by preparative RP-HPLC, all fractions containing the product were combined and concentrated in vacuo to 5 mL, prior to lyophilization, to afford peptide Example 10 (8.2 mg, 6%) as a white powder.

[0394] HRMS (ESI-MS m/z): mass calcd for C.sub.35H.sub.65N.sub.8O.sub.5 [M+H].sup.+, 677.5072; found, 677.5044. Compounds made following the synthesis of example 10:59, 165, 166, 172, 173.

Example 11

##STR00308## ##STR00309##

[0395] Methyl(S)-2-amino-3-(3-nitrophenyl) propanoate hydrochloride (BB7b). To a MeOH (80 mL) solution of H-(m-NO2) Phe-OH BB7a (4.0 g, 19 mmol, 1.0 equiv), SOCl.sub.2 (2.3 mL, 32 mmol, 1.7 equiv) was added at 0 C. The resulting mixture was warmed to RT, then heated to 50 C., and stirred for 6 h, before being concentrated in vacuo. The crude residue was triturated in Et.sub.2O (100 mL), to recover hydrochloride BB7b (4.0 g, 94%) as an off white solid.

[0396] LCMS (ESI-MS m/z): mass calcd for C.sub.10H.sub.13N.sub.2O.sub.4 [M+H].sup.+, 225.09; found, 225.06.

[0397] methyl(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-nitrophenyl) propanoate[Fmoc-(m-NO2)-Phe-OMe (BB7c)]. To a H.sub.2O (50 mL) solution of hydrochloride BB7b (5.0 g, 22 mmo, 1.0 equiv), Na.sub.2CO.sub.3 (4.7 g, 44 mmol, 2.0 equiv) was added, followed by a MeCN (50 mL) solution of Fmoc-OSu (11 g, 33 mmol, 1.5 equiv). The resulting mixture was stirred for 16 h at RT, before it was diluted with H.sub.2O (50 mL), and extracted with EtOAc (3100 mL). The combined organic layer was washed with brine (100 mL), dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, 1:4) to recover Fmoc-(m-NO2)-Phe-OMe BB7c (3.2 g, 33%) as an off-white solid.

[0398] R.sub.f=0.6 (SiO.sub.2, EtOAc/pet-ether, 3:2).

[0399] LCMS (ESI-MS m/z): mass calcd for C.sub.25H.sub.23N.sub.2O.sub.6 [M+H].sup.+, 447.16; found 447.18.

[0400] Methyl(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-aminophenyl) propanoate[Fmoc-(m-NH.sub.2)-Phe-OMe (BB7d)]. To a MeOH/EtOH (1:1, 64 mL) solution of Fmoc-(m-NO2)-Phe-OMe BB7c (3.2 g, 7.2 mmol, 1.0 equiv), 10% wt Pd/C(0.32 g, 10% wt) was added, followed by H.sub.2 (balloon). The resulting mixture was stirred at RT for 4 h, before it was filtered over a CELITE pad, washing with MeOH (20 mL). The filtrate was collected and concentrated in vacuo, to recover amine BB7d (2.9 g, 97%) as a brown oil.

[0401] LCMS (ESI-MS m/z): mass calcd for C.sub.25H.sub.25N.sub.2O.sub.4 [M+H].sup.+, 417.18; found, 417.14.

[0402] Methyl (S,Z)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(2,3-bis(tert-butoxycarbonyl) guanidino)phenyl) propanoate[Fmoc-[m-(bis-Boc) guanidyl]-Phe-OMe (BB7e)]. To a DMF (60 mL) solution of amine BB7d (3.0 g, 7.2 mmol, 1.0 equiv), DIPEA (3.8 mL, 22 mmol, 3.0 equiv) was added at 0 C., followed by SM-1 (4.0 g, 13 mmol, 2.0 equiv) and DMAP (87 mg, 0.72 mmol, 0.1 equiv). The resulting mixture was warmed to RT an stirred for 16h, before in was quenched by the addition of H.sub.2O (45 mL). The aqueous layer was extracted with EtOAc (3 60 mL), and the combined organic layer was washed with brine (60 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, from 1:9 to 1:5) to recover bis-boc-guanidyl BB7e (1.6 g, 34%) as a pale yellow liquid.

[0403] LCMS (ESI-MS m/z): mass calcd for C.sub.36H.sub.43N.sub.4O.sub.8 [M+H].sup.+, 659.31; found, 659.26.

[0404] (S,Z)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-(3-(2,3-bis(tert-butoxycarbonyl) guanidino)phenyl) propanoic acid [Fmoc-[m-(bis-Boc) guanidyl]-Phe-OH(BB7)]. To a H.sub.2O (7.5 mL) solution of NaOH (97 mg, 2.4 mmol, 1.0 equiv), 0.8 M aqueous CaCl.sub.2) (1.6 mL) was added at 0 C., followed by an IPA (16 mL) solution of methyl ester BB7e (1.6 g, 1.4 mL, 1.0 equiv). The resulting mixture was warmed to RT and stirred for 1 h, before it was quenched by the addition of saturated aqueous KHSO.sub.4 (until pH 4). The aqueous layer was extracted with MeOH/DCM (1:9, 350 mL), and the combined organic layer was washed with brine (50 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, from 7:3 to 4:1) to recover Fmoc-AA-OH BB7 (1.6 g, 34%) as a pale yellow liquid.

[0405] R.sub.f=0.3 (SiO.sub.2, DCM/MeOH, 9:1).

[0406] LCMS (ESI-MS m/z): mass calcd for C.sub.35H.sub.41N.sub.4O.sub.8 [M+H].sup.+, 645.29; found, 645.22.

[0407] (S)N.sub.1((S)-1-amino-1-oxo-3-phenylpropan -2-yl)-2-((S)-3-(3-guanidinophenyl)-2-palmitamidopropanamido)pentanediamide bis-TFA [Palmitoyl-(m-guanidyl) Phe-GIn-Phe-NH.sub.2 bis-TFA salt (Example 11)]. was prepared according the general procedure for SPPS using 1.0 g Rink amide AM resin (0.90 mmol), ad exception of the amide coupling of BB7, which was performed using DIC and Oxyma. The crude peptide was purified by preparative RP-HPLC, all fractions containing the product were combined and concentrated in vacuo to 5 mL, prior to lyophilization, to afford peptide Example 11 (33 mg, 12%) as a white powder.

[0408] HRMS (ESI-MS m/z): mass calcd for C.sub.40H.sub.63N.sub.8O.sub.5 [M+H].sup.+, 735.4916; found, 735.4905.

[0409] Compounds made following the synthesis of example 11:67, 68.

Example 12

##STR00310## ##STR00311##

[0410] 1-(Tort-butyl).sub.2-methyl (2S,4R)-4-(pyridin-4-yloxy) pyrrolidine-1,2-dicarboxylate [Boc-trans-(O-pyridinyl) Hyp-OMe (BB8b)]. To a toluene (38 mL) solution of BB8a (1.0 g, 4.1 mmol, 1.0 equiv), 4-hydroxypyridine (580 mg, 6.1 equiv, 1.5 equiv) was added at 0 C., followed by a 40% toluene solution of DEAD (2.4 mL, 6.1 mmol, 1.5 equiv). The resulting mixture was warmed to RT and stirred for 24 h, before it was concentrated in vacuo, and the crude residue was purified via filtration over a silica plug. The plug was washed with Et.sub.2O (100 mL) to remove the impurities, before the product was stripped down by washing the plug with DCM/MeOH (9:1, 200 mL). The DCM/MeOH fraction was concentrated in vacuo, to recover Mitsunobu product BB8b (910 mg, 70%) as a white amorphous solid.

[0411] R.sub.f=0.60 (SiO.sub.2, DCM/MeOH, 9:1).

[0412] .sup.1H NMR (400 MHZ, CDCl.sub.3) 8.47-8.39 (m, 2H), 6.75-6.67 (m, 2H), 4.96 (m, 1H), 4.57*(t, J=5.6 Hz, 0.4H), 4.45 (dd, J=8.9, 2.8 Hz, 0.6H), 3.81-3.63 (m, 5H), 2.61-2.46 (m, 2H), 1.48*(s, 4H), 1.44 (s, 5H)(*=minor rotamer).

[0413] (2S,4R)-1-(tert-butoxycarbonyl)-4-(pyridin-4-yloxy) pyrrolidine-2-carboxylic acid [Boc-trans-(O-pyridinyl) Hyp-OH(BB8)]. To a THF (15 mL) solution of methyl ester BB8b (900 mg, 2.8 mmol, 1.0 equiv), 1.0 M aqueous LiOH (15 mL, 15 mmol, 5.3 equiv) at 0 C. The resulting mixture was stirred for 2 h, before it was quenched by the addition of 1 M aqueous HCl (until pH=1), and it concentrated in vacuo to half its volume. The aqueous layer was extracted with IPA/CHCl.sub.3 (1:3, 330 mL), the combined organic layers were dried over Na.sub.2SO.sub.4, and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, DCM/MeOH/AcOH, from 46:4:0 to 45:4:1) to recover carboxylic acid BB8 (0.15 g, 17%) as a white solid. R.sub.f=0.43 (SiO.sub.2, DCM/MeOH/AcOH, 45:4:1).

[0414] .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 8.35-8.30 (m, 2H), 6.93-6.87 (m, 2H), 5.06-4.98 (m, 1H), 4.15-4.02 (m, 1H), 3.68-3.54 (m, 1H), 3.50-3.41 (m, 1H), 2.36-2.12 (m, 2H), 1.30*(s, 4H), 1.28 (s, 5H)(*=minor rotamer).

[0415] Tert-butyl ((S)-1-(((S)-1-amino-6-(((benzyloxy)carbonyl)amino)-1-oxohexan -2-yl)amino)-1-oxopropan -2-yl)carbamate[Boc-Ala-Lys (Cbz) NH.sub.2 (BB9c)]. To a DCM (25 mL) solution of amine hydrochloride H-Lys (Cbz)-NH.sub.2 BB9b (730 mg, 2.3 mmol, 1.0 equiv), carboxylic acid Boc-Ala-OH BB9a (480 mg, 2.5 mmol, 1.1 equiv) was added at 0 C., followed by HATU (950 mg, 2.5 mmol, 1.1 equiv) and DIPEA (1.4 mL, 8.0 mmol, 3.5 equiv). The resulting mixture was left stirring 2 h, before it was quenched by diluting with 15% wt aqueous citric acid (30 mL) at 0 C. After layer separation, the aqueous layer was further extracted with DCM (330 mL), and the combined organic layers were washed with saturated aqueous NaHCO.sub.3 (30 mL), H.sub.2O (30 mL) and brine (30 mL). The organic layer was dried over Na.sub.2SO.sub.4, and concentrated in vacuo, to recover crude dipeptide BB9c (1.1 g, 97) as an off-white solid, which was used as such in the next synthetic step.

[0416] .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 7.59 (d, J=8.2 Hz, 1H), 7.36-7.20 (m, 5H), 7.29 (d, J=2.3 Hz, 1H), (m, 1H), 7.00-6.93 (m, 2H), 4.95 (s, 2H), 4.11 (m, 1H), 3.89 (m, 1H), 2.91 (q, J=6.6 Hz, 2H), 1.65-1.53 (m, 1H), 1.51-1.38 (m, 1H), 1.32 (s, 11H), 1.25-1.15 (m, 2H), 1.12 (d, J=6.5 Hz, 3H).

[0417] Benzyl ((S)-6-amino-5-((S)-2-aminopropanamido)-6-oxohexyl)carbamate HCl [H-Ala-Lys (Cbz) NH.sub.2 HCl salt (BB9)]. To a 1,4-dioxane (8 mL) solution of dipeptide BB9c (1.0 g, 2.2 mmol, 1.0 equiv), a 4 N 1,4-dioxane solution of HCl (16 mL, 2V) was added at 0 C., and the resulting mixture was stirred for 1 h. The reaction mixture was concentrated in vacuo, and the crude residue was triturated in Et.sub.2O (330 mL), before the so-formed solids were dissolved in DCM (50 mL). The DCM solution was concentrated in vacuo, to recover amine hydrochloride BB9 (880 mg, 90%) as transparent crystals.

[0418] .sup.1H NMR (400 MHZ, DMSO-d.sub.6) 8.53 (d, J=8.1 Hz, 1H), 8.32-8.21 (m, 3H), 7.50-7.40 (m, 1H), 7.30-7.29 (m, 1H), 7.37-7.19 (m, 5H), 7.02-6.96 (m, 1H), 4.95 (s, 1H), 4.18-4.10 (m, 1H), 3.89-3.78 (m, 1H), 2.97-2.87 (m, 2H), 1.74-1.45 (m, 2H), 1.31 (d, J=6.9 Hz, 3H), 1.39-1.16 (m, 4H).

[0419] Tert-butyl (2S,4R)-2-(((S)-1-(((S)-1-amino-6-(((benzyloxy)carbonyl)amino)-1-oxohexan -2-yl)amino)-1-oxopropan -2-yl)carbamoyl)-4-(pyridin-4-yloxy) pyrrolidine-1-carboxylate [Boc-trans-(O-pyridinyl) Hyp-Ala-Lys (Cbz) NH.sub.2 (Intermediate 4b)]. To a DMF (7.0 mL) solution of amine hydrochloride BB9 (190 mg, 0.49 mmol, 1.0 equiv), carboxylic acid BB9 (150 mg, 0.49 mmol, 1.0 equiv) was added at 0 C., followed by HATU (200 mg, 0.53 mmol, 1.1 equiv) and DIPEA (0.34 mL, 1.9 mmol, 4.0 equiv). The resulting mixture was left stirring 3 h, before it was quenched by diluting with H.sub.2O (80 mL) at 0 C. The aqueous layer was extracted with IPA/CHCl.sub.3 (1:3, 3100 mL), and the combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, DCM/MeOH, from 1:0 to 9:1) to recover tripeptide intermediate 4b (0.30 g, 95%) as a white powder. Benzyl ((S)-6-amino-6-oxo-5-((S)-2-((2S,4R)-4-(pyridin-4-yloxy) pyrrolidine-2-carboxamido) propanamido) hexyl)carbamate HCl [H-trans-(O-pyridinyl) Hyp-Ala-Lys (Cbz)-NH.sub.2 HCl salt (Intermediate 4)]. To a 1,4-dioxane (2.5 mL) solution of dipeptide BB9c (0.3 g, 0.42 mmol, 1.0 equiv), a 4 N 1,4-dioxane solution of HCl (5 mL) was added at 0 C., and the resulting mixture was stirred for 1 h. The reaction mixture was concentrated in vacuo, and the crude residue was triturated in Et.sub.2O (330 mL), before the so-formed solids were dissolved in DCM (50 mL). The DCM solution was concentrated in vacuo, to recover amine hydrochloride Intermediate 4 (280 mg, 95%) as a white powder. Methyl palmitoyl-L-alaninate [Palmitoyl-Ala-OMe (Intermediate 3a)]. To a DCM (35 mL) solution of amine hydrochloride H-Ala-OMe BB9d (550 mg, 3.9 mmol, 1.0 equiv), palmitic acid (1.0 g, 3.9 mmol, 1.0 equiv) was added at 0 C., followed by HATU (1.6 g, 4.3 mmol, 1.1 equiv) and DIPEA (2.1 mL, 12 mmol, 3.0 equiv). The resulting mixture was left stirring 2 h, before it was quenched by diluting with 15% wt aqueous citric acid (30 mL) at 0 C. After layer separation, the aqueous layer was further extracted with DCM (330 mL), and the combined organic layers were washed with saturated aqueous NaHCO.sub.3 (30 mL), H.sub.2O (30 mL) and brine (30 mL). The organic layer was dried over Na.sub.2SO.sub.4, and concentrated in vacuo, to recover crude dipeptide BB9c (1.1 g, 97%) as an off-white solid, which was used as such in the next synthetic step.

[0420] .sup.1H NMR (400 MHZ, CDCl.sub.3) 6.07 (d, J=7.4 Hz, 1H), 4.61 (p, J=7.2 Hz, 1H), 3.75 (s, 3H), 2.25-2.16 (m, 2H), 1.69-1.57 (m, 2H), 1.40 (d, J=7.1 Hz, 3H), 1.25 (m, 24H), 0.88 (t, J=7.2, 3H). Palmitoyl-L-alanine[Palmitoyl-Ala-OH(Intermediate 3)]. To a THF (15 mL) solution of methyl ester BB8b (840 mg, 2.5 mmol, 1.0 equiv), 1.0 M aqueous LiOH (13 mL, 13 mmol, 5.2 equiv) at 0 C. The resulting mixture was stirred for 2 h, before it was quenched by the addition of 1 M aqueous HCl (until pH=1), and it concentrated in vacuo to half its volume. The aqueous layer was extracted with CHCl.sub.3 (320 mL), the combined organic layers were dried over Na.sub.2SO.sub.4, and concentrated in vacuo, to recover crude carboxylic acid BB8 (0.72 g, 90%) as a white amorphous solid, which was used as such in the next synthetic step.

[0421] .sup.1H NMR (400 MHZ, CDCl.sub.3) 6.25 (d, J=7.1 Hz, 1H), 4.58 (p, J=7.1 Hz, 1H), 2.27-2.18 (m, 2H), 1.69-1.56 (m, 2H), 1.45 (d, J=7.1 Hz, 3H), 1.33-1.23 (m, 24H), 0.88 (d, J=6.6 Hz, 3H). Benzyl ((S)-6-amino-6-oxo-5-((S)-2-((2S,4R)-1-(palmitoyl-L-alanyl)-4-(pyridin-4-yloxy) pyrrolidine-2-carboxamido) propanamido) hexyl)carbamate[Palmitoyl-Ala-trans-(O-pyridinyl) Hyp-Ala-Lys (Cbz) NH.sub.2 (Intermediate 4c)]. To a DMF (6.0 mL) solution of amine hydrochloride BB9 (300 mg, 0.52 mmol, 1.0 equiv), carboxylic acid BB8 (290 mg, 0.88 mmol, 1.7 equiv) was added at 0 C., followed by HATU (300 mg, 0.78 mmol, 1.5 equiv) and DIPEA (0.32 mL, 0.78 mmol, 3.5 equiv). The resulting mixture was left stirring 24 h, before it was quenched by diluting with H.sub.2O (60 mL) at 0 C. The so-formed precipitate was filtered, washed with H.sub.2O (30 mL) and recovered dissolving in MeOH (30 mL). The methanolic solution was concentrated in vacuo, to recover crude peptide Intermediate 4c (350 mg, 79%) as an off-white solid, which was used as such in the next synthetic step.

[0422] (2S,4R)N((S)-1-(((S)-1,6-diamino-1-oxohexan -2-yl)amino)-1-oxopropan -2-yl)-1-(palmitoyl-L-alanyl)-4-(pyridin-4-yloxy) pyrrolidine-2-carboxamide TFA [Palmitoyl-Ala-trans-(O-pyridinyl) Hyp-Ala-Lys-NH.sub.2 TFA salt (Example 12)].

[0423] To a MeOH (5.0 mL) solution of intermediate 4c (350 mg, 0.41 mmol, 1.0 equiv).sub.10% wt Pd/C (44 mg, 0.041 mmol, 0.1 equiv) was added, followed by H.sub.2 (balloon). The resulting mixture was left stirring for 16 h, before a 4 N 1,4-dioxane solution of HCl (0.067 mL, 0.27 mmol, 2.0 equiv) was added, and the resulting mixture was stirred for 0.5h. Next, the reaction mixture was filtered through a CELITE pad, washing with MeOH (15 mL). The filtrates were combined and concentrated in vacuo, and the crude residue was dissolved in MeOH (10 mL), filtered through a 0.20 m syringe filter, and purified using preparative RP-HPLC(isocratic 30% MeCN in H.sub.2O over 5 min, gradient from 35 to 45% MeCN in H.sub.2O over 20 min, isocratic 45% over 7 min, with a solvent flow rate of 10.0 mL/min, at 30 C., Rt.=25 min). All fractions containing product were combined, concentrated to 5 mL in vacuo, prior to lyophilization, to recover Example 12 (12 mg, 3.5%) as a white powder.

[0424] LCMS (ESI-MS m/z): mass calcd for C.sub.38H.sub.66N.sub.7O.sub.6 [M+H].sup.+, 716.51; found, 716.60.

[0425] Compounds made following the synthesis of example 12:7, 8.

Example 13

##STR00312##

[0426] Lipopeptides where a sulfonamide moiety was installed on C-terminus, yielding compounds such as Example 13, were obtained analogously to examples 5-8, i.e. via CDI mediated coupling of side-chain protected intermediate 2b (whose synthesis was discussed earlier in the document) with a Lysine derivative such as BB14. BB14 was synthesized after HATU coupling of commercially available Boc-(D) Lys (Cbz)-OH with cyclopropyl sulfonamide.

[0427] Compounds made according to the following procedures: 127, 135, 178, 179

Example 13. Experimental Procedures

##STR00313##

[0428] Benzyl tert-butyl (6-(cyclopropanesulfonamido)-6-oxohexane-1,5-diyl)(R)-dicarbamate (BB14a). To a DMF (20 mL) solution of Boc-(D) Lys (Cbz)-OH (1.0 g, 2.6 mmol, 1.0 equiv), HATU (1.3 g, 3.2 mmol, 1.2 equiv) was added at 0 C., followed by DIPEA (1.1 mL, 6.4 mmol, 2.5 equiv). The resulting mixture was left stirring for 10 minutes, before cyclopropyl sulfonamide (0.39 g, 3.2 mmol, 1.2 equiv) was added. The resulting mixture was warmed to 25 C. and left stirring for 16 h, before it was quenched by the addition of water (50 mL), and extracted with EtOAc (3100 mL).

[0429] The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo, to recover BB14a (0.9 g, 73%) as crude material, which was used as such in the next synthetic step. R.sub.f=0.6 (SiO.sub.2, EtOAc).

[0430] LCMS (ESI-MS m/z): mass calcd for C.sub.22H.sub.34N.sub.3O.sub.7S [M+H].sup.+, 484.21; found 484.45.

[0431] Benzyl (R)-(5-amino-6-(cyclopropanesulfonamido)-6-oxohexyl)carbamate TFA (BB14). To a TFA/DMF (8 mL, 1:4) solution, BB14a (0.90 g, 1.9 mmol, 1.0 equiv) was added at 0 C. The resulting mixture was warmed to to 25 C. and left stirring for 16 h, before it was concentrated in vacuo. The crude residue was triturated in Et.sub.2O (50 mL), and the solid was decanted, to recover BB14 (0.8g, quant.) as an off-white solid, which was used as such in the next synthetic step.

[0432] Tert-butyl ((9R,12S,15S,18S)-9-((cyclopropylsulfonyl)carbamoyl)-12,15-dimethyl-3,11,14,17-tetraoxo-18-palmitamido-1-phenyl-2-oxa-4,10,13,16-tetraazadocosan -22-yl)carbamate (Intermediate 9). To a DMF (8 mL) solution of Intermediate 2b (0.40 g, 0.63 mmol, 1.0 equiv), CDI (0.15 g, 0.95 mmol, 1.5 equiv) was added, followed by imidazole (65 mg, 0.95 mmol, 1.5 equiv). The resulting mixture was left stirring for 10 minutes, before BB14 (0.15 g, 0.38 mmol, 0.6 equiv) was added. The resulting mixture was left stirring for 16 h, before it was quenched by the addition of water (50 mL), and extracted with EtOAc (3100 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo, to recover Intermediate 9 (0.5 g, 73%) as crude material, which was used as such in the next synthetic step.

[0433] LCMS (ESI-MS m/z): mass calcd for C.sub.50H.sub.86N.sub.7O.sub.11S [M+H].sup.+, 992.61; found 992.78

[0434] N((S)-6-amino-1-(((S)-1-(((S)-1-(((R)-6-amino-1-(cyclopropanesulfonamido)-1-oxohexan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxopropan -2-yl)amino)-1-oxohexan -2-yl) palmitamide (Example 13). To a TFA/TIPS (95:5, 10 mL) solution, Intermediate 9 (0.50 g, 0.50 mmol, 1.0 equiv) was added at 0 C. The reaction mixture was heated to 50 C. and stirred for 2 h, before it was concentrated in vacuo, and the crude residue was purified via RP-HPLC. All fractions containing product were combined, concentrated to 5 mL in vacuo, prior to lyophilization, to recover Example 13 (28 mg, 6%) as a white powder.

[0435] LCMS (ESI-MS m/z): mass calcd for C.sub.37H.sub.72N.sub.707S [M+H].sup.+, 758.5; found 758.4

Example 14

##STR00314##

[0436] Examples 14a, b and c are palmitoylated tripeptide amides, characterized by a non-natural amino acid in the third position from the C-terminus. The latter amino acid is analogous to Ala, whereas the methyl side-chain is functionalized with a 2-aminoimidazole moiety (through C-4 of the imidazole ring), either directly (n=0, as in Example 14a), or spaced apart by one (n=1,

Example 14b) or two (n=2, Example 14c) methylene groups. Examples 14a, b and c were synthesized via standard SPPS, using Fmoc-amino acids and analogous conditions as before for standard SPPS with Rink AM resin. Non-natural N-Fmoc protected aminoacids as BB12abc, where synthesized via ad hoc synthesis (detailed in the experimental) respectively from Fmoc-Asp-OtBu, Fmoc-Glu-OtBu and Fmoc-hGlu-OBn. In order to be deployed in SPPS, BB12abc had the two basic nitrogens of the 2-aminoimidazole side-chain respectively Boc and Trt protected. Whilst Fmoc-Asp-OtBu and Fmoc-Glu-OtBu are commercially available, Fmoc-hGlu-OBn was synthesized from Fmoc-Glu-OBn via Arndt-Eistert homologation and protecting groups interconversion, as detailed in the experimental section. The carboxylate side chain of Fmoc-Asp-tBu, Fmoc-Glu-OtBu and Fmoc-hGlu-OBn could be converted into the 2-aminoimidazole through the same sequence of transformations, therefore in the experimental section are reported only the synthesis of BB14b as an example, and the synthesis of Fmoc-hGlu-OBn, which is not commercially available.

Example 14.1 Experimental procedure-Synthesis of Fmoc-hGlu-OBn

##STR00315##

[0437] (S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(benzyloxy)-2,6-dioxohexane-1-diazonium (Intermediate 7). To a THF (300 mL) solution of Fmoc-Glu-OBn (30 g, 65 mmol, 1.0 equiv), TEA (27 mL, 200 mmol, 3.0 equiv) was added at 0 C., followed by isobutyl chloroformate (17 mL, 130 mmol, 2.0 equiv). The resulting mixture was stirred at 0 C. for 30 min and the so-formed solids were filtered and washed with THF (50 mL). The filtrates were combined in a round-bottom flask and a ether (150 mL) solution of freshly prepared diazomethane (excess) was added. The reaction mixture was stirred at 0 C. for 1 h, before it was warmed to 25 C. and stirred additional 2 h. The reaction mixture was quenched by the addition of AcOH (2.0 mL), diluted with water (500 mL), and extracted with EtOAc (3500 mL). The combined organic layer was washed with saturated aqueous NaHCO.sub.3 (500 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc,/pet-ether, from 0:1 to 1:4) to recover intermediate 7 (12 g, 38%) as a light-green solid.

[0438] R.sub.f=0.3 (SiO.sub.2, EtOAc/pet-ether, 2:3).

[0439] LCMS (ESI-MS m/z): mass calcd for C.sub.28H.sub.27N.sub.3O.sub.5 [M+H].sup.+, 485.19; found, 484.31.

[0440] (S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(benzyloxy)-6-oxohexanoic acid (Fmoc-hGlu-OBn). To a dioxane/water (1.2 L, 5:1) solution of intermediate 7 (12 g, 25 mmol, 1.0 equiv), PhCO.sub.2Ag (0.56 g, 2.4 mmol, 0.10 equiv) was added. The resulting mixture was sonicated at 25 C. for 30 min, before the reaction was quenched by the addition of 1 N aqueous HCl (1.0 mL), and extracted with EtOAc (3500 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The combined organic layer was washed with saturated aqueous NaHCO.sub.3 (500 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc,/pet-ether, from 0:1 to 3:2) to recover Fmoc-hGlu-OBn (10 g, 87%) as a white solid.

[0441] R.sub.f=0.4 (SiO.sub.2, EtOAc).

[0442] LCMS (ESI-MS m/z): mass calcd for C.sub.28H.sub.28NO6 [M+H].sup.+, 474.19; found, 474.35.

Example 14.2 Experimental procedure-Synthesis of BB12b and Example 14b

##STR00316##

[0443] (S)-5-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-(tert-butoxy)-2,6-dioxohexane-1-diazonium (Intermediate 6a). To a THF (500 mL) solution of Fmoc-Glu-tBu (50 g, 120 mmol, 1.0 equiv), TEA (37 mL, 290 mmol, 2.5 equiv) was added at 10 C., followed by isobutyl chloroformate (30 mL, 230 mmol, 2.0 equiv). The resulting mixture was stirred at 0 C. for 15 min and the so-formed solids were filtered and washed with THF (50 mL). The filtrates were combined in a round-bottom flask and a ether (200 mL) solution of freshly prepared diazomethane (excess) was added. The reaction mixture was stirred at 25 C. for 2 h, before it was quenched by the addition of AcOH (2.0 mL) and diluted with water (500 mL). The resulting mixture was extracted with EtOAc (3500 mL), and the combined organic layer was washed with saturated aqueous NaHCO.sub.3 (500 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc,/pet-ether, from 0:1 to 1:4) to recover intermediate 6a (26 g, 51%) as a yellow liquid.

[0444] R.sub.f=0.6 (SiO.sub.2, EtOAc/pet-ether, 2:3).

[0445] .sup.1H NMR (400 MHZ, DMSO-d.sub.6), : 7.86 (d, J=6.8 Hz, 2H), 7.72 (d, J=7.6 Hz, 2H), 7.78 (d, J=8.0 Hz, 1H), 7.42 (t, J=7.6 Hz, 2H), 7.33 (t, J=7.6 Hz, 2H), 6.07 (bs, 1H), 4.32-4.19 (m, 3H), 3.90-3.85 (m, 1H), 2.47-2.32 (m, 2H), 2.00-1.89 (m, 1H), 1.83-1.73 (m, 1H), 1.39 (s, 9H).

[0446] tert-butyl(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6-bromo-5-oxohexanoate (Intermediate 6b). To a THF (200 mL) solution of intermediate 6a (20 g, 44 mmol, 1.0 equiv), 33% HBr in AcOH (12 mL, 49 mmol, 1.1 equiv) was added at 0 C. The resulting mixture was stirred for 30 min at 0 C., before it was diluted with water (50 mL) and it was extracted with EtOAc (3350 mL). The combined organic layer was washed with brine (500 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc,/pet-ether, from 0:1 to 5:17) to recover intermediate 6b (12 g, 55%) as a pale yellow liquid.

[0447] R.sub.f=0.6 (SiO.sub.2, EtOAc/pet-ether, 2:3).

[0448] .sup.1H NMR (400 MHZ, DMSO-d.sub.6), : 7.90 (d, J=7.6 Hz, 2H), 7.83-7.62 (m, 3H), 7.42 (t, J=7.2 Hz), 7.37-7.28 (m, 2H), 4.48-4.14 (m, 2H), 3.92-3.84 (m, 1H), 2.73-2.62 (m, 1H), 2.09 (s, 1H), 1.99-1.67 (m, 3H), 1.58-1.47 (m, 1H), 1.39 (s, 9H).

[0449] tert-butyl(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(2-((tert-butoxycarbonyl)amino)-1H-imidazol-5-yl) butanoate (Intermediate 6c).

[0450] To a DMF (100 mL) solution of intermediate 6b (10 g, 20 mmol, 1.0 equiv), Boc-guanidine (9.5 g, 60 mmol, 3.0 equiv) was added, followed by powdered molecular sieves (10 g). The resulting mixture was stirred at 25 C. for 16 h, before it was quenched by the addition of water (100 mL), and extracted with EtOAc (3200 mL). The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, from 1:9 to 7:3) to recover intermediate 6c (6.0 g, 53%) as an off-white solid.

[0451] R.sub.f=0.5 (SiO.sub.2, EtOAc).

[0452] LCMS (ESI-MS m/z): mass calcd for C.sub.31H.sub.39N.sub.4O.sub.6 [M+H]+563.3; found 563.3.

[0453] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(2-amino-1H-imidazol-5-yl) butanoic acid (Intermediate 6d). To a TFA/DCM (100 mL, 2:3) solution, intermediate 6c (5.0 g, 8.9 mmol, 1.0 equiv.) was added at 0 C. The resulting mixture was warmed to 25 C. and stirred for 1 h, before it was concentrated in vacuo. The crude residue was co-evaporated with toluene (5 mL) to recover intermediate 6d (4.6 g, quant.) as a crude material, which was used as such in the next synthetic step.

[0454] R.sub.f=0.3 (SiO.sub.2, MeOH/DCM, 1:9).

[0455] LCMS (ESI-MS m/z): mass calcd for C.sub.22H.sub.23N.sub.4O.sub.4 [M+H].sup.+, 407.2; found 407.4.

[0456] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(2-amino-1-trityl-1H-imidazol-5-yl) butanoic acid (Intermediate 6e). To a DCM/DMF (45 mL, 1:1) solution of intermediate 6d (4.5 g, 11 mmol, 1.0 equiv.), TEA (3.8 mL, 28 mmol, 2.5 equiv.) was added at 0 C., followed by Trt-Cl (4.6 g, 16.1 mmol, 1.5 equiv.). The resulting mixture was warmed to 25 C. and stirred for 3 h, before it was concentrated in vacuo. The residue was diluted with water (45 mL) and extracted with EtOAc (3100 mL), and the combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 1:99 to 1:9) to recover intermediate 6e (2.2 g, 38%) as a white solid. R.sub.f=0.5 (SiO.sub.2, MeOH/DCM, 1:9).

[0457] LCMS (ESI-MS m/z): mass calcd for C.sub.41H.sub.37N.sub.4O.sub.4 [M+H].sup.+, 649.28; found 649.27.

[0458] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-(2-((tert-butoxycarbonyl)amino)-1-trityl-1H-imidazol-5-yl) butanoic acid (BB12b). To a THF/water (20 mL, 1:1) solution of intermediate 6d (2.0 g, 3.1 mmol, 2.0 equiv), NaHCO.sub.3 (0.51 g, 6.2 mmol, 2.0 equiv) was added, followed by Boc.sub.2O (1.4 mL, 62 mmol, 20 equiv). The resulting mixture was warmed to 50 C. and stirred for 3 h, before it was quenched by the addition of water (50 mL) and extracted with DCM (2 50 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 1:99 to 1:9) to recover BB12b (500 mg, 47%) as an off-white solid.

[0459] R.sub.f=0.7 (SiO.sub.2, MeOH/DCM, 1:9).

[0460] LCMS (ESI-MS m/z): mass calcd for C.sub.46H.sub.45N.sub.4O.sub.6 [M+H].sup.+, 749.3; found 749.1.

Compounds made following the synthesis of BB12b

[0461] BB12a, starting from Fmoc-Asp-tBu, (used in the synthesis of Example 14a). [0462] BB12c, starting from Fmoc-hGlu-Bn (used in the synthesis of Example 14c).

[0463] (S)N.sub.1((S)-1-amino-1-oxo-3-phenylpropan -2-yl)-2-((S)-4-(2-amino-1H-imidazol-4-yl)-2-palmitamidobutanamido)pentanediamide TFA salt (Example 14b) was prepared according the general procedure for SPPS using 0.7 g Rink amide AM resin. The crude peptide was purified by preparative RP-HPLC, all fractions containing the product were combined and concentrated in vacuo to 5 mL, prior to lyophilization, to afford peptide Example 14b (33 mg, 15%) as a white powder.

[0464] HRMS (ESI-MS m/z): mass calcd for C.sub.37H.sub.61N.sub.8O.sub.5 [M+H].sup.+, 697.4759; found, 697.4726.

Compounds Made Following the Synthesis of Example 14b (55)

[0465] Example 14a (62). [0466] Example 14c (56).

Example 15. Synthetic Approach

##STR00317##

[0467] Examples 15a and b are palmitoylated tripeptide amides, characterized by a non-natural amino acid in the third position from the C-terminus. The latter amino acid is analogous to Ala, whereas the methyl side-chain is functionalized with a 2-aminoimidazole moiety (through the 2-amino substituent of the imidazole ring), either spaced apart by one (n=1, Example 15a) or two (n=2, Example 15b) methylene groups. Examples 15a and b were synthesized via standard SPPS, using Fmoc-amino acids and analogous conditions as before for standard SPPS with Rink AM resin. Non-natural N-Fmoc protected aminoacids as BB13ab, where synthesized via ad hoc synthesis (detailed in the experimental) respectively from commercially available(S)(N-Cbz).sub.2-aminoGBL or Boc-Glu-OtBu, and the ad hoc synthesized BB14. In order to be deployed in SPPS, BB13ab, other than the alpha-nitrogen Fmoc-protected, had the basic nitrogen of the imidazole ring Trt protected. After several protecting group interconversions, the sidechain of(S)(N-Cbz).sub.2-aminoGBL and Boc-Glu-OtBu were converted to aldehyde, prior to reacting with BB14 via reductive amination. BB14 was obtained one protecting group interconversion and Trt protection, from commercially available 1H-imidazol-2-amine sulfate.

Example 15.1 Experimental Procedure-Synthesis of BB14

##STR00318##

[0468] 1H-imidazol-2-amine (BB14a). To an aqueous (100 mL) solution of 1H-imidazol-2-amine sulfate (10 g, 38 mmol, 1.0 equiv), Na.sub.2CO.sub.3 (12 g, 110 mmol, 3.0 equiv) was added. The resulting mixture was stirred at 25 C. for 1 h, before it was concentrated in vacuo. The residue was dissolved in EtOH (200 mL), as the resulting mixture was stirred for at 25 C. for 1 h, before filtering over Celite. The filtrate was concentrated in vacuo, to recover BB14a (5.0 g, quant.) as a crude material, which was used as such in the next synthetic step.

[0469] (E)-N-(1H-imidazol-2-yl)-N,N-dimethylformimidamide (BB14b). A DMFDMA/EtOAc (100 mL, 1:1) solution of crude BB14a (5.0 g, 38 mmol, 1.0 equiv) was stirred at 25 C. for 16 h, before it was concentrated in vacuo, to recover BB14b (10 g, quant.) as a crude material, which was used as such in the next synthetic step.

[0470] LCMS (ESI-MS m/z): mass calcd for C.sub.6H.sub.11N.sub.4 [M+H].sup.+, 139.10; found 138.97.

[0471] (E)-N,N-dimethyl-N-(1-trityl-1H-imidazol-2-yl)formimidamide (BB14c). To a DCM (100 mL) solution of crude BB14b (10 g, 38 mmol, 1.0 equiv), TEA (15 mL, 110 mmol, 2.9 equiv) was added at 0 C., followed by Trt-Cl (10 g, 36 mmol, 0.9 equiv). The resulting mixture was warmed to 25 C. and stirred for 16 h, before it was quenched by the addition of water (100 mL), and extracted with EtOAc (3200 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 1:99 to 1:9) to recover BB14c (7.0 g, 49%) as a white solid.

[0472] R.sub.f=0.6 (SiO.sub.2, EtOAc).

[0473] LCMS (ESI-MS m/z): mass calcd for C.sub.25H.sub.25N.sub.4 [M+H].sup.+, 381.21; found 381.31.

[0474] 1-trityl-1H-imidazol-2-amine (BB14). To a EtOH (70 mL) solution of BB14c (7.0 g, 18 mmol, 1.0 equiv), AcOH (5.1 mL, 92 mmol, 5.0 equiv) was added at 0 C., followed by hydrazine hydrate (4.5 mL, 92 mmol, 5.0 equiv). The resulting mixture was warmed to 50 C. and was stirred for 5 h, before it was concentrated in vacuo. The residue was diluted with DCM (100 mL) and 1 N aqueous NaOH (50 mL), and the resulting mixture was stirred for 30 min at 25 C. After layer-separation, the organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo, to recover BB14 (5.5 g, 93%) as a white solid.

[0475] R.sub.f=0.3 (SiO.sub.2, EtOAc).

[0476] LCMS (ESI-MS m/z): mass calcd for C.sub.22H.sub.20N.sub.3 [M+H].sup.+, 326.17; found 326.26.

Example 15. Experimental Procedure-BB13a and Example 15a

##STR00319##

[0477] Benzyl ((benzyloxy)carbonyl)-L-homoserinate (Intermediate 7a). To a EtOH (360 mL) solution of(S)(N-Cbz).sub.2-aminoGBL (30 g, 120 mmol, 1.0 equiv), an aqueous (50 mL) solution of NaOH (4.7 g, 120 mmol, 1.0 equiv) was added at 0 C. The resulting mixture was warmed to 25 C. and stirred for 16 h, before it was concentrated in vacuo, and the residue was triturated in EtOH (3100 mL). The so-formed solid was filtered and dissolved in DMF (300 mL), before BnBr (14 mL, 120 mmol, 1.0 equiv) was added at at 0 C. The resulting mixture was warmed to 25 C. and stirred for 16 h, before it was quenched by the addition of water (500 mL), and extracted with EtOAc (3750 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, from 0:1 to 3:7) to recover Intermediate 7a (30 g, quant.) as a white solid.

[0478] R.sub.f=0.4 (SiO.sub.2, EtOAc/pet-ether, 3:2).

[0479] LCMS (ESI-MS m/z): mass calcd for C.sub.19H.sub.22NNaO.sub.5 [M+H].sup.+, 366.13; found 366.30. Benzyl(S)-2-(((benzyloxy)carbonyl)amino)-4-oxobutanoate (intermediate 7b). To a DCM (200 mL) solution of Intermediate 7a (10 g, 29 mmol, 1.0 equiv), DMP (18 g, 43 mmol, 1.5 equiv) was added at 0 C. The resulting mixture was warmed to 25 C. and stirred for 4 h, before it was quenched by the addition of 10% aqueous sodium thiosulfate (100 mL) and stirred additional 15 min. The resulting mixture was extracted with DCM (3250 mL), and the combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, from 0:1 to 1:3) to recover Intermediate 7b (7.1 g, 71%) as a white solid.

[0480] R.sub.f=0.6 (SiO.sub.2, EtOAc/pet-ether, 3:2).

[0481] .sup.1H NMR (400 MHZ, CDCl.sub.3), : 9.62 (s, 1H), 7.38-7.28 (m, 10H), 5.69 (d, J=7.6 Hz, 1H), 5.20-5.08 (m, 4H), 4.73-4.65 (m, 1H), 3.19-3.04 (m, 2H).

[0482] Benzyl(S)-2-(((benzyloxy)carbonyl)amino)-4-((1-trityl-1H-imidazol-2-yl)amino) butanoate (Intermediate 7c). To a toluene (40 mL) solution of Intermediate 7b (2.0 g, 5.9 mmol, 1.0 equiv), BB14 (1.9 g 5.9 mmol, 1.0 equiv) was added. The resulting mixture was heated to 120 C. and stirred for 5 h, before it was cooled to 0 C., and NaCNBH.sub.3 (1.1 g, 18 mmol, 3.0 equiv) was added. The resulting mixture was warmed to 25 C. and stirred for 48 h, before it was quenched by the addition of saturated aqueous ammonium chloride (50 mL), and extracted with EtOAc (3200 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 1:99 to 3:97) to recover Intermediate 7c (1.2 g, 31%) as a light-yellow gum.

[0483] R.sub.f=0.4 (SiO.sub.2, MeOH/DCM, 1:19).

[0484] LCMS (ESI-MS m/z): mass calcd for C.sub.41H.sub.39N.sub.4O.sub.4 [M+H].sup.+, 651.30; found 651.20.

[0485] (S)-2-amino-4-((1-trityl-1H-imidazol-2-yl)amino) butanoic acid (Intermediate 7d). To a MeOH (20 mL) solution of Intermediate 7c (1.0 g, 1.5 mmol, 1.0 equiv), 10% Pd(OH).sub.2 (0.40 g, 40% wt) was added, and the resulting mixture was stirred under H.sub.2 (balloon) atmosphere for 16 h, before it was filtered through Celite. The filtrate was concentrated in vacuo to recover intermediate 7d (0.6 g, 91%) as an off-white solid.

[0486] LCMS (ESI-MS m/z): mass calcd for C.sub.26H.sub.27N.sub.4O.sub.2 [M+H].sup.+, 427.21; found 427.50.

[0487] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-4-((1-trityl-1H-imidazol-2-yl)amino) butanoic acid (BB13a). To a water/acetone (9 mL, 3:1) solution of Intermediate 7d (0.60 g, 1.4 mmol, 1.0 equiv), NaHCO.sub.3 (0.23 g, 2.8 mmol, 2.0 equiv) was added at 0 C., followed by Fmoc-OSu (0.47 g, 1.4 mmol, 1.0 equiv). The resulting mixture was warmed to 25 C. and stirred for 4 h, before it was quenched by the addition of 10% aqueous citric acid (50 mL), and extracted with EtOAc (3100 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 1:99 to 1:24) to recover BB13a (0.45 g, 50%) as a white solid.

[0488] R.sub.f=0.3 (SiO.sub.2, MeOH/DCM, 1:19).

[0489] LCMS (ESI-MS m/z): mass calcd for C.sub.41H.sub.37N.sub.4O.sub.4 [M+H].sup.+, 649.28; found 649.20.

[0490] (S)-2-((S)-4-((1H-imidazol-2-yl)amino)-2-palmitamidobutanamido)-N.sub.1((S)-1-amino-1-oxo-3-phenylpropan -2-yl)pentanediamide TFA salt (Example 15a) was prepared according the general procedure for SPPS using 0.7 g Rink amide AM resin. The crude peptide was purified by preparative RP-HPLC, all fractions containing the product were combined and concentrated in vacuo to 5 mL, prior to lyophilization, to afford peptide Example 15a (16 mg, 6.5%) as a white powder.

[0491] HRMS (ESI-MS m/z): mass calcd for C.sub.37H.sub.61N.sub.8O.sub.5 [M+H].sup.+, 697.4759; found, 697.4742. Compounds made according to example 15 are 65 and 66

Example 15. Experimental procedure-BB13b and Example 15b

##STR00320##

[0492] 1-(Tert-butyl).sub.5-methyl (tert-butoxycarbonyl)-L-glutamate (Intermediate 8a). To a DCM (500 mL) solution of Boc-Glu-OtBu (25 g, 82 mmol, 1.0 equiv), TEA (23 mL, 160 mmol, 2.0 equiv) was added at 0 C., followed by DMAP (1.0 g, 8.2 nnol, 0.1 equiv) and CICOOMe (9.4 mL, 120 mmol, 1.5 equiv). The resulting mixture was warmed to 25 C. and stirred for 3 h, before it was quenched by the addition of water (200 mL), and extracted with DCM (3500 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo, to recover Intermediate 8a (25 g, quant.) as a crude material, which was used as such in the next synthetic step.

[0493] R.sub.f=0.5 (SiO.sub.2, EtOAc/pet-ether, 1:4).

[0494] LCMS (ESI-MS m/z): mass calcd for C.sub.15H.sub.28NO6 [M+H].sup.+, 318.19; found 318.23.

[0495] 1-(Tert-butyl).sub.5-methyl N,N-bis(tert-butoxycarbonyl)- L-glutamate (Intermediate 8b). To a THF (500 mL) solution of Intermediate 8a (25 g, 79 mmol, 1.0 equiv), DMAP (4.8 g, 39 mmol, 1.0 equiv) was added at 0 C., followed by Boc.sub.2O (34 mL, 160 mmol, 2.0 mmol). The resulting mixture was warmed to 25 C. and stirred for 36 h, before with was quenched by the addition of water (300 mL), and extracted with DCM (3500 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, from 0:1 to 1:9) to recover Intermediate 8b (19 g, 60%) as a white solid.

[0496] R.sub.f=0.6 (SiO.sub.2, EtOAc/pet-ether, 1:4).

[0497] LCMS (ESI-MS m/z): mass calcd for C.sub.20H.sub.35NO8 [M+H].sup.+, 418.24; found 418.34.

[0498] Tert-butyl(S)-2-(bis(tert-butoxycarbonyl)amino)-5-oxopentanoate (Intermediate 8c). To a diethyl ether (100 mL) solution of Intermediate 8b (5.0 g, 12 mmol, 1.0 equiv), a 1 M solution of DIBAL-H in hexane (18 mL, 18 mmol, 1.5 equiv) was added drop-wise, over 5 min, at 78 C. The resulting mixture was quenched by the addition of 10% aqueous sodium potassium tartrate (25 mL), and stirred additional 30 min, before it was extracted with EtOAc (3100 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, from 0:1 to 1:4) to recover Intermediate 8c (4.0 g, 86%) as a white solid.

[0499] R.sub.f=0.4 (SiO.sub.2, EtOAc/pet-ether, 1:4).

[0500] .sup.1H NMR (400 MHZ, CDCl.sub.3), : 9.80-9.72 (m, 1H), 4.80-4.72 (m, 1H), 2.63-2.38 (m, 4H), 1.58-1.38 (m, 27H).

[0501] Tert-butyl(S)-2-(bis(tert-butoxycarbonyl)amino)-5-((1-trityl-1H-imidazol-2-yl)amino)pentanoate (Intermediate 8d). To a toluene (50 mL) solution of Intermediate 8c (2.5 g, 6.4 mmol, 1.0 equiv), BB14 (2.1 g, 6.4 mmol, 1.0 equiv) was added. The resulting mixture was heated to 120 C. and stirred for 5 h, before it was cooled to 0 C., and NaCNBH.sub.3 (0.73 g, 20 mmol, 3.0 equiv) was added. The resulting mixture was warmed to 25 C. and stirred for 48 h, before it was quenched by the addition of saturated aqueous ammonium chloride (50 mL), and extracted with EtOAc (3200 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, EtOAc/pet-ether, from 1:4 to 7:3) to recover Intermediate 8d (0.9 g, 20%) as a white solid. R.sub.f=0.4 (SiO.sub.2, MeOH/DCM, 1:19).

[0502] LCMS (ESI-MS m/z): mass calcd for C.sub.41H.sub.53N.sub.4O.sub.6 [M+H].sup.+, 697.40; found 697.56.

[0503] (S)-2-amino-5-((1-trityl-1H-imidazol-2-yl)amino)pentanoic acid (Intermediate 8e). To a TFA/DCM (18 mL, 1:1) solution, Intermediate 8d (0.9 g, 1.3 mmol, 1.0 equiv) was added at 0 C. The resulting mixture was warmed to 25 C. and stirred fot 4 h, before it was concentrated in vacuo.

[0504] The crude residue was triturated in diethyl ether (50 mL) to recover crude Intermediate 8e (0.5 g, quant.) as a light-brown solid, which was used as such in the next synthetic step.

[0505] LCMS (ESI-MS m/z): mass calcd for C.sub.27H.sub.29N.sub.4O.sub.2 [M+H].sup.+, 441.23; found 441.35.

[0506] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-((1-trityl-1H-imidazol-2-yl)amino)pentanoic acid (BB13b). To a water/acetone (7.5 mL, 2:1) solution of intermediate 8e (0.50 g, 1.1.mmol, 1.0 equiv), NaHCO.sub.3 (0.19 g, 2.3 mmol, 2.0 equiv) was added at 0 C., followed by Fmoc-OSu (0.38 g, 1.1 mmol, 1.0 equiv). The resulting mixture was warmed to 25 C. and stirred for 4 h, before it was quenched by the addition of 10% aqueous citric acid (50 mL), and extracted with EtOAc (3100 mL). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated in vacuo. The crude residue was purified via flash column chromatography (SiO.sub.2, MeOH/DCM, from 1:99 to 3:47) to recover BB13b (0.9 g, 20%) as an off-white solid.

[0507] R.sub.f=0.3 (SiO.sub.2, MeOH/DCM, 1:19).

[0508] LCMS (ESI-MS m/z): mass calcd for C.sub.42H.sub.39N.sub.4O.sub.4 [M+H].sup.+, 663.30; found 663.47.

[0509] (S)-2-((S)-5-((1H-imidazol-2-yl)amino)-2-palmitamidopentanamido)-N.sub.1((S)-1-amino-1-oxo-3-phenylpropan -2-yl)pentanediamide TFA (Example 15b) was prepared according the general procedure for SPPS using 0.7 g Rink amide AM resin. The crude peptide was purified by preparative RP-HPLC, all fractions containing the product were combined and concentrated in vacuo to 5 mL, prior to lyophilization, to afford peptide Example 15b (75 mg, 30%) as a white powder.

[0510] HRMS (ESI-MS m/z): mass calcd for C.sub.38H.sub.62N.sub.8O.sub.5 [M+H].sup.+, 711.4916; found, 711.4890

REFERENCES

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M., N-methoxy-n-methylamides as effective acylating agents. Tetrahedron Letters 1981, 22 (39), 3815-3818. [0516] 6. Kwon, H.; Kim, Y.; Park, K.; Choi, S. A.; Son, S.-H.; Byun, Y., Structure-based design, synthesis, and biological evaluation of Leu-Arg dipeptide analogs as novel hepsin inhibitors. Bioorganic & Medicinal Chemistry Letters 2016, 26 (2), 310-314. [0517] 7. Venkatraman, S.; Bogen, S. L.; Arasappan, A.; Bennett, F.; Chen, K.; Jao, E.; Liu, Y.-T.; Lovey, R.; Hendrata, S.; Huang, Y.; Pan, W.; Parekh, T.; Pinto, P.; Popov, V.; Pike, R.; Ruan, S.; Santhanam, B.; Vibulbhan, B.; Wu, W.; Yang, W.; Kong, J.; Liang, X.; Wong, J.; Liu, R.; Butkiewicz, N.; Chase, R.; Hart, A.; Agrawal, S.; Ingravallo, P.; Pichardo, J.; Kong, R.; Baroudy, B.; Malcolm, B.; Guo, Z.; Prongay, A.; Madison, V.; Broske, L.; Cui, X.; Cheng, K.-C.; Hsieh, Y.; Brisson, J.-M.; Prelusky, D.; Korfmacher, W.; White, R.; Bogdanowich-Knipp, S.; Pavlovsky, A.; Bradley, P.; Saksena, A. K.; Ganguly, A.; Piwinski, J.; Girijavallabhan, V.; Njoroge, F. G., Discovery of (1R,5S)N-[3-Amino-1-(cyclobutylmethyl)-2,3-dioxopropyl]-3-[2 (S)-[[(1,1-dimethylethyl)amino]carbonyl]amino]-3,3-dimethyl-1-oxobutyl]-6,6-dimethyl-3-azabicyclo[3.1.0]hexan -2 (S)-carboxamide (SCH 503034), a Selective, Potent, Orally Bioavailable Hepatitis C Virus NS3 Protease Inhibitor: A Potential Therapeutic Agent for the Treatment of Hepatitis C Infection. Journal of Medicinal Chemistry 2006, 49 (20), 6074-6086. [0518] 8. Rakesh; Sun, D.; Lee, R. B.; Tangallapally, R. P.; Lee, R. E., Synthesis, optimization and structure-activity relationships of 3,5-disubstituted isoxazolines as new anti-tuberculosis agents. European Journal of Medicinal Chemistry 2009, 44 (2), 460-472. [0519] 9. Saitoh, M.; Kunitomo, J.; Kimura, E.; Iwashita, H.; Uno, Y.; Onishi, T.; Uchiyama, N.; Kawamoto, T.; Tanaka, T.; Mol, C. D.; Dougan, D. R.; Textor, G. P.; Snell, G. P.; Takizawa, M.; Itoh, F.; Kori, M., 2-{3-[4-(Alkylsulfinyl)phenyl]-1-benzofuran -5-yl}-5-methyl-1,3,4-oxadiazole Derivatives as Novel Inhibitors of Glycogen Synthase Kinase-3B with Good Brain Permeability. Journal of Medicinal Chemistry 2009, 52 (20), 6270-6286. [0520] 10. Dekhane, D. V.; Pawar, S. S.; Gupta, S.; Shingare, M. S.; Patil, C. R.; Thore, S. N., Synthesis and anti-inflammatory activity of some new 4,5-dihydro-1,5-diaryl-1H-pyrazole-3-substituted-heteroazole derivatives. Bioorganic & Medicinal Chemistry Letters 2011, 21 (21), 6527-6532. [0521] 11. Matteson, D. S.; Sadhu, K. M., Boronic ester homologation with 99% chiral selectivity and its use in syntheses of the insect pheromones (3S,4S)-4-methyl-3-heptanol and exo-brevicomin. Journal of the American Chemical Society 1983, 105 (7), 2077-2078. [0522] 12. Nitsche, C.; Zhang, L.; Weigel, L. F.; Schilz, J.; Graf, D.; Bartenschlager, R.; Hilgenfeld, R.; Klein, C. D., Peptide-Boronic Acid Inhibitors of Flaviviral Proteases: Medicinal Chemistry and Structural Biology. Journal of Medicinal Chemistry 2017, 60 (1), 511-516. [0523] 13. Galemmo, R. A.; Fevig, J. M.; Carini, D. J.; Cacciola, J.; Wells, B. L.; Hillyer, G. L.; Buriak, J.; Rossi, K. A.; Stouten, P. F. W.; Alexander, R. S.; Hilmer, R.; Bostrom, L.; Abelman, M. M.; Lee, S.-L.; Weber, P. C.; Kettner, C. A.; Knabb, R. M.; Wexler, R. R., (N-acyl-N-alkyl) glycyl borolysine analogs: A new class of potent thrombin inhibitors. Bioorganic & Medicinal Chemistry Letters 1996, 6 (24), 2913-2918. [0524] 14. Dess, D. B.; Martin, J. C., Readily accessible 12-1-5 oxidant for the conversion of primary and secondary alcohols to aldehydes and ketones. The Journal of Organic Chemistry 1983, 48 (22), 4155-4156. [0525] 15. Behnam, M. A. M.; Graf, D.; Bartenschlager, R.; Zlotos, D. P.; Klein, C. D., Discovery of Nanomolar Dengue and West Nile Virus Protease Inhibitors Containing a 4-Benzyloxyphenylglycine Residue. Journal of Medicinal Chemistry 2015, 58 (23), 9354-9370. [0526] 16. Khl, N.; Graf, D.; Bock, J.; Behnam, M. A. M.; Leuthold, M.-M.; Klein, C. D., A New Class of Dengue and West Nile Virus Protease Inhibitors with Submicromolar Activity in Reporter Gene DENV-2 Protease and Viral Replication Assays. Journal of Medicinal Chemistry 2020, 63 (15), 8179-8197. [0527] 17. Bloemhoff, W.; Kerling, K. E. T., Studies on polypeptides XV Synthesis of L- and D-homohistidine. Recueil des Travaux Chimiques des Pays-Bas 1975, 94 (8), 182-185. [0528] 18. Elliott, J. T.; Hoekstra, W. J.; Maryanoff, B. E.; Prestwich, G. D., Photoactivatable peptides based on BMS-197525: A potent antagonist of the human thrombin receptor (PAR-1). Bioorganic & Medicinal Chemistry Letters 1999, 9 (2), 279-284. [0529] 19. Albericio, F.; Bofill, J. M.; El-Faham, A.; Kates, S. A., Use of Onium Salt-Based Coupling Reagents in Peptide Synthesis1. The Journal of Organic Chemistry 1998, 63 (26), 9678-9683. [0530] 20. Mitsunobu, O., The Use of Diethyl Azodicarboxylate and Triphenylphosphine in Synthesis and Transformation of Natural Products. Synthesis 1981, 1981 (01), 1-28. [0531] 21. Bellier, B.; McCort-Tranchepain, I.; Ducos, B.; Danascimento, S.; Meudal, H.; Noble, F.; Garbay, C.; Roques, B. P., Synthesis and Biological Properties of New Constrained CCK-B Antagonists: Discrimination of Two Affinity States of the CCK-B Receptor on Transfected CHO Cells. Journal of Medicinal Chemistry 1997, 40 (24), 3947-3956. [0532] 22. Bailey, M. D.; Bordeleau, J.; Garneau, M.; Leblanc, M.; Lemke, C. T.; O'Meara, J.; White, P. W.; Llinas-Brunet, M., Peptide backbone replacement of hepatitis C virus NS3 serine protease C-terminal cleavage product analogs: Discovery of potent succinamide inhibitors. Bioorganic & Medicinal Chemistry Letters 2013, 23 (15), 4447-4452. [0533] 23. Lampa, A.; Ehrenberg, A. E.; Gustafsson, S. S.; Vema, A.; kerblom, E.; Lindeberg, G.; Karlen, A.; Danielson, U. H.; Sandstrm, A., Improved P2 phenylglycine-based hepatitis C virus NS3 protease inhibitors with alkenylic prime-side substituents. Bioorganic & Medicinal Chemistry 2010, 18 (14), 5413-5424. [0534] 24. Lindgren, C.; Tyagi, M.; Viljanen, J.; Toms, J.; Ge, C.; Zhang, N.; Holmdahl, R.; Kihlberg, J.; Linusson, A., Dynamics Determine Signaling in a Multicomponent System Associated with Rheumatoid Arthritis. Journal of Medicinal Chemistry 2018, 61 (11), 4774-4790.

Example 16-In Vitro Protease Assays

16.1 Materials and Methods

[0535] Single dose screening of compounds and/or IC.sub.50 determination was performed by a fluorimetric assay for DENV protease as described before (Nitsche and Klein, Fluorimetric and HPLC-Based Dengue Virus Protease Assays Using a FRET Substrate. In Antiviral Methods and Protocols, Gong, E. Y., Ed. Humana Press: Totowa, New Jersey, 2013; pp 221-236-Nitsche et al., J. Med. Chem. 2013, 56 (21), 8389-8403.) The FRET substrate was an internally quenched anthranilamide/nitrotyrosine substrate as described by Steuer et al. (DOI: 10.1177/1087057109344115)(Km=105 M). By catalytic activity of the protease, the cleaved substrate results in increased fluorescence that can be monitored using a BMG Labtech Fluostar OPTIMA Microtiter fluorescence plate reader (excitation wavelength of 320 nm and a monitored emission wavelength of 405 nm). Dilutions were made starting from 10 mM stock solutions in DMSO and final concentrations were measured in triplicates. The inhibitors were preincubated for 15 min with DENV protease (100 nM) in the assay buffer (50 mM Tris-HCl pH 9.0, ethylene glycol (10% v/v), and 0.0016% Brij 58). The enzymatic reaction was initiated by the addition of the FRET substrate (final concentration 50 M) to obtain a final assay volume of 100 L per well. The enzymatic activity was monitored for 15 min and determined as a slope of relative fluorescence units per second (RFU/s) for each concentration. The mean of the triplicates were plotted against the corresponding concentration in CDDVault to determine the IC.sub.50-values. Analogous procedures were used to determine the inhibitory potency on WNV (Khl et al., J. Med. Chem. 2020, 63 (15): 8179-8197) and ZIKA protease (Nitsche et al., ACS Med. Chem. Lett. 2019 Feb. 14; 10 (2): 168-174). For selectivity screening of active candidates, similar biochemical assays with human serine proteases trypsin (Weigel et al., J. Med. Chem. 2015, 58 (19), 7719-7733.) and/or thrombin (Nitsche et al., J. Med. Chem. 2013, 56 (21), 8389-8403) were used.

16.2 Inhibition of Dengue

[0536] The following compounds showed at least 25% inhibition of DENV at 50 M: 1-17, 19, 21, 23, 25, 29, 32, 36-45, 48-68, 72, 78, 82, 83, 89-93, 97, 98, 110-123, 125-130, 132-139, 146-152, 156, 158-168, 170-190, 192, 193, 196

[0537] The following compounds showed at least 20% inhibition of DENV at 1 M: 39, 41, 48-51, 114, 125, 129, 132, 136, 137, 148, 167, 172, 173

[0538] The following compounds had IC.sub.50 of 10 UM or less for DENV: 1, 2, 4, 7, 9-17, 24, 25, 32, 39-41, 43, 44, 48-51, 53, 54, 72, 82, 83, 90, 92, 97, 98, 110-119, 121-123, 125, 128, 129, 132, 133, 134, 136, 137, 139, 146-150, 156, 158-161, 167, 168, 170-177, 180-183, 186, 187, 189, 190, 192, 193, 196

[0539] The following compounds had EC.sub.50 of 20 M or less for DENV: 9, 11, 32, 38, 43, 44, 69, 70, 71, 72, 73, 75, 77, 79, 81, 82, 83, 86, 87, 88, 89, 92, 94, 98, 113, 114, 115, 116, 117, 118, 122, 123, 129, 136, 137, 156, 158, 162, 167, 168, 170, 171, 174, 175, 176, 190, 192, 196.

16.3 Inhibition of West Nile virus

[0540] The following compounds showed at least 25% inhibition of WNV at 50 M: 5, 6, 8-11, 13, 16, 17, 43, 44, 48, 90, 92, 115, 116, 117, 118, 122, 123, 125, 129, 132, 136, 137, 167, 168, 170, 171, 174, 175, 176, 180-186, 190, 191, 192, 193, 196

[0541] The following compounds showed at least 10% inhibition of WNNV at 1 M: 132, 137, 186

[0542] The following compounds had IC.sub.50 of 40 M or less for WNNV: 5, 6, 8, 9, 10, 11, 13, 16, 17, 39, 41, 43, 44, 48, 90, 92, 113, 114, 115, 116, 117, 118, 122, 123, 125, 129, 132, 136, 137, 156, 167, 168, 170, 171, 174, 175, 176, 180, 181, 182, 183, 186, 189, 190, 193, 196.

[0543] The following compounds had EC.sub.50 of 25 M or less for WNNV: 9, 10, 43, 44, 50, 51, 53, 54, 113, 114, 118, 136, 170, 189, 196.

16.4 Inhibition of Zika Virus

[0544] The following compounds showed at least 65% inhibition of ZIKV at 50 M: 5, 6, 8, 9, 10, 11, 39, 43, 44, 48, 49, 50, 51, 53, 54, 92, 113, 114, 115, 116, 117, 118, 122, 123, 125, 129, 136, 137, 167, 168, 170, 171, 175, 176, 189, 196.

[0545] The following compounds showed at least 10% inhibition of ZIKV at 1 M: 9, 11, 39, 43, 44, 48, 49, 50, 51, 54, 92, 113, 114, 115, 116, 117, 118, 123, 125, 129, 136, 137, 167, 168, 189.

[0546] The following compounds had IC.sub.50 of 25 M or less for ZIKV: 5, 6, 8, 9, 10, 11, 39, 43, 44, 48, 49, 50, 51, 53, 54, 92, 113, 114, 115, 116, 117, 118, 122, 123, 125, 129, 136, 137, 167, 168, 170, 171, 175, 176, 189, 196.

[0547] The following compounds had EC.sub.50 of 10 M or less for ZIKV: 44, 49, 50, 51, 53, 54, 113, 114, 118, 136, 137, 170, 189, 196.

16.5 Selectivity of Compounds

[0548] This data shows the selectivity of the compounds, in low activity with regard to host proteases.

[0549] The following compounds showed less than 20% inhibition of human Thrombin at 25 M: 5, 6, 8, 9, 10, 11, 13, 16, 17, 43, 44, 90, 92, 115, 116, 117, 118, 122, 123, 125, 129, 132, 136, 137, 167, 168, 170, 171, 174, 175, 176, 180, 181, 186, 189, 190, 196.

[0550] The following compounds showed less than 5% inhibition of human Thrombin at 5 M: 5, 6, 8, 9, 10, 11, 13, 16, 17, 43, 44, 90, 92, 115, 116, 117, 118, 122, 123, 125, 129, 132, 136, 137, 167, 168, 170, 171, 174, 175, 176, 180, 181, 186, 189, 190, 196.

[0551] The following compounds showed less than 20% inhibition of human Trypsin at 25 M: 5, 6, 8, 11, 13, 16, 17, 43, 44, 90, 92, 114, 115, 116, 117, 118, 122, 123, 125, 129, 132, 136, 137, 168, 170, 171, 174, 175, 181, 189, 190, 196.

[0552] The following compounds showed less than 10% inhibition of human Trypsin at 5 M: 5, 6, 8, 10, 11, 13, 16, 17, 43, 44, 90, 92, 114, 115, 116, 117, 118, 122, 123, 125, 129, 136, 137, 168, 170, 171, 174, 175, 189, 190, 196.

Example 17-In Vivo Assay

17.1 Materials and Methods

[0553] The purpose of this study was to determine the efficacy of the compounds on Dengue virus 2 (DENV2, ATCC VR-1584) infection in AG129 mice.

17.1.1 Animal Welfare and Husbandry

[0554] Six groups of AG129 mice, 6-8 weeks old, with 9 mice per group were used to test compounds according to table 3.1.1. Animal room environment was monitored for temperature and relative humidity twice per day. The temperature range was 22 C.3 C. and humidity range was 30-70%. The animals were provided with 12 hours light and 12 hours darkness. Feed and drinking water was provided ad libitum. Compounds 43 and 113 were tested.

TABLE-US-00004 TABLE 3.1.1 Experimental groups Group # of ID Treatment Dosing and administration route animals G1 Infection Control Vehicle (IP) 9 G2 43 (Dose1) 20 mg/kg body weight (I.P, b.i.d) 9 G3 43 (Dose2) 10 mg/kg body weight (I.P, b.i.d) 9 G4 113 (Dose1) 20 mg/kg body weight (I.P, b.i.d) 9 G5 113 (Dose2) 10 mg/kg body weight (I.P, b.i.d) 9 G6 Positive control Ribavirin (100 mg/kg, SC) 9

[0555] 17.1.2 Virus Production

[0556] C6/36 (ATCC(CRL-1660) cell lines were grown in RPMI 1640 medium (Gibco/HiMedia) plus 5% fetal bovine serum at 28 C. Dengue virus type 2 (ATCCVR-1584) New Guinea C(NGC, DENV2) was passaged in BHK-21 cell cultures to make sufficient stocks for assay. Virus titer was determined by TCID.sub.50 method using BHK-21 cells.

17.1.3 Induction of Infection

[0557] Twenty-four hours post treatment (Day 0), animals were injected intraperitoneally (IP) with 0.2 ml of virus suspension (510.sup.7/animal).

17.1.4 Formulations

[0558] The vehicle for tested compounds was 5% DMSO+95% (20%) hydroxypropylcyclodextrin. Saline (0.9%) was used as vehicle for Ribavirin. Formulations were prepared daily immediately before dosing.

17.1.5 Treatment, Sampling and Clinical Observations

[0559] Administration of compounds and vehicle to animals started twenty four hours before infection (Day 1). Animals were treated daily (From Day-1 to day 4) as per the experimental design shown below in table 3.1.5. Animals were monitored for general clinical signs, morbidity and mortality. Body weights were measured daily.

TABLE-US-00005 TABLE 3.1.5 treatment and sampling schedule DAY Treatment & sampling details Day 1 Administration Vehicle/Compounds Blood sampling (Animal #1, 2, 3) 1 hr post 1.sup.st dose Day 0 Administration Vehicle/Compounds Blood sample (Animal #4, 5, 6) & Infection Day 1 Administration Vehicle/Compounds Blood sample (Animal #7, 8, 9) Day 2 Administration Vehicle/Compounds Blood sample (Animal #1, 2, 3) Day 3 Administration Vehicle/Compounds Blood sample (Animal #4, 5, 6) Day 4 Administration Vehicle/Compounds Blood sample (Animal #7, 8, 9) Day 5 Terminal Sacrifice Blood sample & spleen collection (12 hr post last dose)

17.1.6 Blood Collection

[0560] Blood was collected on days-1, 0, 1, 2, 3, 4 to 5.

17.1.7 Plaque Assay

[0561] BHK-21 cells were cultured to approximately 80% confluency in 24-well plates (NUNC, NY, USA). Plasma samples were 10-fold serially diluted in RPMI 1640 (GIBCO). BHK-21 monolayers were infected with 100 l of each virus dilution. After incubation at 37 C. and 5% CO.sub.2 atmosphere for one hour with rocking at 15 min intervals, the medium was decanted and 1 ml of 1% (w/v)carboxymethyl cellulose in RPMI supplemented with 2% FCS was added to each well. After four days incubation at 37 C. in 5% CO.sub.2, the cells were fixed with 4% paraformaldehyde and stained for 30 min with 200 ml of 1% crystal violet dissolved in 37% formaldehyde. After thorough rinsing with water, plates were dried and the plaques were scored visually.

17.1.8 Infection Markers

[0562] Cytokine levels (TNF alpha, IL-6 and IL-12) in plasma samples were measured using Enzyme-Linked Immunosorbent Assay (ELISA). Mouse TNF alpha ELISA Kit (Abcam: ab208348), Mouse IL-6 ELISA Kit (Abcam: ab100712), and Mouse IL-12 p40+IL-12 p70 ELISA Kit (Abcam: ab100699) were used to estimate TNF alpha, IL-6 and IL-12, respectively. The assays were performed according to the manufacturer's instructions and was analysed on a TECAN-NanoQuant Plate system.

17.1.9 Termination

[0563] Animals were sacrificed on day 5 post infection with Isoflurane followed by an overdose of CO.sub.2. Spleens were excised, measured and weighed.

17.1.10 Data Analysis

[0564] The MeanSD Log10PFU/ml, relative MeanSD spleen weight, MeanSD biomarker concentrations were estimated in each group. Significant differences between group means and control were analyzed by one-way ANOVA, followed by a Dunnett's multiple comparison test, using GraphPad Prism 5 at 95% confidence levels. A p value of <0.05 was considered as significant.

17.2 Results

[0565] Animals treated with 20 and 10 mg/kg of compounds 43 or 113 were apparently normal. Although there were differences in mean body at the end of treatment (Table 3.2.1 and FIG. 1), they were not statistically significant. 43 (10 mg/kg and 20 mg/kg) and 113 (10 and 20 mg/kg) showed significant reduction in spleen weights (an indication of lowered infection) when compared to the vehicle control (Table 3.2.2 and FIG. 2). No significant reduction was observed with Ribavirin.

TABLE-US-00006 TABLE 3.2.1 Body weights (g) of mice at the end of the study (Mean SD) G1 G2 G3 G4 G5 G6 (Vehicle 43 43 113 113 (Ribavirin Control), (20 mg/kg) (10 mg/kg) (20 mg/kg) (10 mg/kg) 100 mg/kg) n = 9 n = 9 n = 9 n = 9 n = 9 n = 9 19.44 2.24 18.11 1.45 18.78 2.28 16.89 1.36 17.22 1.39 20.11 1.05

TABLE-US-00007 TABLE 3.2.2 Spleen weights (g) (Mean SD) G1 G2 G3 G4 G5 G6 (Vehicle 43 43 113 113 (Ribavirin Control), (20 mg/kg) (10 mg/kg) (20 mg/kg) (10 mg/kg) 100 mg/kg) n = 9 n = 9 n = 9 n = 9 n = 9 n = 9 0.191 0.012 0.121 0.018* 0.159 0.008* 0.166 0.010* 0.164 0.011* 0.176 0.010 *Significantly different from vehicle control (p < 0.05)

17.2.3 Viral Reduction in Plasma (Plaque Assay)

[0566] Compound 43 (Intraperitoneal Twice daily, at doses 20 mg/kg and10 mg/kg) showed significant dose dependent anti-viral effect in plasma, determined by Plaque assay, when compared to vehicle control (p<0.05) on day 3 and 4 (Table 3.2.3 and FIG. 3). Compound 113 showed significant dose dependent anti-viral effect in plasma on day 3 (Intraperitoneal Twice daily, at doses 20 mg/kg and10 mg/kg) and 4 (Intraperitoneal Twice daily, at doses 20 mg/kg)(Table 3.2.3 and FIG. 3). Ribavirin (100 mg/kg) did not show significant antiviral activity when compared to vehicle control on both days.

TABLE-US-00008 TABLE 3.2.3 Mean + SD Log.sub.10PFU/ml Plasma Experimental Group Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 G1 NA NA 3.15 0.43 5.37 0.12 4.87 0.04 3.77 0.10 G2 NA NA 3.48 0.25 4.34 0.07* 3.94 0.05* 3.06 0.31 G3 NA NA 3.36 0.41 4.91 0.06* 4.11 0.10* 3.28 0.21 G4 NA NA 3.07 0.28 4.25 0.05* 4.29 0.05* 3.77 0.11 G5 Na NA 3.79 0.10 5.05 0.23* 4.65 0.08 3.66 0.11 G6 NA NA 3.34 0.29 5.12 0.18 4.86 0.06 3.50 0.13

17.2.4 Biomarkers

[0567] Compounds 43 (20 mg/kg) and 113 (20 mg/kg) showed significant reduction in TNF- levels when compared to the vehicle control (p<0.05). No significant reduction was observed with Ribavirin (p>0.05) when compared to the vehicle control (Table 3.2.4 and FIG. 4).

[0568] Compound 113 (20 mg/kg) showed significant reduction in IL-6 levels when compared to the vehicle control (p<0.05). No significant reduction was observed with compound 43 (20 mg/kg, 10 mg/kg), compound 113 (10 mg/kg), and Ribavirin (p>0.05) when compared to the vehicle control (Table 3.2.4 and FIG. 4).

[0569] Compound 43 (10 mg/kg, 20 mg/kg), compound 113 (20 mg/kg), and Ribavirin showed significant reduction in IL-12 levels when compared to the vehicle control (p<0.05)(Table 3.2.4 and FIG. 4).

TABLE-US-00009 TABLE 3.2.4 Plasma concentrations of TNF-alpha, IL-6 and IL-12 in mouse plasma after treatment G2 G3 G4 G5 G1 43 43 113 113 G6 (Vehicle Control), (20 mg/kg) (10 mg/kg) (20 mg/kg) (10 mg/kg) (Ribavirin 100 mg/kg) Cytokine n = 9 n = 9 n = 9 n = 9 n = 9 n = 9 TNF-alpha 37.85 14.35 18.20 13.26* 32.45 12.30 19.55 10.23* 25.99 9.19 31.09 16.91 IL-6 21.76 11.21 13.92 7.59 16.08 6.47 9.62 3.02* 18.92 10.14 13.13 5.34 IL-12 95.69 28.29 51.67 16.83* 67.27 29.47* 53.06 16.62* 81.79 14.30 54.56 9.39* *Significantly different from vehicle control (p < 0.05)

Example 18-in vivo assay using subcutaneous administration

18.1 Introduction

[0570] These studies were largely performed as in Example 17, with a difference found in the administration of compounds of the invention. In Example 17, a comparative control using known antiviral drug ribavirin was administered via subcutaneous administration, and this comparative control was outperformed by the intraperitoneally administered compounds of the invention. In this example the compounds of the invention are administered using subcutaneous administration, and compound 113 was used as exemplary for compounds according to the invention.

18.2 Results

[0571] Subcutaneous administration showed slow elimination of compound 113 in blood plasma and improved efficacy against dengue virus infection in mice over other routes of administration.

18.2.1 Compounds Show Good Pharmacokinetics

[0572] As described in Example 17, compounds of the invention such as compound 113 give potent and specific inhibition of DENV protease in biochemical and cellular infection assays with minimal cytotoxicity. In vivo pharmacokinetic experiments for compound 113 showed reliable pharmacokinetic profiles with limited inter animal variability and dose proportionality following three routes: intravenous, intraperitoneal and subcutaneous (FIG. 5A). Similar data was obtained with compound 43 (FIG. 5D)

[0573] Low oral bioavailability of drugs such as peptidomimetics can be due to instability in the gastrointestinal tract, first pass elimination, or low permeability over biological membranes. The first two are not applicable to compounds of the invention: physical and chemical stability was found to be preserved in simulated intestinal fluids containing the appropriate number of metabolizing enzymes (pancreatin/pepsin), while permeability in Caco-2 cells was found to be low. The rate of elimination via systemic administration routes was also found to be slow, in tune with the limited hepatic clearance observed in vitro in both hepatocytes and liver microsomes. Furthermore, in vitro ADMET experiments showed no CYP inhibition/induction liability, indicating a low to absent risk of drug-drug interactions.

18.2.2 Subcutaneous Administration Increases Log-Reduction

[0574] The experiments described in Example 17 also confirmed excellent efficacy in mice (>92% viral load reduction on day 3 post-infection), with a treatment window up to 48 hours post-infection. It was found that with subcutaneous administration, viral load reduction was improved by more than one additional log-unit (>98% and >99% viral load reduction on day 3 and 4 after infection respectively; FIG. 5B). Via subcutaneous route, measured plasma levels over time were higher than via intraperitoneal administration (FIG. 5A and FIG. 5D).

[0575] Compounds 113 and 43 also showed good therapeutic efficacy as evidenced by plaque assays of the compounds in mouse plasma through the treatment period. Results are shown in the table below, where compounds were used at 20 mg/kg (5h post infection). Antiviral activity is much improved after subcutaneous administration, with a longer lasting effect.

TABLE-US-00010 G1 G2 G3 G4 G5 G6 Administration IP SC Compound Vehicle 113 43 Vehicle 113 43 Day 3 5.66 0.19 4.70 0.75* 4.32 0.45* 5.43 0.12 3.58 0.34* 3.43 0.304* Day 4 5.02 + 0.23 4.23 + 0.52* 4.38 + 0.41 4.64 + 0.39 3.26 + 0.49* 3.34 + 0.40*

18.2.3 Compounds Show Good Organ Targeting

[0576] Following administration, molecules reach the systemic circulation via either the blood capillaries or the lymphatic system. Biodistribution in rats showed that compound 113 could reach the main target sites of dengue (liver and spleen, FIG. 5C). These data indicate that subcutaneous administration can enhance delivery to target organs, further reducing viral load.

18.3 Conclusion

[0577] For dengue there is currently no traveller vaccine or antiviral on the market. For travellers visiting endemic countries, it is currently common practice for other infectious diseases (like hepatitis A or yellow fever) that they are vaccinated by single or repeated injection. In the case of malaria, travellers use daily oral drugs to prevent infection. Both treatment modalities are generally well accepted. Vaccinations are mostly given by (intramuscular) injection and are generally well accepted to obtain protection in this population.

[0578] At present there is no dengue prophylaxis on the market. The invention can provide such a prophylaxis or traveller vaccine. Subcutaneous administration was found to have specific dengue-relevant advantages for efficient systemic delivery and patient/traveller compliance and ease. Preclinical pharmacokinetic data showed slow elimination of the compounds, indicating that a single dose subcutaneous administration can be sufficient for one or two weeks of treatment in a therapeutic and prophylactic setting.