Macrocyclization of peptidomimetics

Abstract

A method of macrocyclization of peptidomimetics is described which comprises substitution of one or more of the backbone amide C═O bonds with a turn-inducing motif. The method is general with enhancements seen across a range of ring sizes (e.g. tri-, tetra-, penta- and hexapeptides). Specifically, a peptidomimetic macrocycle is described comprising a carbonyl bioisosteric turn-inducing element having the structure: ##STR00001## wherein X is a heteroatom; and wherein R.sub.1 to R.sub.6 are each independently selected from alkyl, aryl, heteroaryl and H.

Claims

1. A method of synthesizing a peptidomimetic macrocycle which is a continuous loop of 11 atoms or more and which comprises a carbonyl bioisosteric turn-inducing element comprising the steps: (a) synthesizing a linear peptidomimetic comprising the carbonyl bioisosteric turn-inducing element and (b) performing a cyclisation reaction of the linear peptidomimetic; wherein the turn-inducing element is: ##STR00123## wherein X is a secondary or tertiary amine; and wherein R.sub.1 to R.sub.6 are each independently selected from the group consisting of alkyl, aryl, heteroaryl and H.

2. The method according to claim 1, wherein the cyclisation reaction is selected from the group consisting of: a head-to-tail reaction; a sidechain-to-sidechain reaction; a head-to-sidechain reaction; and a sidechain-to-tail reaction.

3. The method according to claim 2, wherein the cyclization reaction is a sidechain-to-sidechain reaction and is achieved by an amide, ester, thioester, or disulfide bond formation.

4. The method according to claim 1, which comprises solution-phase or solid-phase peptide synthesis.

5. The method according to claim 1, wherein a compound of formula (III) is employed in synthesizing the linear peptidomimetic comprising the carbonyl bioisosteric turn-inducing element: ##STR00124## wherein X is a secondary or tertiary amine; and wherein R.sub.1 to R.sub.5 are each independently selected from the group consisting of alkyl, aryl, heteroaryl and H.

6. The method according to claim 5, wherein X is NH.

7. The method according to claim 5, wherein the compound of formula (III) is: ##STR00125## wherein R.sup.b is H, alkyl, aryl, heteroaryl, or a removable protecting group.

8. The method according to claim 7, wherein the removable protecting group is tert-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc), or carboxybenzyl (Cbz).

Description

(1) The invention will now be described in more detail by way of the following non-limiting Examples and with reference to the accompanying figures, in which:

(2) FIG. 1—Extracted ion chromatograms of peptide macrocyclizations for 1 to 3 (top) and 2 to 4 (bottom);

(3) FIG. 2—Spectra of Cyclo(Trp-Leu-Gly-Gly) (33);

(4) FIG. 3—Spectra of Cyclo(Trp-Leu-GOx-Gly) (5);

(5) FIG. 4—Spectra for Cyclo(Ala-GOx-Ala-Tyr-Leu) (8);

(6) FIG. 5—Spectra for Cyclo(Ala-Gly-Ala-Tyr-Leu) (10);

(7) FIG. 6—Spectra of Cyclo(H-Cys-Asn-GOx-Arg-Cys-OH (12);

(8) FIG. 7—Spectra of Cyclo(Boc-Cys-GOx-Cys-OtBu) (13);

(9) FIG. 8—a. Inhibition of APN by oxetane modified peptide 12. The shown data are the average of two independent experiments performed in duplicate. Error bars are displaying standard deviations. b. Inhibition of APN by parent peptide 15. The shown data are the average of two independent experiments performed in duplicate and triplicate, respectively. Error bars are displaying standard deviations;

(10) FIG. 9—Conversion of linear peptides 6 & 7 to cyclic peptides 8 & 9 and dimer 42 over 74 hours;

(11) FIG. 10—LCMS traces for oxetane and azetidine-containing cyclic peptides in TFA after 24 hours.

EXAMPLES

General Experimental Information

(12) Reaction mixtures were stirred magnetically. All chemicals were purchased from Acros Organics, Alfa Aesar, Fluorochem or Sigma-Aldrich and used as received unless otherwise mentioned. Preloaded 2-chlorotrityl resins were purchased from Merck, the polymer matrix is copoly (styrene-1% DVB), 200-400 mesh. TNBS test kit picrylsulfonic acid (ca. 1% in DMF) 10 mL/N,N-diisopropylethylamine (ca. 10% in DMF) 10 mL for detection of primary amines was purchased from TC Chemicals. Anhydrous solvents were purchased from Sigma-Aldrich or Acros Organics in Sure-Seal™ bottles. All other solvents were reagent grade and used as received. Petroleum ether refers to the fraction that boils in the range of 40-60° C. .sup.1H Nuclear Magnetic Resonance (NMR) spectra were recorded in CDC.sub.3, CD.sub.3OD or DMSO-d.sub.6, using a Bruker HD400 (400 MHz), AV500 (500 MHz) or AV600 (600 MHz) Fourier transform spectrometer. Chemical shifts (δH) are quoted in parts per million (ppm) and referred to the residual protic solvent signals of CDCl3 (7.26 ppm), CD.sub.3OD (3.31 ppm) or DMSO-d.sub.6 (2.50 ppm). 1H NMR coupling constants are reported in hertz and refer to apparent multiplicities. Data are reported as follows: chemical shift, multiplicity (s=singlet, br. s=broad singlet, d=doublet, t=triplet, q=quartet, quint=quintet, sext=sextet, sept=septet, m=multiplet, dd=doublet of doublet, etc.), coupling constant, integration, and assignment. .sup.13C NMR spectra were recorded at 101, 126 or 151 MHz. Chemical shifts (SC) are quoted in ppm referenced to CHCl.sub.3 (77.16 ppm), CD.sub.3OD (49.00 ppm) or DMSO-d6 (39.52 ppm). NMR assignments were deduced using 2D experiments (COSY, HSQC and HMBC). NH and OH are not visible in protic solvents (CD.sub.3OD). In most cases, Azetidinyl amine NH (in the .sup.1H) and the quaternary azetidine carbon (in the .sup.13C) is missing, and other selected missing peaks are noted on a compound by compound basis. Low-resolution mass spectra were recorded on an Agilent 6130B single Quad (ESI) instrument. High resolution mass spectra were recorded using a Bruker MaXis Impact. All infrared spectra were recorded on the neat compounds using a Bruker ALPHA-Platinum FTIR spectrometer, irradiating between 4000 cm.sup.−1 and 600 cm.sup.−1. Only strong and selected absorbances (ν.sub.max) are reported. Analytical TLC was performed on aluminium backed silica plates (Merck, Silica Gel 60 F254, 0.25 mm). Compounds were visualised by fluorescence quenching or by staining the plates with 10% solution of phosphomolybdic S3 acid (H.sub.3PMo.sub.12O.sub.40) in EtOH or 1% solution of potassium permanganate (KMnO.sub.4) in water followed by heating. Flash column chromatography was performed on silica gel (Aldrich or Fluorochem, Silica Gel 60, 40-63 μm). All mixed solvent eluents are reported as v/v solutions. Optical rotations were obtained using an AA1000 polarimeter at 589 nm (Na D-line) in a cell with a path length of 2 dm. Specific rotation values are given in (deg mL)/(g dm). Melting points were measured with a Gallenkamp melting point apparatus.

Example 1—Synthesis of Head-to-Tail Cyclisations

(13) Initially, a general route for the synthesis of linear peptides containing the carbonyl bioisoteric turn-inducing element in the backbone was developed as illustrated below for pentapeptide 1:

(14) ##STR00060##

(15) Turn-inducing element introduction was achieved by: (i) conjugate addition of the N-terminus of the growing peptide to the appropriately substituted nitroalkene (e.g. 3-(nitromethylene)oxetane); (ii) nitro group reduction and in situ coupling of the resulting amine to the next protected amino acid, preactivated as its succinyl ester..sup.7,11 Conventional peptides (e.g. 2) were made for comparison purposes. For all head-to-tail cyclizations, Bn ester/Z-protection of the C- and N-termini was used to allow synthesis of salt-free precursors by use of a final hydrogenolysis step. This enabled reliable comparisons in product yields to be made across different substrates.

(16) The impact of the introduction of this turn-inducing element on macrocyclization, ring closure of 1 to 3 was studied under a variety of conditions (see Table 1, entries 1-5). This substrate was chosen because the cyclization of the corresponding unmodified pentapeptide 2 to 4 is very low yielding even under high dilution (Table 1, entries 6-8)..sup.12 Upon introduction of the turn-inducing modification, a 3-fold yield improvement was observed. Best results were obtained using PyBOP (Table 1, entry 3), although DEPBT and HATU are also effective. Of practical importance, the reaction can be conducted on a larger scale (0.5 mmol) under less dilute conditions (0.005 M) without loss of yield (Table 1, entry 1).

(17) TABLE-US-00002 TABLE 1 Impact of oxetane modification on the cyclization efficiency of a pentapeptide. embedded image entry substrate.sup.a coupling reagent.sup.b product yield (%).sup.c 1 1 DEPBT 3 48 (50).sup.d 2 1 DEPBT.sup.e 3 33 3 1 PyBOP 3 60 4 1 HATU 3 53 5 1 T3P 3 28 6 2 DEPBT 4 .sup. 13.sup.f 7 2 PyBOP 4 23 8 2 HATU 4 15 .sup.b3-(Diethoxy-phosphoryloxy)-1,2,3,-benzotriazin-4(3H)-one (DEPBT); O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU); benzotriazol-1-yloxytri(pyrrolidino) phosphonium hexafluorophosphate (PyBOP); 1-propylphosphonic anhydride (T3P). .sup.cIsolated yield after column chromatography. .sup.dYield in parenthesis relates to larger scale reaction (0.5 mmol) run at higher concentration (0.005M). .sup.eiPr.sub.2EtN omitted. .sup.fTaken from ref 12.

(18) Insights into the impact of the turn-inducing element introduction on the efficiency of this macrocydization were revealed by LC-MS analysis (see FIG. 1). In the formation of 4, there are significant quantities of unreacted linear pentapeptide 2 even after 48 h. In contrast, for turn-inducing element containing 1, essentially complete consumption of starting material is seen on this timescale. The reaction is also much cleaner with fewer by-products. In the cyclization to 4, linear and cyclic decapeptides arising from substrate dimerization alongside a second cyclic pentapeptide are evident (FIG. 1, bottom). In contrast, these by-products are seen in only trace quantities in the formation of 3 (FIG. 1, top). Thus, significant improvements in this difficult macrocydization can be realized through introduction of the turn-inducing element.

(19) To determine the scope of this chemistry, other head-to-tail macrocyclizations were studied:

(20) ##STR00063##

(21) Oxetane modified cyclic peptides by head-to-tail macrocyclization. .sup.b Bond formed in macrocyclization indicated in bold in the ring; yield in parenthesis for the cyclization of linear peptide with C═O rather than oxetane in backbone. .sup.c PyBOP as activator. .sup.dDMTMM tetrafluoroborate as activator.

(22) Data taken from ref 13.

(23) Tetrapeptides 5-7, pentapeptide 8 and hexapeptide 9 all cyclize in higher yields than the corresponding unmodified peptides.

Example 1a—Detailed Example for Previous Cyclization Methods without the Carbonyl Bioisosteric Turn-Inducing Element

(24) Preparation of Cyclic Tetrapeptides 33, 36, and 39:

(25) ##STR00064##

(26) Boc-Leu-Gly-Gly-OBn (30): To a solution of dipeptide TsOH.Math.H-Gly-Gly-OBn.sup.[3] (3.94 g, 10.0 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (100 mL) was added Boc-Leu-OH (2.43 g, 10.5 mmol, 1.05 equiv), EDC.Math.HCl (2.01 g, 10.5 mmol, 1.05 equiv), HOBt-H.sub.2O (1.42 g, 10.5 mmol, 1.05 equiv) and NMM (4.40 mL, 40.0 mmol, 4.0 equiv), and the mixture was stirred at room temperature for 24 h. The mixture was diluted with EtOAc (100 mL) and washed with brine (3×100 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/EtOAc 1:1.fwdarw.EtOAc) to give Boc-Leu-Gly-Gly-OBn (30) (2.98 g, 6.84 mmol, 68%) as a white solid.

(27) Cbz-Trp-Leu-Gly-Gly-OBn (31): To a solution of Boc-Leu-Gly-Gly-OBn (30) (0.93 g, 2.15 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (2.5 mL) was added TFA (2.5 mL) and the mixture was stirred at room temperature for 1 h (Gas evolution!). The mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×25 mL) and concentrated in vacuo to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (22 mL), Cbz-Trp-OH (0.73 g, 2.15 mmol, 1.0 equiv), EDC.Math.HCl (0.41 g, 2.15 mmol, 1.0 equiv), HOBt-H.sub.2O (0.29 g, 2.15 mmol, 1.0 equiv) and NMM (0.95 mL, 8.60 mmol, 4.0 equiv) were added, and the mixture was stirred at room temperature for 24 h. The mixture was diluted with EtOAc (25 mL) and washed with brine (3×25 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 40:1) to give Cbz-Trp-Leu-Gly-Gly-OBn (31) (1.21 g, 1.89 mmol, 88%) as a white solid.

(28) H-Trp-Leu-Gly-Gly-OH (32): To a solution of tetrapeptide Cbz-Trp-Leu-Gly-Gly-OBn (31) (900 mg, 1.37 mmol, 1.0 equiv) in MeOH (15 mL) was added 10 wt % Pd/C (90 mg, 10 wt %) and the reaction flask was evacuated, filled with nitrogen, evacuated, and placed under an atmosphere of hydrogen (balloon). The reaction mixture was stirred at room temperature for 16 h, placed under nitrogen and filtered through a plug of Celite, which was washed with MeOH (3×). The filtrate was concentrated in vacuo to give H-Trp-Leu-Gly-Gly-OH (32) as a white solid (590 mg, 1.37 mmol, quant. yield), which required no further purification.

(29) Cyclo(Trp-Leu-Gly-Gly) (33): To a solution of H-Trp-Leu-Gly-Gly-OH (32) (86 mg, 0.20 mmol, 1.0 equiv) in anhydrous DMF (200 mL, 0.001 M) under an atmosphere of nitrogen was added PyBOP (208 mg, 0.40 mmol, 2.0 equiv) and DIPEA (70 μL, 0.40 mmol, 2.0 equiv) and the mixture was stirred for 48 h at room temperature. The solvent was removed under reduced pressure, and the residue was purified twice by column chromatography (SiO.sub.2, DCM/MeOH 92.5:7.5.fwdarw.4:1) to give cyclic tetrapeptide 33 as a yellow solid (13 mg, 31 μmol, 15%). R.sub.f (CH.sub.2Cl.sub.2/MeOH 9:1) 0.20; mp 216-219° C.; .sup.1H NMR (500 MHz, CD.sub.3OD) δ.sub.H 7.59 (d, J=7.9 Hz, 1H, ArH), 7.36 (d, J=8.1 Hz, 1H, ArH), 7.20 (s, 1H, ArH), 7.11 (t, J=7.5 Hz, 1H, ArH), 7.02 (t, J=7.5 Hz, 1H, ArH), 4.55 (dd, J=8.2, 5.2 Hz, 1H, CHα-Trp), 4.20 (dd, J=10.6, 4.4 Hz, 1H, CHα-Leu), 4.01 (d, J=16.8 Hz, 1H, CHHGly), 3.86 (d, J=16.1 Hz, 1H, CHHGly), 3.74 (d, J=16.1 Hz, 1H, CHHGly), 3.65 (d, J=16.8 Hz, 1H, CHHGly), 3.39-3.34 (m, 1H, CHHβ-Trp), 3.23 (dd, J=14.9, 8.3 Hz, 1H, CHHβ-Trp), 1.73-1.65 (m, 1H, CHHβ-Leu), 1.54 (ddd, J=13.9, 9.6, 4.5 Hz, 1H, CHHβ-Leu), 1.36-1.26 (m, 1H, CHγ-Leu), 0.84 (d, J=6.6 Hz, 3H, CH.sub.3δ-Leu), 0.78 (d, J=6.6 Hz, 3H, CH.sub.3δ-Leu); .sup.13C NMR (126 MHz, CD.sub.3OD) Sc 175.7 (C═O), 174.9 (C═O), 173.0 (C═O), 172.6 (C═O), 138.1 (C), 128.6 (C), 124.7 (CH), 122.6 (CH), 120.0 (CH), 119.2 (CH), 112.4 (CH), 110.5 (C), 56.8 (CH, α-Trp), 54.0 (CH, α-Leu), 44.1 (CH.sub.2, Gly), 43.4 (CH.sub.2, Gly), 40.6 (CH.sub.2, β-Leu), 27.9 (CH.sub.2, β-Trp), 25.7 (CH, γ-Leu), 23.7 (CH.sub.3, δ-Leu), 21.6 (CH.sub.3, δ-Leu); ν.sub.max (neat)=3298, 2869, 1645, 1522, 1234, 742 cm.sup.−1; MS (ESI.sup.+) m/z 849 [2M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.42H.sub.54N.sub.10NaO.sub.8 [2M+Na].sup.+ 849.4018. found 849.4020. [α].sub.D.sup.27 −16.6 (c 0.04, MeOH). Spectra in FIG. 2.

Example 1b—Detailed Example of a Head-to-Tail Cyclisation for a Tetrapeptide

(30) ##STR00065##

(31) Boc-Leu-GOx-Gly-OBn (23): To a solution of NO.sub.2-GOx-Gly-OBn.sup.[2] (1.70 g, 6.06 mmol, 1.0 equiv) in THF (60 mL) was added Boc-Leu-OSu (2.98 g, 9.09 mmol, 1.5 equiv) and Raney Ni (slurry in H.sub.2O, 6.0 mL). The solution was placed under an atmosphere of nitrogen, evacuated and filled with hydrogen (balloon). The reaction mixture was stirred vigorously for 4.0 h at room temperature. Then, the mixture was filtered through a plug of Celite eluting with EtOAc, concentrated under reduced pressure, the filtrate was suspended in EtOAc (50 mL), washed with saturated Na.sub.2CO.sub.3 (3×50 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Boc-Leu-GOx-Gly-OBn (23) was afforded after purification by column chromatography (SiO.sub.2, EtOAc/PE 3:2) as a colourless viscous oil (1.91 g, 4.13 mmol, 68%).

(32) Cbz-Trp-Leu-GOx-Gly-OBn (24): To a solution of tripeptide Boc-Leu-GOx-Gly-OBn 23 (1.29 g, 2.79 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (3.0 mL) was added TFA (3.0 mL) and the mixture was stirred at room temperature for 1 h (Caution—gas evolution!). The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×25 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in a mixture of CH.sub.2Cl.sub.2 (30 mL), Cbz-Trp-OH (0.94 g, 2.79 mmol, 1.0 equiv), EDC.Math.HCl (0.53 g, 2.79 mmol, 1.0 equiv), HOBt-H.sub.2O (0.38 g, 2.79 mmol, 1.0 equiv) and NMM (1.23 mL, 11.2 mmol, 4.0 equiv) were added subsequently, and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with EtOAc (30 mL) and washed with brine (30 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, 49:1 CH.sub.2Cl.sub.2/MeOH) to give tetrapeptide Cbz-Trp-Leu-GOx-Gly-OBn (24) (834 mg, 1.22 mmol, 44%) as a colourless viscous oil.

(33) H-Trp-Leu-GOx-Gly-OH (25): To a solution of tetrapeptide Cbz-Trp-Leu-GOx-Gly-OBn (24) (683 mg, 1.00 mmol, 1.0 equiv) in MeOH (10 mL) was added 10 wt % Pd/C (68 mg, 10 wt %) and the reaction flask was evacuated, filled with nitrogen, evacuated, and placed under an atmosphere of hydrogen (balloon). The reaction mixture was stirred at room temperature for 16 h, placed under nitrogen and filtered through a plug of Celite, which was washed with MeOH (3×). The filtrate was concentrated in vacuo to give tetrapeptide H-Trp-Leu-GOx-Gly-OH (25) as a yellow solid (458 mg, 1.00 mmol) in quantitative yield.

(34) Cyclo(Trp-Leu-GOx-Gly) (5): To a solution of tetrapeptide H-Trp-Leu-GOx-Gly-OH (25) (46 mg, 0.10 mmol, 1.0 equiv) in anhydrous DMF (100 mL, 0.001 M) under an atmosphere of nitrogen was added DEPBT (60 mg, 0.10 mmol, 2.0 equiv) and DIPEA (35 μL, 0.10 mmol, 2.0 equiv) and the reaction mixture was stirred for 48 h at room temperature. The solvent was removed under reduced pressure at 60° C. over 30 min, and the residue was dried in vacuo. The residue was analysed by LCMS and purified twice by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 19:1.fwdarw.9:1) to give cyclic tetrapeptide (5) as a yellow solid (29 mg, 65 μmol, 65%). R.sub.f (CH.sub.2Cl.sub.2/MeOH 9:1) 0.41; mp 200-203° C.; .sup.1H NMR (500 MHz, DMSO-d6) δ.sub.H 10.87 (s, 1H, NH), 8.25 (d, J=10.4 Hz, 1H, NH), 7.97 (d, J=9.1 Hz, 1H, NH), 7.56-7.50 (m, 2H, NH, ArH), 7.34 (d, J=8.0 Hz, 1H, ArH), 7.11 (s, 1H, ArH), 7.08 (t, J=7.5 Hz, 1H, ArH), 7.00 (t, J=7.4 Hz, 1H, ArH), 4.59 (q, J=9.3 Hz, 1H, CHα-Trp), 4.41 (d, J=6.3 Hz, 1H, OCHH-Ox), 4.18 (d, J=6.9 Hz, 1H, OCHH-Ox), 4.15 (d, J=6.3 Hz, 1H, OCHH-Ox), 4.04-3.97 (m, 1H, CHα-Leu), 3.90 (d, J=6.9 Hz, 1H, OCHH-Ox), 3.81 (dd, J=13.3, 7.7 Hz, 1H, CHHGly or CHHGOx), 3.43-3.37 (m, 1H, CHHGly or CHHGOx), 3.26-3.18 (m, 2H, CHHGly or CHHGOx, CHHβ-Trp), 3.07-2.97 (m, 2H, CHHGly or CHHGOx, CHHβ-Trp), 1.65-1.57 (m, 2H, CHHβ-Leu, CHγ-Leu), 1.53-1.45 (m, 1H, CHHβ-Leu), 0.92 (d, J=6.1 Hz, 3H, CH.sub.3δ-Leu), 0.79 (d, J=6.1 Hz, 3H, CH.sub.3δ-Leu). N.B. Secondary amine NH not observed; .sup.13C NMR (126 MHz, DMSO-d6) δ.sub.C 173.2 (C═O), 172.9 (C═O), 171.4 (C═O), 136.1 (C), 127.1 (C), 123.0 (CH), 121.0 (CH), 118.3 (CH), 118.1 (CH), 111.4 (CH), 109.5 (C), 78.1 (OCH.sub.2), 76.3 (OCH.sub.2), 60.3 (C, Ox), 56.0 (CH, α-Trp), 54.0 (CH, α-Leu), 47.3 (CH.sub.2, GOx or CH.sub.2, Gly), 44.2 (CH.sub.2, GOx or CH.sub.2, Gly), 39.2 (CH.sub.2, β-Leu), 26.5 (CH.sub.2, β-Trp), 24.6 (CH, γ-Leu), 22.8 (CH.sub.3, δ-Leu), 21.3 (CH.sub.3, δ-Leu); ν.sub.max (neat)=3278, 2954, 1660, 1516, 740 cm.sup.−1; MS (ESI.sup.+) m/z 464 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.23H.sub.31N.sub.5NaO.sub.4 [M+Na].sup.+ 464.2268. found 464.2270. [α].sub.D.sup.26 −60.3 (c 0.01, MeOH). See FIG. 3.

Example 2—the Effect of the Location of the Turn-Inducing Motif on Macrocyclic Yield

(35) Macrocyclization to pentapeptide 8 (from pentapeptides 44, 49, 54, and 61) by formation of all four possible amide bonds was examined to see whether the location of the turn-inducing element relative to the amide bond being formed is important:

(36) ##STR00066## Macrocyclization efficiency as a function of location of oxetane relative to forming amide bond..sup.b Average of two runs. .sup.c Taken from ref 12

(37) The yields were compared with those obtained making pentapeptide 10 by the same disconnections. This approach removes inherent differences in cyclization efficiency associated with amide bond formation between different amino acid residues. For all four pairs of cyclization studied, the turn-inducing-modified system outperformed the C═O system leading to higher product yields. However, it appears that larger improvements are seen when the modification is more centrally located along the precursor backbone.

(38) Details of Macrocyclisation to Pentapeptide 8

(39) Preparation of Pentapeptide 44:

(40) ##STR00067##

(41) NO.sub.2-GOx-Ala-OBn (40): To a solution of Boc-Ala-OBn (3.36 g, 12.0 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (12 mL) was added TFA (12 mL) and the mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated in vacuo to give the crude amine. In a second reaction vessel, oxetane-3-one (1.54 mL, 24.0 mmol, 2.0 equiv), nitromethane (1.82 mL, 33.6 mmol, 2.8 equiv) and triethylamine (670 μL, 4.80 mmol, 0.4 equiv) were combined at 0° C. and stirred for 1 h at room temperature. The mixture was dissolved in anhydrous CH.sub.2Cl.sub.2 (40 mL), cooled to −78° C., and triethylamine (6.70 mL, 48.0 mmol, 4.0 equiv) was added followed by dropwise addition of a solution of methanesulfonyl chloride (1.86 mL, 24.0 mmol, 2.0 equiv) in anhydrous CH.sub.2Cl.sub.2 (12 mL). The reaction mixture was stirred at −78° C. for 1.5 h and a solution of the crude amine and triethylamine (2.52 mL, 18.0 mmol, 1.5 equiv) in anhydrous CH.sub.2Cl.sub.2 (40 mL) was added slowly via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. A saturated solution of NH.sub.4Cl (50 mL) was added and stirred for 10 min. The layers were separated and the aqueous one extracted with CH.sub.2Cl.sub.2 (2×30 mL) and EtOAc (2×30 mL). The combined organic phases were washed with sat. NaHCO.sub.3 solution (50 mL), brine (50 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 2:1.fwdarw.1:1) to give 40 (3.28 g, 11.1 mmol, 93%) as an orange oil.

(42) Boc-Ala-GOx-Ala-OBn (41): To a solution of NO.sub.2-GOx-Ala-OBn (40) (3.18 g, 10.8 mmol, 1.0 equiv) in THF (108 mL) was added Boc-Ala-OSu (6.18 g, 21.6 mmol, 2.0 equiv), NaHCO.sub.3 (3.63 g, 43.2 mmol, 4.0 equiv) and Raney Ni (slurry in H.sub.2O, 22 mL). The solution was placed under an atmosphere of nitrogen, evacuated and filled with hydrogen (balloon). The reaction mixture was stirred vigorously for 4.0 h at room temperature. Then, the mixture was filtered through a plug of Celite eluting with EtOAc, the filtrate was washed with saturated Na.sub.2CO.sub.3 (3×50 mL) and concentrated in vacuo. Boc-Ala-GOx-Ala-OBn (41) was afforded after purification by column chromatography (SiO.sub.2, PE/EtOAc 1:1.fwdarw.EtOAc) as a pale-yellow oil (3.20 g, 7.34 mmol, 68%).

(43) Boc-Leu-Ala-GOx-Ala-OBn (42): To a solution of Boc-Ala-GOx-Ala-OBn (41) (915 mg, 2.10 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (2.5 mL) was added TFA (2.5 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (21 mL), Boc-Leu-OH (583 mg, 2.50 mmol, 1.2 equiv), HATU (958 mg, 2.50 mmol, 1.2 equiv) and DIPEA (1.46 mL, 8.40 mmol, 4.0 equiv) were added subsequently, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (30 mL) and washed with 10% citric acid solution (2×50 mL) and saturated NaHCO.sub.3 solution (2×50 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiO.sub.2, EtOAc) to give tetrapeptide 42 (855 mg, 1.56 mmol, 74%) as an off-white solid.

(44) Cbz-Tyr(Bn)-Leu-Ala-GOx-Ala-OBn (43): To a solution of Boc-Leu-Ala-GOx-Ala-OBn (42) (637 mg, 1.16 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (2.0 mL) was added TFA (2.0 mL) and the mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (12 mL), Cbz-Tyr(Bn)-OH (565 mg, 1.39 mmol, 1.2 equiv), HATU (565 mg, 1.39 mmol, 1.2 equiv) and DIPEA (808 μL, 4.64 mmol, 4.0 equiv) were added, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (20 mL) and washed with 10% citric acid solution (2×30 mL) and saturated NaHCO.sub.3 solution (2×30 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 19:1.fwdarw.9:1) to give pentapeptide 43 (463 mg, 0.55 mmol, 48%) as an off-white solid.

(45) H-Tyr-Leu-Ala-GOx-Ala-OH (44): To a solution of pentapeptide 43 (348 mg, 0.42 mmol) in anhydrous MeOH (4.0 mL) was added 10 wt % Pd/C (35 mg, 10 wt %) and the reaction flask was evacuated, filled with nitrogen, evacuated, and placed under an atmosphere of hydrogen (balloon). The reaction mixture was stirred at room temperature for 16 h, placed under nitrogen and filtered through a plug of Celite, which was washed with MeOH (3×). The filtrate was concentrated in vacuo to give 44 as an off-white solid (273 mg, >99%), which required no further purification.

(46) Preparation of Pentapeptide 49:

(47) ##STR00068##

(48) Boc-Tyr(Bn)-Leu-OBn (45): To a solution of Boc-Leu-OBn (8.04 g, 25.0 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (25 mL) was added TFA (25 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (250 mL), Boc-Tyr(Bn)-OH (11.1 g, 30.0 mmol, 1.2 equiv), HATU (11.4 g, 30.0 mmol, 1.2 equiv) and DIPEA (17.4 mL, 100 mmol, 4.0 equiv) were added subsequently, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was washed with 10% citric acid solution (2×100 mL) and saturated NaHCO.sub.3 solution (2×100 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 2:1.fwdarw.1:1) to give dipeptide 45 (14.1 g, 24.5 mmol, 98%) as a white solid.

(49) Boc-Ala-Tyr(Bn)-Leu-OBn (46): To a solution of Boc-Tyr(Bn)-Leu-OBn (45) (12.9 g, 22.5 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (22.5 mL) was added TFA (22.5 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated in vacuo to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (225 mL), Boc-Ala-OH (5.11 g, 27.0 mmol, 1.2 equiv), HATU (10.3 g, 27.0 mmol, 1.2 equiv) and DIPEA (15.7 mL, 90.0 mmol, 4.0 equiv) were added, and the mixture was stirred at room temperature for 16 h. The reaction mixture was washed with 10% citric acid solution (2×100 mL) and saturated NaHCO.sub.3 solution (2×100 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 1:1) to give tripeptide 46 (13.4 g, 20.7 mmol, 92%) as a white foam.

(50) NO.sub.2-GOx-Ala-Tyr(Bn)-Leu-OBn (47): To a solution of Boc-Ala-Tyr(Bn)-Leu-OBn (46) (3.87 g, 6.00 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (6.0 mL) was added TFA (6.0 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure to give the crude amine. In a second reaction vessel, oxetane-3-one (770 μL, 12.0 mmol, 2.0 equiv), nitromethane (910 μL, 16.8 mmol, 2.8 equiv) and triethylamine (335 μL, 2.40 mmol, 0.4 equiv) were combined at 0° C. and stirred for 1 h at room temperature. The mixture was dissolved in anhydrous CH.sub.2Cl.sub.2 (20 mL), cooled to −78° C., and triethylamine (3.35 mL, 24.0 mmol, 4.0 equiv) was added followed by dropwise addition of a solution of methanesulfonyl chloride (930 μL, 12.0 mmol, 2.0 equiv) in anhydrous CH.sub.2Cl.sub.2 (6.0 mL). The reaction mixture was stirred at −78° C. for 1.5 h and a solution of the crude amine and triethylamine (1.26 mL, 9.0 mmol, 1.5 equiv) in anhydrous CH.sub.2Cl.sub.2 (20 mL) was added slowly via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. A saturated solution of NH.sub.4Cl (30 mL) was added and stirred for 10 min. The layers were separated and the aqueous one extracted with CH.sub.2Cl.sub.2 (2×20 mL) and EtOAc (2×20 mL). The combined organic phases were washed with saturated aqueous NaHCO.sub.3 solution (30 mL), brine (30 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 1:1.fwdarw.EtOAc) to give 47 (1.90 g, 2.88 mmol, 48%) as an off-white solid.

(51) Cbz-Ala-GOx-Ala-Tyr(Bn)-Leu-OBn (48): To a solution of NO.sub.2-GOx-Ala-Tyr(Bn)-Leu-OBn (47) (1.26 g, 1.90 mmol, 1.0 equiv) in THF (20 mL) was added Cbz-Ala-OSu (1.22 g, 3.80 mmol, 2.0 equiv), NaHCO.sub.3 (638 mg, 7.60 mmol, 4.0 equiv) and Raney Ni (slurry in H.sub.2O, 4.0 mL). The solution was placed under an atmosphere of nitrogen, evacuated and filled with hydrogen (balloon). The mixture was stirred for 2.5 h at room temperature, filtered through a plug of Celite eluting with EtOAc, and the filtrate was concentrated in vacuo. Pentapeptide 48 was afforded after purification by column chromatography (SiO.sub.2, EtOAc-CH.sub.2Cl.sub.2/MeOH 9:1) as an off-white foam (989 mg, 1.18 mmol, 83%).

(52) H-Ala-GOx-Ala-Tyr-Leu-OH (49): To a solution of pentapeptide 48 (1.75 g, 2.09 mmol) in anhydrous MeOH (21 mL) was added 10 wt % Pd/C (175 mg, 10 wt %) and the reaction flask was evacuated, filled with nitrogen, evacuated, and placed under an atmosphere of hydrogen (balloon). The reaction mixture was stirred at room temperature for 16 h, placed under nitrogen and filtered through a plug of Celite, which was washed with MeOH (3×). The filtrate was concentrated in vacuo to give 49 as a white solid (1.14 g, >99%), which required no further purification.

(53) Preparation of Pentapeptide 54:

(54) ##STR00069##

(55) Boc-Ala-Tyr(Bn)-OBn (50): To a solution of Boc-Tyr(Bn)-OBn (11.3 g, 25.0 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (25 mL) was added TFA (25 mL) and the mixture was stirred at room temperature for 30 min (Caution—gas evolution!). The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (250 mL), Boc-Ala-OH (5.68 g, 30.0 mmol, 1.2 equiv), HATU (11.4 g, 30.0 mmol, 1.2 equiv) and DIPEA (17.4 mL, 100 mmol, 4.0 equiv) were added subsequently, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was washed with 10% citric acid solution (2×100 mL) and saturated NaHCO.sub.3 solution (2×100 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 2:1.fwdarw.1:1) to give dipeptide 50 (13.1 g, 24.5 mmol, 98%) as a white solid.

(56) O.sub.2N-GOx-Ala-Tyr(Bn)-OBn (51): To a solution of Boc-Ala-Tyr(Bn)-OBn (50) (3.20 g, 6.00 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (6.0 mL) was added TFA (6.0 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure to give the crude amine. In a second reaction vessel, oxetane-3-one (770 μL, 22.0 mmol, 2.0 equiv), nitromethane (910 μL, 16.8 mmol, 2.8 equiv) and triethylamine (335 μL, 2.40 mmol, 0.4 equiv) were combined at 0° C. and stirred for 1 h at room temperature. The mixture was dissolved in anhydrous CH.sub.2Cl.sub.2 (40 mL), cooled to −78° C., and triethylamine (3.35 mL, 24.0 mmol, 4.0 equiv) was added followed by dropwise addition of a solution of methanesulfonyl chloride (930 μL, 12.0 mmol, 2.0 equiv) in anhydrous CH.sub.2Cl.sub.2 (12 mL). The reaction mixture was stirred at −78° C. for 1.5 h and a solution of the crude amine and triethylamine (1.26 mL, 9.00 mmol, 1.5 equiv) in anhydrous CH.sub.2Cl.sub.2 (20 mL) was added slowly via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. A saturated solution of NH.sub.4Cl (50 mL) was added and stirred for 10 min. The layers were separated and the aqueous one extracted with CH.sub.2Cl.sub.2 (2×30 mL) and EtOAc (2×30 mL). The combined organic phases were washed with saturated aqueous NaHCO.sub.3 solution (50 mL), brine (50 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 1:1.fwdarw.EtOAc) to give 51 (2.79 g, 5.09 mmol, 85%) as an orange wax-like solid.

(57) Boc-Ala-GOx-Ala-Tyr(Bn)-OBn (52): To a solution of NO.sub.2-GOx-Ala-Tyr(Bn)-OBn (51) (2.66 g, 4.86 mmol, 1.0 equiv) in THF (48 mL) was added Boc-Ala-OSu (2.78 g, 9.71 mmol, 2.0 equiv), NaHCO.sub.3 (1.63 g, 19.4 mmol, 4.0 equiv) and Raney Ni (slurry in H.sub.2O, 10 mL). The solution was placed under an atmosphere of nitrogen, evacuated and filled with hydrogen (balloon). The reaction mixture was stirred vigorously for 4.0 h at room temperature. Then, the mixture was filtered through a plug of Celite eluting with EtOAc, the filtrate was washed with saturated Na.sub.2CO.sub.3 (3×50 mL) and concentrated under reduced pressure. Boc-Ala-GOx-Ala-Tyr(Bn)-OBn (52) was afforded after purification by column chromatography (SiO.sub.2, EtOAc) as an off-white foam (1.96 g, 2.85 mmol, 59%).

(58) Cbz-Leu-Ala-GOx-Ala-Tyr(Bn)-OBn (53): To a solution of Boc-Ala-GOx-Ala-Tyr(Bn)-OBn (52) (1.81 g, 2.63 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (3.0 mL) was added TFA (3.0 mL) and the mixture was stirred at room temperature for 10 min. The mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated in vacuo to give the crude amine. The residue was dissolved in a mixture of CH.sub.2Cl.sub.2 (26 mL) and DMF (5.0 mL), Cbz-Leu-OH (837 mg, 3.15 mmol, 1.2 equiv), HATU (1.20 g, 3.15 mmol, 1.2 equiv) and DIPEA (1.83 mL, 10.5 mmol, 4.0 equiv) were added subsequently, and the reaction mixture was stirred at room temperature for 16 h. The mixture was diluted with CH.sub.2Cl.sub.2 (30 mL) and washed with 10% citric acid solution (2×50 mL) and saturated NaHCO.sub.3 solution (2×50 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 98:2.fwdarw.97:3.fwdarw.96:4) to give pentapeptide 53 (1.21 g, 1.45 mmol, 55%) as a white foam.

(59) H-Leu-Ala-GOx-Ala-Tyr-OH (54): To a solution of pentapeptide 53 (1.14 g, 1.36 mmol) in anhydrous MeOH (14 mL) was added 10 wt % Pd/C (114 mg, 10 wt %) and the reaction flask was evacuated, filled with nitrogen, evacuated, and placed under an atmosphere of H.sub.2 (balloon). The reaction mixture was stirred at room temperature for 16 h, placed under N.sub.2 and filtered through a plug of Celite, which was washed with MeOH (3×). The filtrate was concentrated in vacuo to give 54 as an off-white solid (742 mg) in quantitative yield.

(60) Preparation of Pentapeptide 61:

(61) ##STR00070## ##STR00071##

(62) Fmoc-Ala-OCumyl (55): To sodium hydride (60% dispersion in mineral oil, 280 mg, 7.00 mmol, 0.5 equiv) in anhydrous diethyl ether (28 mL) was added freshly distilled 2-phenyl-2-propanol (4.20 g, 30.8 mmol, 2.2 equiv) at 0° C. and the mixture was stirred for 1 h at room temperature. The reaction mixture was cooled to 0° C., 2,2,2-trichloroacetonitrile (2.80 mL, 28.0 mmol, 2.0 equiv) were added slowly and stirring was continued for 3 h at ambient temperature. The solvent was removed under reduced pressure and the residue re-dissolved in PE (7.0 mL), anhydrous MeOH (283 μL, 7.00 mmol, 0.5 equiv) was added and the solution was stirred for 10 min at room temperature. The mixture was filtered through a plug of Celite eluting with PE and the filtrate was concentrated in vacuo to give the crude imidate. To a suspension of Fmoc-Ala-OH (4.36 g, 14.0 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (80 mL) was added a solution of the imidate in CH.sub.2Cl.sub.2 (15 mL) and the mixture was stirred for 16 h at room temperature. The reaction mixture was filtered through a plug of Celite eluting with CH.sub.2Cl.sub.2, the solvent was removed in vacuo, and the residue was purified by column chromatograph (SiO.sub.2, PE/EtOAc 4:1) to give Fmoc-Ala-OCumyl (55) (6.00 g, 14.0 mmol, quant. yield) contaminated with small amounts of 2-phenyl-2-propanol (85:15 by .sup.1H NMR) as a pale-yellow oil.

(63) O.sub.2N-GOx-Ala-OCumyl (56): To a solution of Fmoc-Ala-OCumyl (55) (3.70 g, 8.60 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (9.0 mL) was added diethylamine (9.0 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×15 mL) and concentrated under reduced pressure to give the crude amine. In a second reaction vessel, oxetane-3-one (1.10 mL, 17.2 mmol, 2.0 equiv), nitromethane (1.30 mL, 24.1 mmol, 2.8 equiv) and triethylamine (480 μL, 3.44 mmol, 0.4 equiv) were combined at 0° C. and stirred for 1 h at room temperature. The mixture was dissolved in anhydrous CH.sub.2Cl.sub.2 (60 mL), cooled to −78° C., and triethylamine (4.80 mL, 34.4 mmol, 4.0 equiv) was added followed by dropwise addition of a solution of methanesulfonyl chloride (1.33 mL, 17.2 mmol, 2.0 equiv) in anhydrous CH.sub.2Cl.sub.2 (18 mL). The reaction mixture was stirred at −78° C. for 1.5 h and the solution of the crude amine in anhydrous CH.sub.2Cl.sub.2 (30 mL) was added slowly via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. A saturated solution of NH.sub.4Cl (50 mL) was added and stirred for 10 min. The layers were separated and the aqueous one extracted with CH.sub.2Cl.sub.2 (2×30 mL) and EtOAc (2×30 mL). The combined organic phases were washed with saturated aqueous NaHCO.sub.3 solution (50 mL), brine (50 mL), dried over MgSO.sub.4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 2:1.fwdarw.1:1) to give 56 (2.04 g, 6.33 mmol, 74%) as a pale-yellow oil.

(64) Cbz-GOx-Ala-OCumyl (57): To a solution of NO.sub.2-GOx-Ala-OCumyl (56) (2.04 g, 6.32 mmol, 1.0 equiv) in THF (65 mL) was added N-(benzyloxycarbonyloxy) succinimide (3.15 g, 12.6 mmol, 2.0 equiv), NaHCO.sub.3 (2.12 g, 25.2 mmol, 4.0 equiv) and Raney Ni (slurry in H.sub.2O, 6.3 mL). The mixture was placed under an atmosphere of nitrogen, evacuated and filled with hydrogen (balloon). The reaction mixture was stirred vigorously for 2.5 h at room temperature. Then, the mixture was filtered through a plug of Celite eluting with EtOAc, the filtrate was washed with sat. Na.sub.2CO.sub.3 (3×50 mL) and concentrated under reduced pressure. Cbz-GOx-Ala-OCumyl (57) was afforded after purification by column chromato-graphy (SiO.sub.2, PE/EtOAc 2:1.fwdarw.1:1-EtOAc) as a pale yellow oil (1.55 g, 3.63 mmol, 58%).

(65) Boc-Leu-Ala-OBn (58): To a solution of Boc-Ala-OBn (4.56 g, 16.3 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (16 mL) was added TFA (16 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (160 mL), Boc-Leu-OH (4.53 g, 19.6 mmol, 1.2 equiv), HATU (7.45 g, 19.6 mmol, 1.2 equiv) and DIPEA (11.4 mL, 65.2 mmol, 4.0 equiv) were added subsequently, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (50 mL) and washed with 10% citric acid solution (2×100 mL) and sat. NaHCO.sub.3 solution (2×100 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiO.sub.2, PE/EtOAc 2:1.fwdarw.1:1) to give dipeptide 58 (6.40 g, 16.3 mmol, >99%) as a colourless viscous oil.

(66) Boc-Tyr(Bn)-Leu-Ala-OBn (59): To a solution of Boc-Leu-Ala-OBn (58) (5.85 g, 14.9 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (15 mL) was added TFA (15 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (150 mL), Boc-Tyr(Bn)-OH (6.40 g, 17.2 mmol, 1.2 equiv), HATU (6.54 g, 17.2 mmol, 1.2 equiv) and DIPEA (10.4 mL, 59.6 mmol, 4.0 equiv) were added subsequently, and the reaction mixture was stirred at room temperature for 16 h. The mixture was diluted with CH.sub.2Cl.sub.2 (50 mL) and washed with 10% citric acid solution (2×100 mL) and saturated NaHCO.sub.3 solution (2×100 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiO.sub.2, PE/EtOAc 2:1.fwdarw.1:1) to give tripeptide 59 (9.60 g, 14.9 mmol, >99%) as a white solid.

(67) Cbz-GOx-Ala-Tyr(Bn)-Leu-Ala-OBn (60): Cbz-GOx-Ala-OCumyl (57) (853 mg, 2.00 mmol, 1.0 equiv) was stirred in 2% TFA/CH.sub.2Cl.sub.2 (40 mL) at room temperature for 90 min. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated under reduced pressure to give the crude acid. In a separate reaction flask Boc-Tyr(Bn)-Leu-Ala-OBn (59) (1.29 g, 2.00 mmol, 1.0 equiv) was dissolved in CH.sub.2Cl.sub.2 (2.0 mL), TFA (2.0 mL) was added and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in a mixture of CH.sub.2Cl.sub.2 (20 mL) and DMF (1.0 mL) and added to the crude acid. HATU (760 mg, 2.00 mmol, 1.0 equiv) and DIPEA (1.39 mL, 8.00 mmol, 4.0 equiv) were added subsequently, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with EtOAc (50 mL), washed with brine (2×50 mL), 1.0 M HCl (3×50 mL), saturated NaHCO.sub.3 solution (3×50 mL), brine (50 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiO.sub.2, EtOAc) to give pentapeptide 60 (1.15 g, 1.37 mmol, 68%) as a white foam.

(68) H-GOx-Ala-Tyr-Leu-Ala-OH (61): To a solution of pentapeptide 60 (316 mg, 0.38 mmol) in anhydrous MeOH (4.0 mL) was added 10 wt % Pd/C (32 mg, 10 wt %) and the reaction flask was evacuated, filled with nitrogen, evacuated, and placed under an atmosphere of hydrogen (balloon). The reaction mixture was stirred at room temperature for 5 h, placed under nitrogen and filtered through a plug of Celite, which was washed with MeOH (3×). The filtrate was concentrated in vacuo to give 61 as an off-white solid (189 mg, 0.36 mmol, 95%), which required no further purification.

(69) Representative example for cyclisation: Cyclo(Ala-GOx-Ala-Tyr-Leu) (8): To a solution of H-Ala-GOx-Ala-Tyr-Leu-OH (49) (52 mg, 0.10 mmol, 1.0 equiv) in anhydrous DMF (100 mL, 0.001 M) under an atmosphere of nitrogen was added DEPBT (60 mg, 0.20 mmol, 2.0 equiv) and DIPEA (35 μL, 0.20 mmol, 2.0 equiv) and the reaction mixture was stirred for 24 h at room temperature. The solvent was removed under reduced pressure, and the residue was purified twice by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 9:1.fwdarw.4:1) to give the cyclic pentapeptide (8) as a white solid (27 mg, 54 μmol, 54%). R.sub.f (CH.sub.2Cl.sub.2/MeOH 4:1) 0.57; mp 213-217° C.; .sup.1H NMR (400 MHz, CD.sub.3OD) δ.sub.H ppm 7.06 (d, J=8.3 Hz, 2H, ArH), 6.72 (d, J=8.3 Hz, 2H, ArH), 4.53 (d, J=7.8 Hz, 1H, OCHH-Ox), 4.49 (q, J=7.1 Hz, 1H, CHα-Ala), 4.46 (d, J=6.2 Hz, 1H, OCHH-Ox), 4.32-4.25 (m, 3H, 2×OCHH-Ox, CHα-Leu), 4.19 (dd, J=9.9, 6.1 Hz, 1H, CHα-Tyr), 3.70 (d, J=14.0 Hz, 1H, CHHGOx), 3.49 (q, J=6.9 Hz, 1H, CHα-Ala), 3.43 (d, J=14.0 Hz, 1H, CHHGOx), 3.20 (dd, J=13.4, 10.4 Hz, 1H, CHHβ-Tyr), 3.07 (dd, J=13.4, 6.1 Hz, 1H, CHHβ-Tyr), 1.79 (ddd, J=13.9, 11.2, 4.7 Hz, 1H, CHHβ-Leu), 1.70-1.62 (m, 1H, CHHβ-Leu), 1.57-1.48 (m, 1H, CHγ-Leu), 1.38 (d, J=7.1 Hz, 3H, CH.sub.3β-Ala), 1.20 (d, J=6.9 Hz, 3H, CH.sub.3β-Ala), 0.94 (d, J=6.6 Hz, 3H, CH.sub.3δ-Leu), 0.86 (d, J=6.5 Hz, 3H, CH.sub.3δ-Leu); .sup.13C NMR (101 MHz, CD.sub.3OD) δ.sub.C ppm 178.7 (C═O), 175.6 (C═O), 174.3 (C═O), 173.9 (C═O), 157.4 (C), 131.3 (CH), 129.1 (C), 116.3 (CH), 81.6 (OCH.sub.2), 79.0 (OCH.sub.2), 62.0 (C, Ox), 57.9 (CH, α-Tyr), 55.2 (CH, α-Leu), 53.7 (CH, α-Ala), 50.7 (CH, α-Ala), 46.2 (CH.sub.2, GOx), 40.7 (CH.sub.2, β-Leu), 35.7 (CH.sub.2, β-Tyr), 25.9 (CH, γ-Leu), 23.5 (CH.sub.3, δ-Leu), 21.5 (CH.sub.3, β-Ala), 21.2 (CH.sub.3, δ-Leu), 17.9 (CH.sub.3, β-Ala); ν.sub.max (neat)=3260, 2958, 1647, 1513, 1232, 965, 828 cm.sup.−1; MS (ESI.sup.+) m/z 504 [M+H].sup.+, 526 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.25H.sub.38N.sub.5O.sub.6 [M+H].sup.+ 504.2817. found 504.2818. [α].sub.D.sup.28 −69.7 (c 0.06, MeOH). See FIG. 4 for spectra.

(70) Details of Macrocyclisation of Pentapeptide 10

(71) Preparation of Pentapeptide 64:

(72) ##STR00072##

(73) Boc-Gly-Ala-Tyr(Bn)-Leu-OBn (62): To a solution of Boc-Ala-Tyr(Bn)-Leu-OBn (46) 1.00 g, 1.55 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (10 mL) was added TFA (2.0 mL) and the mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (16 mL), Boc-Gly-OH (326 mg, 1.86 mmol, 1.2 equiv), HATU (707 mg, 1.86 mmol, 1.2 equiv) and DIPEA (1.08 mL, 6.20 mmol, 4.0 equiv) were added subsequently, and the mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (20 mL) and washed with 10% citric acid solution (2×20 mL) and saturated NaHCO.sub.3 solution (2×20 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 1:1.fwdarw.EtOAc) to give tetrapeptide 62 (912 mg, 1.30 mmol, 84%) as a white solid.

(74) Cbz-Ala-Gly-Ala-Tyr(Bn)-Leu-OBn (63): To a solution of Boc-Gly-Ala-Tyr(Bn)-Leu-OBn (62) (861 mg, 1.23 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (10 mL) was added TFA (1.5 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (13 mL), Cbz-Ala-OH (328 mg, 1.47 mmol, 1.2 equiv), HATU (559 mg, 1.47 mmol, 1.2 equiv) and DIPEA (853 μL, 4.90 mmol, 4.0 equiv) were added subsequently, and the mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with CH.sub.2Cl.sub.2 (20 mL) and washed with 10% citric acid solution (2×20 mL) and saturated NaHCO.sub.3 solution (2×20 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 19:1.fwdarw.9:1) to give pentapeptide 63 (928 mg, 1.15 mmol, 93%) as a white solid.

(75) H-Ala-Gly-Ala-Tyr-Leu-OH (64): To a solution of pentapeptide 63 (820 mg, 1.01 mmol) in anhydrous DMF (20 mL) was added 10 wt % Pd/C (82 mg, 10 wt %) and the reaction flask was evacuated, filled with nitrogen, evacuated, and placed under an atmosphere of hydrogen (balloon). The reaction mixture was stirred at room temperature for 24 h, placed under nitrogen and filtered through a plug of Celite, which was washed with MeOH (3×). The filtrate was concentrated in vacuo to give 64 as a tan-coloured solid (418 mg, 65%), which required no further purification. N.B. The product was isolated as a complex with 2.0 equiv of DMF.

(76) Preparation of Pentapeptide 68:

(77) ##STR00073##

(78) Boc-Gly-Ala-Tyr(Bn)-OBn (65): To a solution of Boc-Ala-Tyr(Bn)-OBn (50) (2.66 g, 5.00 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (5.0 mL) was added TFA (5.0 mL) and the mixture was stirred at room temperature for 30 min (Caution—gas evolution!). The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in a mixture of CH.sub.2Cl.sub.2 (50 mL) and DMF (10 mL), Boc-Gly-OH (1.05 g, 6.00 mmol, 1.2 equiv), HATU (2.28 g, 6.00 mmol, 1.2 equiv) and DIPEA (3.48 mL, 20.0 mmol, 4.0 equiv) were added subsequently, and the mixture was stirred at room temperature for 16 h. The reaction mixture was washed with 10% citric acid solution (2×50 mL) and saturated NaHCO.sub.3 solution (2×50 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 1:1.fwdarw.EtOAc) to give tripeptide Boc-Gly-Ala-Tyr(Bn)-OBn (65) (2.57 g, 4.36 mmol, 87%) as a white foam.

(79) Boc-Ala-Gly-Ala-Tyr(Bn)-OBn (66): To a solution of Boc-Gly-Ala-Tyr(Bn)-OBn (65) (887 mg, 1.50 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (2.0 mL) was added TFA (2.0 mL) and the mixture was stirred at room temperature for 30 min (Caution—gas evolution!). The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in a mixture of CH.sub.2Cl.sub.2 (15 mL) and DMF (5.0 mL), Boc-Ala-OH (341 mg, 1.80 mmol, 1.2 equiv), HATU (684 mg, 1.80 mmol, 1.2 equiv) and DIPEA (1.05 mL, 6.00 mmol, 4.0 equiv) were added subsequently, and the mixture was stirred at room temperature for 16 h. The reaction mixture was washed with 10% citric acid solution (2×50 mL) and saturated NaHCO.sub.3 solution (2×50 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, EtOAc) to give tetrapeptide Boc-Ala-Gly-Ala-Tyr(Bn)-OBn (66) (725 mg, 1.10 mmol, 73%) as a white solid.

(80) Cbz-Leu-Ala-Gly-Ala-Tyr(Bn)-OBn (67): To a solution of Boc-Ala-Gly-Ala-Tyr(Bn)-OBn (66) (642 mg, 0.97 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (2.0 mL) was added TFA (1.0 mL) and the mixture was stirred at room temperature for 30 min (Caution—gas evolution!). The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in a mixture of CH.sub.2Cl.sub.2 (5.0 mL) and DMF (5.0 mL), Cbz-Leu-OH (309 mg, 1.16 mmol, 1.2 equiv), HATU (441 mg, 1.16 mmol, 1.2 equiv) and DIPEA (696 μL, 3.88 mmol, 4.0 equiv) were added subsequently, and the mixture was stirred at room temperature for 16 h. The reaction mixture was washed with 10% citric acid solution (2×50 mL) and saturated NaHCO.sub.3 solution (2×50 mL), dried over MgSO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 98:2-496:4) to give pentapeptide Cbz-Leu-Ala-Gly-Ala-Tyr(Bn)-OBn (67) (496 mg, 0.61 mmol, 63%) as a white solid.

(81) H-Leu-Ala-Gly-Ala-Tyr-OH (68): To a solution of pentapeptide 67 (402 mg, 0.50 mmol) in a mixture of anhydrous MeOH (15 mL) and DMF (5.0 mL) was added 10 wt % Pd/C (40 mg, 10 wt %) and the reaction flask was evacuated, filled with nitrogen, evacuated, and placed under an atmosphere of hydrogen (balloon). The reaction mixture was stirred at room temperature for 16 h, placed under nitrogen and filtered through a plug of Celite, which was washed with MeOH (3×). The filtrate was concentrated in vacuo to give 68 as an off-white solid (199 mg) in 64% yield. N.B. The product was obtained as a complex with 2.0 equiv of DMF.

(82) Representative example for cyclisation: Cyclo(Ala-Gly-Ala-Tyr-Leu) (10): To a solution of H-Ala-Gly-Ala-Tyr-Leu-OH (64) (50 mg, 0.10 mmol, 1.0 equiv) in anhydrous DMF (100 mL, 0.001 M) under an atmosphere of nitrogen was added DEPBT (60 mg, 0.20 mmol, 2.0 equiv) and DIPEA (35 μL, 0.20 mmol, 2.0 equiv) and the mixture was stirred for 24 h at room temperature. The solvent was removed in vacuo and the residue was purified twice by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 9:1.fwdarw.4:1) to give the cyclic pentapeptide 10 as a white solid (20 mg, 42 μmol, 42%). R.sub.f (CH.sub.2Cl.sub.2/MeOH 4:1) 0.48; mp 294-296° C. (decomposition). Lit. 284-286° C.;.sup.[4] 1H NMR (400 MHz, CD.sub.3OD) δ.sub.H ppm 7.08 (d, J=8.4 Hz, 2H, ArH), 6.70 (d, J=8.4 Hz, 2H, ArH), 4.51 (t, J=8.0 Hz, 1H, CHα-Tyr), 4.34 (q, J=7.0 Hz, 1H, CHα-Ala), 4.19 (q, J=7.3 Hz, 1H, CHα-Ala), 4.04 (dd, J=10.5, 5.2 Hz, 1H, CHα-Leu), 3.98 (d, J=14.7 Hz, 1H, CHH-Gly), 3.59 (d, J=14.7 Hz, 1H, CHH-Gly), 3.06 (dd, J=12.3, 6.1 Hz, 1H, CHHβ-Tyr), 3.01 (dd, J=12.3, 7.5 Hz, 1H, CHHβ-Tyr), 1.86 (ddd, J=13.5, 10.7, 4.8 Hz, 1H, CHHβ-Leu), 1.57-1.48 (m, 1H, CHHβ-Leu), 1.47-1.40 (m, 1H, CHγ-Leu), 1.37 (d, J=7.1 Hz, 3H, CH.sub.3β-Ala), 1.28 (d, J=7.3 Hz, 3H, CH.sub.3β-Ala), 0.93 (d, J=6.5 Hz, 3H, CH.sub.3δ-Leu), 0.86 (d, J=6.4 Hz, 3H, CH.sub.3δ-Leu); .sup.13C NMR (101 MHz, CD.sub.3OD) δ.sub.C ppm 175.5 (C═O), 175.03 (C═O), 174.98 (C═O), 173.7 (C═O), 172.0 (C═O), 157.4 (C), 131.3 (CH), 128.8 (C), 116.2 (CH), 57.2 (CH, α-Tyr), 56.2 (CH, α-Leu), 51.8 (CH, α-Ala), 50.6 (CH, α-Ala), 44.4 (CH.sub.2, Gly), 40.4 (CH.sub.2, β-Leu), 37.0 (CH.sub.2, β-Tyr), 25.9 (CH, γ-Leu), 23.4 (CH.sub.3, δ-Leu), 21.7 (CH.sub.3, δ-Leu), 17.7 (CH.sub.3, β-Ala), 17.1 (CH.sub.3, β-Ala); ν.sub.max (neat)=3279, 1648, 1631, 1530, 1514, 1440, 1384, 1226, 1087 cm.sup.−1; MS (ESI.sup.+) m/z 498 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.23H.sub.33N.sub.5NaO.sub.6 [M+Na].sup.+ 498.2323. found 498.2322. [α].sub.D.sup.26 −82.8 (c 0.20, MeOH). Lit. [α].sub.D.sup.20 −104 (c 0.10, C.sub.2H.sub.5OH)..sup.[4] See FIG. 5 for spectra.

(83) The preparation of cyclic pentapeptides 8 and 10 by different bond formations is summarized below:

(84) TABLE-US-00003 8 embedded image 10 Cyclic Average Linear pentapeptide peptide 1.sup.st run 2.sup.nd run yield H-GOx-Ala-Tyr-Leu-Ala-OH (61) 8 43% 43% 43% H-Ala-GOx-Ala-Tyr-Leu-OH (49) 8 54% 60% 57% H-Leu-Ala-GOx-Ala-Tyr-OH (54) 8 56% 46% 51% H-Tyr-Leu-Ala-GOx-Ala-OH (44) 8 32% 30% 31% H-Ala-Gly-Ala-Tyr-Leu-OH (64) 10 42% 38% 40% H-Leu-Ala-Gly-Ala-Tyr-OH (68) 10 33% 29% 31%

Example 3—Synthesis of Sidechain-to-Sidechain Cyclisations

(85) The methodology can be used to make disulfide containing macrocycles through oxidative cyclization of cysteine side chains:

(86) ##STR00076## Oxetane modified cyclic peptides by disulfide bond formation

(87) Treatment of 11 with iodine provided the 17-membered macrocycle by way of trityl deprotection and disulfide bond formation. Subsequent reaction with TFA facilitated removal of the side chain protecting groups providing 12 after reverse-phase HPLC purification. Importantly, the four-membered turn-inducing element is sufficiently stable to survive the strongly acid conditions required to globally deprotect the side-chains. Smaller 11- and 14-membered macrocycles 13 and 14 were also conveniently produced in high yields using this chemistry.

Example 3a—Detailed Example of a Sidechain-to-Sidechain Cyclisation for a Pentapeptide

(88) ##STR00077## ##STR00078##

(89) Fmoc-Arg(Pbf)-OCumyl (76): To sodium hydride (60% dispersion in mineral oil, 200 mg, 5.00 mmol, 0.5 equiv) in anhydrous diethyl ether (20 mL) was added freshly distilled 2-phenyl-2-propanol (3.00 g, 22.0 mmol, 2.2 equiv) at 0° C. and the mixture was stirred for 1 h at room temperature. The reaction mixture was cooled to 0° C., 2,2,2-trichloroacetonitrile (2.00 mL, 20.0 mmol, 2.0 equiv) were added slowly and stirring was continued for 3 h at ambient temperature. The solvent was removed under reduced pressure and the residue re-dissolved in petroleum ether (5.0 mL), anhydrous MeOH (202 μL, 5.00 mmol, 0.5 equiv) was added and the solution was stirred for 10 min at room temperature. The mixture was filtered through a plug of Celite eluting with PE and the filtrate was concentrated under reduced pressure to give the crude imidate. To a suspension of Fmoc-Arg(Pbf)-OH (6.49 g, 10.0 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (60 mL) was added a solution of the imidate in CH.sub.2Cl.sub.2 (15 mL) and the mixture was stirred for 16 h at room temperature. The reaction mixture was filtered through a plug of Celite eluting with CH.sub.2Cl.sub.2, the solvent was removed in vacuo, and the residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 1:1.fwdarw.EtOAc) to give Fmoc-Arg(Pbf)-OCumyl (76) (2.10 g, 2.74 mmol, 27%) as a white solid.

(90) NO.sub.2-GOx-Arg(Pbf)-OCumyl (77): To a solution of Fmoc-Arg(Pbf)-OCumyl (76) (2.40 g, 3.13 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (3.5 mL) was added diethylamine (3.5 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure to give the crude amine. In a second reaction vessel, oxetane-3-one (410 μL, 6.26 mmol, 2.0 equiv), nitromethane (475 μL, 8.76 mmol, 2.8 equiv) and triethylamine (174 μL, 1.25 mmol, 0.4 equiv) were combined at 0° C. and stirred for 1 h at room temperature. The mixture was dissolved in anhydrous CH.sub.2Cl.sub.2 (24 mL), cooled to −78° C., and triethylamine (1.74 mL, 12.5 mmol, 4.0 equiv) was added followed by dropwise addition of a solution of methanesulfonyl chloride (485 μL, 6.26 mmol, 2.0 equiv) in anhydrous CH.sub.2Cl.sub.2 (6.0 mL). The reaction mixture was stirred at −78° C. for 1.5 h and a solution of the crude amine in anhydrous CH.sub.2Cl.sub.2 (12 mL) was added slowly via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. A saturated solution of NH.sub.4Cl (30 mL) was added and stirred for 10 min. The layers were separated and the aqueous layer was extracted with CH.sub.2Cl.sub.2 (2×20 mL) and EtOAc (2×20 mL). The combined organic phases were washed with saturated aqueous NaHCO.sub.3 solution (30 mL), brine (30 mL), dried over MgSO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 1:1.fwdarw.EtOAc) to give 77 (1.41 g, 2.14 mmol, 68%) as an orange foam.

(91) Fmoc-GOx-Arg(Pbf)-OCumyl (78): To a solution of NO.sub.2-GOx-Arg(Pbf)-OCumyl (77) (1.41 g, 2.14 mmol, 1.0 equiv) in THF (22 mL) was added Fmoc N-hydroxysuccinimide ester (1.44 g, 4.28 mmol, 2.0 equiv), NaHCO.sub.3 (719 mg, 8.56 mmol, 4.0 equiv) and Raney Ni (slurry in H.sub.2O, 2.2 mL). The reaction mixture was placed under an atmosphere of nitrogen, evacuated and filled with hydrogen (balloon). The reaction mixture was stirred vigorously for 2.5 h at room temperature, filtered through a plug of Celite eluting with EtOAc, and the filtrate was concentrated under reduced pressure. Fmoc-GOx-Arg(Pbf)-OCumyl (78) was afforded after purification by column chromatography (SiO.sub.2, PE/EtOAc 1:1.fwdarw.EtOAc) as a white foam (1.29 g, 1.51 mmol, 71%).

(92) Fmoc-GOx-Arg(Pbf)-Cys(Trt)-OtBu (79): Fmoc-GOx-Arg(Pbf)-OCumyl (78) (256 mg, 0.30 mmol, 1.0 equiv) was dissolved in 2% TFA/CH.sub.2Cl.sub.2 (0.05 M) and stirred at room temperature for 2 h following a procedure from Beadle et al..sup.[5] The reaction mixture was concentrated under reduced pressure, and the resulting residue was repeatedly re-suspended in CH.sub.2Cl.sub.2 (3×15 mL) and the solvent removed under reduced pressure. Meanwhile, diethylamine (2.0 mL) was added to a solution of Fmoc-Cys(Trt)-OtBu (81) (298 mg, 0.45 mmol, 1.5 equiv) in CH.sub.2Cl.sub.2 (2.0 mL) and the reaction mixture was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×15 mL) and concentrated under reduced pressure to give the crude amine. The crude Fmoc-GOx-Arg(Pbf)-OH was dissolved in DMF (5.0 mL) and HATU (125 mg, 0.33 mmol, 1.1 equiv), diisopropylethyl-amine (204 μL, 1.20 mmol, 4.0 equiv) and the crude amine in DMF (2.0 mL) were added successively. The reaction mixture was stirred at room temperature for 48 h and the solvent removed under reduced pressure. The residue was purified by flash column chromatography (SiO.sub.2, PE/EtOAc 1:1.fwdarw.5% MeOH in CH.sub.2Cl.sub.2) to give tripeptide 79 (268 mg, 0.24 mmol, 79%) as a white foam;

(93) Fmoc-Asn(Trt)-GOx-Arg(Pbf)-Cys(Trt)-OtBu (80): Diethylamine (2.0 mL) was added to a solution of Fmoc-GOx-Arg(Pbf)-Cys(Trt)-OtBu (79) (262 mg, 0.23 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (2.0 mL) and the reaction mixture was stirred at room temperature for 1 h. The mixture was concentrated in vacuo and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×15 mL) and concentrated under reduced pressure to give the crude amine. HATU (132 mg, 0.35 mmol, 1.5 equiv), diisopropylethylamine (181 μL, 1.10 mmol, 3.0 equiv) and Fmoc-Asn(Trt)-OH (208 mg, 0.35 mmol, 1.5 equiv) were added to the crude amine in CH.sub.2Cl.sub.2 (5.0 mL). The reaction mixture was stirred at room temperature for 16 h and the solvent was removed in vacuo. The residue was purified by flash column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2.fwdarw.5% MeOH in CH.sub.2Cl.sub.2) to give tetrapeptide 80 (262 mg, 0.18 mmol, 78%) as a white foam;

(94) Fmoc-Cys(Trt)-Asn(Trt)-GOx-Arg(Pbf)-Cys(Trt)-OtBu (11): Diethylamine (1.0 mL) was added to a solution of Fmoc-GOx-Arg(Pbf)-Cys(Trt)-OtBu (80) (200 mg, 0.14 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (1.0 mL) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×15 mL) and concentrated under reduced pressure to give the crude amine. HATU (76 mg, 0.20 mmol, 1.4 equiv), diisopropylethylamine (105 μL, 0.60 mmol, 3.0 equiv) and Boc-Cys(Trt)-OH (94 mg, 0.20 mmol, 1.4 equiv) were added to the crude amine in CH.sub.2Cl.sub.2 (5.0 mL). The reaction mixture was stirred at room temperature for 16 h and the solvent removed under reduced pressure. The residue was purified by flash column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2.fwdarw.5% MeOH in CH.sub.2Cl.sub.2) to give pentapeptide 11 (191 mg, 0.11 mmol, 81%) as a white foam;

(95) Cyclo(H-CVs-Asn-GOx-Arg-CVs-OH) (12): The fully protected pentapeptide 11 (66.5 mg, 0.04 mmol, 1.0 equiv) was dissolved in MeOH (2.0 mL) and slowly added to a solution of iodine (30 mg, 0.12 mmol, 3.0 equiv) in MeOH (2.0 mL). The mixture was stirred for 1 h at room temperature, cooled to 0° C. and saturated aqueous Na.sub.2S.sub.2O.sub.2 was added until a nearly colourless solution was obtained. The mixture was concentrated in vacuo to a volume of ca. 0.5 mL, EtOAc (10 mL) was added, the solution was washed with 0.1 M aqueous Na.sub.2S.sub.2O.sub.2 solution (5.0 mL), dried over Na.sub.2SO.sub.4 and filtered. The crude product was treated with 70% TFA/20% CH.sub.2Cl.sub.2/10% TIS under anhydrous conditions for 2.5 h at room temperature. The cleavage cocktail was removed under a steam of nitrogen and the crude peptide precipitated in cold diethyl ether. After centrifugation, the peptide was dissolved in water and further purified by HPLC (0-3 min 3%, 3-10 min 25%, 10-15 min 100%, R.sub.t=7.32 min) to give the cyclic peptide 12 as a white solid (7.4 mg, 32% yield over two steps). mp 161-165° C. (decomposition); .sup.1H NMR (500 MHz, D.sub.2O @ 323 K) δ.sub.H ppm 4.70 (m, 1H, CHα-Asn) 4.56-4.51 (m, 2H, OCHH-Ox, CHα-Cys), 4.45-4.36 (m, 3H, OCH.sub.2—Ox, OCHH-Ox), 4.14 (t, J=4.9 Hz, 1H, CHα-Cys), 3.98 (d, J=14.6 Hz, 1H, CHHGOx), 3.64 (dd, J=14.7, 5.5 Hz, 1H, CHHβ-Cys), 3.45 (t, J=6.2 Hz, 1H, CHα-Arg), 3.35 (dd, J=14.7, 4.5 Hz, 1H, CHHβ-Cys), 3.21 (d, J=14.6 Hz, 2H, CHHβ-Cys, CHHGOx), 3.12 (t, J=6.6 Hz, 2H, CH.sub.2δ-Arg), 2.81 (dd, J=14.6, 10.6 Hz, 1H, CHHβ-Cys), 2.78-2.68 (m, 2H, CH.sub.2β-Asn), 1.70-1.61 (m, 2H, CH.sub.2β-Arg), 1.59-1.49 (m, 2H, CH.sub.2γ-Arg). N.B. CHα-Asn underwater peak; .sup.13C NMR (126 MHz, D.sub.2O @ 323 K) δ.sub.C ppm 176.4 (C═O), 176.1 (C═O), 174.4 (C═O), 172.7 (C═O), 170.2 (C═O), 156.7 (C═NH), 80.2 (OCH.sub.2), 78.7 (OCH.sub.2), 60.4 (C, Ox), 56.1 (CH, α-Arg), 54.3 (CH, α-Cys), 52.6 (CH, α-Cys), 50.8 (CH, α-Asn), 44.0 (CH.sub.2 β-Cys), 43.6 (CH.sub.2, GOx), 42.7 (CH.sub.2 β-Cys), 40.7 (CH.sub.2, δ-Arg), 36.2 (CH.sub.2, β-Asn), 31.3 (CH.sub.2, β-Arg), 24.2 (CH.sub.2, γ-Arg); ν.sub.max (neat)=2943, 1660, 1409, 1285, 1170, 697, 466 cm.sup.−1; MS (ESI.sup.+) m/z 578 [M+H].sup.+, 600 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.2H.sub.36N.sub.9O.sub.7S.sub.2 [M+H].sup.+ 578.2174. found 578.2178. [α].sub.D.sup.28 −81.3 (c 0.0004, DMF). See FIG. 6.

Example 3b—Detailed Example of a Sidechain-to-Sidechain Cyclisation for a Tripeptide

(96) ##STR00079##

(97) Fmoc-Cys(Trt)-OtBu (81): To a suspension of Fmoc-Cys(Trt)-OH (11.7 g, 20.0 mmol, 1.0 equiv) in anhydrous CH.sub.2Cl.sub.2 (160 mL) was added tert-butyl 2,2,2-trichloroacetimidate (8.74 g, 40.0 mmol, 2.0 equiv) and the mixture was stirred at ambient temperature for 3 d. The mixture was filtered through a pad of Celite and the solids were washed with EtOAc. The filtrate was concentrated in vacuo and the residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 5:1) to give 81 (11.6 g, 18.1 mmol, 90%) as a white solid.

(98) NO.sub.2-GOx-Cys(Trt)-OtBu (82): To a solution of Fmoc-Cys(Trt)-OtBu (81) (3.85 g, 6.00 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (6.0 mL) was added diethylamine (6.0 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated under reduced pressure to give the crude amine. In a second reaction vessel, oxetane-3-one (770 μL, 12.0 mmol, 2.0 equiv), nitromethane (910 μL, 16.8 mmol, 2.8 equiv) and triethylamine (335 μL, 2.40 mmol, 0.4 equiv) were combined at 0° C. and stirred for 1 h at room temperature. The mixture was dissolved in anhydrous CH.sub.2Cl.sub.2 (40 mL), cooled to −78° C., and triethylamine (3.35 mL, 24.0 mmol, 4.0 equiv) was added followed by dropwise addition of a solution of methanesulfonyl chloride (930 μL, 12.0 mmol, 2.0 equiv) in anhydrous CH.sub.2Cl.sub.2 (12 mL). The reaction mixture was stirred at −78° C. for 1.5 h and a solution of the crude amine in anhydrous CH.sub.2Cl.sub.2 (20 mL) was added slowly via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. A saturated solution of NH.sub.4Cl (50 mL) was added and stirred for 10 min. The layers were separated and the aqueous one extracted with CH.sub.2Cl.sub.2 (2×40 mL) and EtOAc (2×40 mL). The combined organic phases were washed with saturated aqueous NaHCO.sub.3 solution (30 mL), brine (30 mL), dried over MgSO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 4:1.fwdarw.2:2.fwdarw.1:1) to give 82 (2.18 g, 4.08 mmol, 68%) as an orange foam.

(99) Boc-Cys(Trt)-GOx-Cys(Trt)-OtBu (83): To a solution of 82 (534 mg, 1.00 mmol, 1.0 equiv) in THF (20 mL) was added zinc powder (196 mg, 3.00 mmol, 3.0 equiv) and acetic acid (458 μL, 8.00 mmol, 8.0 equiv) and the reaction mixture was vigorously stirred with a glass-coated magnetic stir bar at room temperature for 1 h. Additional zinc powder (196 mg, 3.00 mmol, 3.0 equiv) and acetic acid (458 μL, 8.00 mmol, 8.0 equiv) were added and the mixture was stirred at ambient temperature for 1 h (repeat 3×). The mixture was cooled to 0° C. and saturated aqueous NaHCO.sub.3 solution (20 mL) was added followed by Boc-Cys-OSu (841 mg, 1.50 mmol, 1.5 equiv) and the solution was stirred for 16 h at room temperature. Brine (20 mL) was added and the mixture as extracted with EtOAc (3×15 mL). The combined organic layers were washed with saturated aqueous NaHCO.sub.3 solution (30 mL) and brine (30 mL), dried over MgSO.sub.4, filtered and concentrated in vacuo. Purification by column chromatography (SiO.sub.2, PE/EtOAc 2:1.fwdarw.1:1) gave tripeptide 83 (391 mg, 0.41 mmol, 41%) as a white foam.

(100) Cyclo(Boc-Cys-GOx-Cys-OtBu) (13): To a solution of iodine (305 mg, 1.20 mmol, 3.0 equiv) in anhydrous MeOH (40 mL) was slowly added a solution of Boc-Cys(Trt)-GOx-Cys(Trt)-OtBu (83) (380 mg, 0.40 mmol, 1.0 equiv) in anhydrous MeOH (40 mL). The mixture was stirred for 1 h at room temperature, cooled to 0° C. and a saturated aqueous solution of Na.sub.2S.sub.2O.sub.2 was added until a nearly colourless solution was obtained. The mixture was concentrated in vacuo to a volume of ca. 5 mL, EtOAc (25 mL) was added, the solution was washed with 0.1 M aqueous Na.sub.2S.sub.2O.sub.2 solution (10 mL), dried over Na.sub.2SO.sub.4 and filtered. The solvent was removed in vacuo and the residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 1:1.fwdarw.EtOAc) to afford cyclic tripeptide 13 (119 mg, 0.26 mmol, 64%) as a white foam. R.sub.f (EtOAc) 0.55; mp 98-100° C.; .sup.1H NMR (600 MHz, CDCl.sub.3 @ 323 K) δ.sub.H ppm 6.58-6.54 (m, 1H, NH), 5.43 (d, J=4.2 Hz, 1H, NH), 4.59 (d, J=6.5 Hz, 1H, OCHH-Ox), 4.48 (d, J=6.5 Hz, 1H, OCHH-Ox), 4.43 (d, J=6.5 Hz, 2H, 2×OCHH-Ox), 4.27 (t, J=7.2 Hz, 1H, CHα-Cys), 4.04 (dd, J=14.0, 6.8 Hz, 1H, CHHGOx), 3.69 (dd, J=14.0, 4.5 Hz, 1H, CHHGOx), 3.63 (t, J=5.8 Hz, 1H, CHα-Cys), 3.39 (d, J=13.6 Hz, 1H, CHHβ-Cys), 3.20 (br. m, 1H, CHHβ-Cys), 2.94 (dd, J=14.0, 5.1 Hz, 1H, CHHβ-Cys), 2.82 (dd, J=14.0, 5.6 Hz, 1H, CHHβ-Cys), 2.45 (br. s, 1H, NH), 1.48 (s, 9H, 3×CH.sub.3, tBu), 1.45 (s, 9H, 3×CH.sub.3, tBu); .sup.13C NMR (151 MHz, CDCl.sub.3 @ 323 K) δ.sub.C ppm 172.7 (C═O), 171.5 (C═O), 155.1 (C═O, Boc), 82.8 (C, tBu), 82.3 (OCH.sub.2), 80.9 (C, Boc), 79.8 (OCH.sub.2), 60.0 (C, Ox), 57.1 (CH, α-Cys), 55.7 (CH, α-Cys), 46.4 (CH.sub.2, GOx), 44.9 (CH.sub.2, β-Cys), 28.5 (CH.sub.3, tBu), 28.2 (CH.sub.3, tBu). N.B. One carbon signal for CH.sub.2, 3-Cys not visible; ν.sub.max (neat)=3306, 2931, 1714, 1657, 1490, 1366, 1247, 1149, 971, 843, 751 cm.sup.−1; MS (ESI.sup.+) m/z 464 [M+H].sup.+, 486 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.19H.sub.33N.sub.3NaO.sub.6S.sub.2 [M+Na].sup.+ 486.1703. found 486.1705; [β].sub.D.sup.28 +81.4 (c 1.04, CHCl.sub.3). See FIG. 7.

Example 4—the Impact of the Turn-Inducing Motif on the Properties of Cyclic Peptides

(101) Initial studies to explore the impact of the turn-inducing element modification on the properties of the derived cyclic peptides were undertaken. cCNGRC (15) is known to target Aminopeptidase N (APN), a transmembrane zinc-dependent metalloprotease involved in a variety of processes, including blood pressure regulation, cell migration, viral uptake, cell survival, and angiogenesis..sup.14,15 Both the modified derivative 12 and cCNGRC (15) were examined for their inhibitory activity toward porcine APN using a spectrophotometric assay (see Table 2)..sup.15 Similar IC.sub.50 values were observed for 12 and 15, (see FIGS. 8a and 8b) suggesting that the turn-inducing motif is an excellent bioisostere of the amide bond in this system.

(102) TABLE-US-00004 TABLE 2 Relative inhibitory effects of 12 and cCNGRC (15) against porcine Aminopeptidase N (APN). entry compound IC.sub.50 (μM) 1 12 175 2 cCNGRC (15) 212.sup.b 3 bestatin.sup.c 4.1.sup.b .sup.bValues slightly lower than those reported in ref 15. .sup.cPositive control.

(103) The assay used was an in vitro inhibition assay of aminopeptidase N with oxetane modified peptide 12 and parent peptide 15. Peptide 15 was synthesised following a procedure by Piras et al..sup.[7] using HCTU as coupling reagent and NMM as base. Oxidation was performed by on-resin cyclization as described for the biotin labelled compound. Analytical data were in accordance with the literature.

(104) For the determination of IC.sub.50 values of the modified peptide 12 and parent peptide 15, a protocol published by Piras et al..sup.[7] was followed using L-leucine-p-nitroanilide as substrate and microsomal aminopeptidase from porcine kidney (pAPN, Sigma Aldrich) (18 units/mg protein). IC.sub.50 values were calculated by following the formation of p-nitroaniline. Formation of p-nitroaniline was monitored by measurement of the UV absorption at 405 nm on a Hidex Sense plate reader. The assay was performed in a 96-well plate in PBS buffer (pH 7.2, 1.47 mM KH.sub.2PO.sub.4, 7.8 mM Na.sub.2HPO.sub.4, 137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl.sub.2, 1.8 mM MgCl.sub.2) at 37° C. with a total volume of 100 μL. Bestatin hydrochloride was used as positive control. Peptides were used in gradient concentrations between 2.5 μM and 3.5 mM, and bestatin in concentrations between 50 nM and 25 μM. The peptides were incubated with the enzyme (1.0 μg/mL) for 5 min. before a solution of L-leucine-p-nitroanilide was added with a final concentration of 250 μM. The plate was incubated at 37° C. for 1 hour before the p-nitroaniline was detected. IC.sub.50 was defined as the concentration that led to 50% of maximal pAPN catalytic activity. For the calculation of the IC.sub.50 values log of the concentration was plotted against the UV absorption in GraphPad Prism 5 using nonlinear regression (variable slope (four parameters) with interpolation) for analysis.

Example 5—Preparation of Oxetane Modified Cyclic Peptides 18-21 Via Solid Phase Peptide Synthesis (SPPS)

(105) In an alternative method to make the linear peptides, preformed Fmoc-protected dipeptide building blocks containing the oxetane modification can be incorporated into the growing peptide chain. This method is fully compatible with standard Fmoc/.sup.tBu SPPS and avoids the need to subject the growing peptide chain to strongly reductive conditions.

(106) Synthesis by SPPS avoids the need to purify any of the intermediates, simplifying and accelerating the process and enabling automation. This was realised through the synthesis of cyclic tetrapeptide 18 in an impressive 39% yield from commercial H-Trp(Boc)-2-ClTrt (15). Macrocycles 19-21 based on different ring sizes were readily made through further generalisation of this SPPS approach.

(107) ##STR00080##
Preparation of Fmoc-GOx-Gly-OH (16):

(108) ##STR00081##

(109) To a solution of Fmoc-Gly-OH (2.00 g, 6.72 mmol, 1.0 equiv) in anhydrous CH.sub.2Cl.sub.2 (30 mL) were added 2-phenyl-2-propanol (3.36 g, 24.7 mmol, 3.7 equiv), DCC (1.70 g, 8.24 mmol, 1.2 equiv) and DMAP (167 mg, 1.37 mmol, 0.2 equiv) and the mixture was stirred for 24 h at room temperature. The solvent was removed in vacuo, the residue was diluted with diethyl ether (100 mL) and filtered through a plug of Celite eluting with diethyl ether. The filtrate was washed with saturated NaHCO.sub.3 solution (100 mL), dried over MgSO.sub.4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 9:1.fwdarw.4:1) to give Fmoc-Gly-OCumyl (120) (1.43 g, 3.44 mmol, 51%) as a white solid. R.sub.f (PE/EtOAc 2:1) 0.46; mp 109-111° C.; H NMR (500 MHz, CDCl.sub.3) δ.sub.H ppm 7.78 (d, J=7.5 Hz, 2H, ArH), 7.60 (d, J=7.5 Hz, 2H, ArH), 7.43-7.26 (m, 9H, ArH), 5.31 (s, 1H, NH), 4.40 (d, J=7.2 Hz, 2H, CH.sub.2—Fmoc), 4.23 (t, J=7.2 Hz, 1H, CH-Fmoc), 4.01 (d, J=5.3 Hz, 2H, CH.sub.2Gly), 1.84 (s, 6H, 2×CH.sub.3, cumyl); .sup.13C NMR (126 MHz, CDCl.sub.3) δ.sub.C ppm 168.7 (C═O), 156.3 (C═O, Fmoc), 145.1 (C), 143.9 (C), 141.3 (C), 128.5 (CH), 127.8 (CH), 127.4 (CH), 127.1 (CH), 125.2 (CH), 124.4 (CH), 120.1 (CH), 83.4 (C, cumyl), 67.2 (CH.sub.2, Fmoc), 47.2 (CH, Fmoc), 43.5 (CH.sub.2, Gly), 28.6 (CH.sub.3, cumyl); ν.sub.max (neat)=3308, 2938, 1738, 1687, 1546, 1214, 1053, 760, 697 cm.sup.−1; MS (ESI.sup.+) m/z 438 [M+Na].sup.+, 454 [M+K].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.26H.sub.25NNaO.sub.4 [M+Na].sup.+ 438.1676. found 438.1674.

(110) O.sub.2N-GOx-Gly-OCumyl (121)

(111) ##STR00082##

(112) To a solution of Fmoc-Gly-OCumyl (120) (1.29 g, 3.10 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (4.0 mL) was added diethylamine (4.0 mL) and the mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×15 mL) and concentrated under reduced pressure to give the crude amine. In a second reaction vessel, oxetane-3-one (398 μL, 6.21 mmol, 2.0 equiv), nitromethane (470 μL, 8.68 mmol, 2.8 equiv) and trimethylamine (173 μL, 1.24 mmol, 0.4 equiv) were combined at 0° C. and stirred for 1 h at room temperature. The mixture was dissolved in anhydrous CH.sub.2Cl.sub.2 (20 mL), cooled to −78° C., and trimethylamine (1.73 mL, 12.4 mmol, 4.0 equiv) was added followed by dropwise addition of a solution of methanesulfonyl chloride (481 μL, 6.21 mmol, 2.0 equiv) in anhydrous CH.sub.2Cl.sub.2 (6.0 mL). The reaction mixture was stirred at −78° C. for 1.5 h and a solution of the crude amine in anhydrous CH.sub.2Cl.sub.2 (20 mL) was added slowly via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. A saturated solution of NH.sub.4Cl (20 mL) was added and stirred for 10 min. The layers were separated and the aqueous one extracted with CH.sub.2Cl.sub.2 (2×30 mL) and EtOAc (2×30 mL). The combined organic phases were concentrated under reduced pressure and the residue was purified by column chromatography (SiO.sub.2, PE/EtOAc 4:1.fwdarw.2:1.fwdarw.1:1) to give 121 (940 mg, 3.05 mmol, 98%) as an off-white solid. R.sub.f (PE/EtOAc 2:1) 0.15; mp 71-72° C.; .sup.1H NMR (400 MHz, CDCl.sub.3) δ.sub.H ppm 7.39-7.27 (m, 5H, ArH), 4.77 (s, 2H, NO.sub.2CH.sub.2), 4.57 (d, J=7.2 Hz, 2H, OCH.sub.2—Ox), 4.53 (d, J=7.2 Hz, 2H, OCH.sub.2—Ox), 3.53 (s, 2H, CH.sub.2Gly), 2.29 (s, 1H, NH), 1.80 (s, 6H, 2×CH.sub.3, cumyl); .sup.13C NMR (101 MHz, CDCl.sub.3) δ.sub.C ppm 170.6 (C═O), 145.1 (C), 128.5 (CH), 127.5 (CH), 124.4 (CH), 83.3 (C, cumyl), 78.9 (NO.sub.2CH.sub.2), 78.3 (2×OCH.sub.2), 59.6 (C, Ox), 45.6 (CH.sub.2, Gly), 28.6 (CH.sub.3, cumyl); ν.sub.max (neat)=3293, 2979, 1736, 1545, 1364, 1215, 1140, 1101, 978, 762, 695 cm.sup.−1; MS (ESI.sup.+) m/z 331 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.15H.sub.20N.sub.2NaO.sub.5 [M+Na].sup.+ 331.1264. found 331.1268.

(113) Fmoc-GOx-Gly-OCumyl (122)

(114) ##STR00083##

(115) To a solution of NO.sub.2-GOx-Gly-OCumyl (121) (928 mg, 3.00 mmol, 1.0 equiv) in THF (30 mL) was added Fmoc N-hydroxysuccinimide ester (2.02 g, 6.00 mmol, 2.0 equiv), NaHCO.sub.3 (1.01 g, 12.0 mmol, 4.0 equiv) and Raney Ni (slurry in H.sub.2, 3.0 mL). The reaction mixture was placed under an atmosphere of nitrogen, evacuated and filled with hydrogen (balloon). The mixture was stirred vigorously for 4 h at room temperature. Then, the mixture was filtered through a plug of Celite eluting with EtOAc, the filtrate was washed with saturated Na.sub.2CO.sub.3 (3×50 mL) and concentrated under reduced pressure. Fmoc-GOx-Gly-OCumyl (122) was afforded after purification by column chromatography (SiO.sub.2, PE/EtOAc 2:1.fwdarw.1:1.fwdarw.EtOAc) as a white sticky foam (897 mg, 1.79 mmol, 60%). R.sub.f (PE/EtOAc 1:1) 0.21; .sup.1H NMR (400 MHz, CDCl.sub.3) δ.sub.H ppm 7.72 (d, J=7.4 Hz, 2H, ArH), 7.54 (d, J=7.4 Hz, 2H, ArH), 7.38-7.21 (m, 9H, ArH), 5.26 (s, 1H, NH), 4.48-4.24 (m, 6H, CH.sub.2—Fmoc, 2×OCH.sub.2—Ox), 4.16 (t, J=6.2 Hz, 1H, CH-Fmoc), 3.50 (d, J=5.1 Hz, 2H, CH.sub.2GOx), 3.41 (s, 2H, CH.sub.2Gly), 1.92 (br. s, 1H, NH), 1.76 (s, 6H, 2×CH.sub.3, cumyl); .sup.13C NMR (101 MHz, CDCl.sub.3) δ.sub.C ppm 171.4 (C═O), 157.0 (C═O, Fmoc), 145.1 (C), 144.0 (C), 141.4 (C), 128.5 (CH), 127.8 (CH), 127.5 (CH), 127.2 (CH), 125.2 (CH), 124.4 (CH), 120.1 (CH), 83.2 (C, cumyl), 79.2 (2×OCH.sub.2), 66.9 (CH.sub.2, Fmoc), 59.7 (C, Ox), 47.3 (CH, Fmoc), 45.5 (CH.sub.2, GOx or Gly), 45.4 (CH.sub.2, GOx or Gly), 28.6 (CH.sub.3, cumyl); ν.sub.max (neat)=3309, 2941, 1716, 1535, 1448, 1214, 1134, 974, 758, 739, 698 cm.sup.−1; MS (ESI.sup.+) m/z 501 [M+H].sup.+, 523 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.30H.sub.32N.sub.2NaO.sub.5 [M+Na].sup.+ 523.2203. found 523.2197.

(116) Solid-Phase Peptide Synthesis of Cyclo(Leu-GOx-Gly-Trp(Boc)) (18):

(117) ##STR00084##

(118) Fmoc-GOx-Gly-Cumyl (122) (200 mg, 0.40 mmol, 4.0 equiv) was stirred at room temperature in 2% TFA in CH.sub.2Cl.sub.2 (8.0 mL) for 2-3 h until complete deprotection of the cumyl ester was observed by TLC. The solvent was removed under reduced pressure and the resulting residue was repeatedly dissolved in CH.sub.2Cl.sub.2 (3×10 mL) and concentrated under reduced pressure. The crude Fmoc-GOx-Gly-OH (16) was used for coupling without further purification.

(119) H-Trp(Boc)-2-chlorotrityl resin (15) (145 mg, 0.10 mmol, 1.0 equiv) was placed in a 10 mL reaction vessel and the resin was pre-swollen in DMF (2.0 mL) for 30 min. Fmoc-GOx-Gly-OH (16) was dissolved in DMF (4.0 mL). HATU (72 mg, 0.19 mmol, 1.9 equiv) and DIPEA (70 μL, 0.40 mmol, 4.0 equiv) were added to 2.0 mL solution of 16 and the coupling solution was added to the resin. The coupling reaction was allowed to proceed for 2 h at room temperature under slight agitation. The resin was filtered, washed with DMF (1×2.0 mL) and the coupling step was repeated before the Fmoc group was removed with 20% piperidine in DMF (2.0 mL) for 20 min at room temperature. After washing the resin with DMF (5×2.0 mL), Fmoc-Leu-OH (177 mg, 0.50 mmol, 5.0 equiv) was coupled with HATU (186 mg, 0.49 mmol, 4.9 equiv), DIPEA (174 μL, 1.00 mmol, 10 equiv) in DMF (2.0 mL) for 1 h at room temperature. In case of a positive TNBS test, the coupling step was repeated. The resin was washed with DMF (5×2.0 mL) before the Fmoc-group was removed as described before. The tetrapeptide was then cleaved from the resin with TFE in CH.sub.2Cl.sub.2 (1:4, 1.0 mL) for 1 h at room temperature. This was repeated twice and the combined cleavage solutions were evaporated to dryness under reduced pressure. Success of the synthesis was confirmed by mass spectrometry and NMR. The crude yield of the solid phase synthesis was approximately 70-80%.

(120) The crude peptide was dissolved in DMF (76 mL, 1 mM) and DEPBT (45 mg, 0.15 mmol, 2.0 equiv) and DIPEA (26 μL, 0.15 mmol, 2.0 equiv) were added. The reaction mixture was stirred at room temperature for 64 h before, the solvent was removed under reduced pressure and the residue purified twice by column chromatography (5-12% MeOH in CH.sub.2Cl.sub.2). Cyclic tetrapeptide 18 was obtained as a sticky white/colourless solid in 39% yield (21.1 mg, 39 μmol) over the complete reaction sequence.

(121) R.sub.f (CH.sub.2Cl.sub.2/MeOH 9:1) 0.55; H NMR (500 MHz, DMSO-d6) δ.sub.H 8.25 (d, J=10.3 Hz, 1H, NH), 8.03 (d, J=8.1 Hz, 1H, ArH), 7.95 (d, J=9.1 Hz, 1H, NH), 7.63 (d, J=7.7 Hz, 1H, ArH), 7.48 (s, 1H, ArH), 7.47-7.43 (m, 1H, NH), 7.34 (t, J=7.7 Hz, 1H, ArH), 7.27 (t, J=7.5 Hz, 1H, ArH), 4.61 (q, J=8.8 Hz, 1H, CHα-Trp), 4.40 (d, J=6.3 Hz, 1H, OCHH-Ox), 4.17 (d, J=6.9 Hz, 1H, OCHH-Ox), 4.15 (d, J=6.3 Hz, 1H, OCHH-Ox), 4.01 (td, J=9.8, 5.2 Hz, 1H, CHα-Leu), 3.89 (d, J=6.9 Hz, 1H, OCHH-Ox), 3.80 (dd, J=13.4, 7.8 Hz, 1H, CHHGOx), 3.44-3.35 (m, 1H, CHHGly), 3.24 (d, J=15.2 Hz, 1H, CHHGly), 3.18 (dd, J=15.0, 5.6 Hz, 1H, CHHβ-Trp), 3.07-3.03 (m, 1H, CHHβ-Trp), 3.01 (d, J=11.3 Hz, 1H, CHHGOx), 1.62 (s, 9H, 3×CH.sub.3, Boc), 1.61-1.56 (m, 2H, CHH-Leu, CHγ-Leu), 1.52-1.44 (m, 1H, CHHβ-Leu), 0.91 (d, J=6.2 Hz, 3H, CH.sub.3δ-Leu), 0.78 (d, J=6.2 Hz, 3H, CH.sub.3δ-Leu). N.B. One NH not observed; .sup.13C NMR (126 MHz, DMSO-d6) δ.sub.C 173.13 (C═O), 173.09 (C═O), 171.0 (C═O), 148.9 (C═O, Boc), 134.7 (C), 130.0 (C), 124.5 (CH), 123.4 (CH), 122.6 (CH), 119.1 (CH), 116.1 (C), 114.8 (CH), 83.7 (C, Boc), 78.1 (OCH.sub.2), 76.3 (OCH.sub.2), 60.3 (C, Ox), 55.2 (CH, α-Trp), 54.0 (CH, α-Leu), 47.2 (CH.sub.2, Gly), 44.3 (CH.sub.2, GOx), 39.6 (CH.sub.2, β-Leu), 27.7 (CH.sub.3, Boc), 25.8 (CH.sub.2, β-Trp), 24.6 (CH, γ-Leu), 22.9 (CH.sub.3, δ-Leu), 21.1 (CH.sub.3, β-Leu); ν.sub.max (neat)=3266, 2925, 1672, 1532, 1225, 704 cm.sup.−1; MS (ESI.sup.+) m/z 564 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.28H.sub.39N.sub.5NaO.sub.6 [M+Na].sup.+ 564.2793. found 564.2791. [α].sub.D.sup.27+10.0 (c 0.05, CHCl.sub.3).

(122) Solid-Phase Peptide Synthesis of Cyclo(Ala-Trp-GOx-Gly-Leu) (19):

(123) ##STR00085##

(124) Cyclic peptide 19 was synthesised as described above starting from H-Leu-2-chlorotrityl resin (67.5 mg, 0.05 mmol). Tryptophan was incorporated without side chain protecting group. The crude cyclic peptide was purified by preparative HPLC (solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in MeCN; gradient: 0-3 min, 5% B; 3-28 min, 3-40% B; 28-32 min, 40-100% B; retention time: 28.9 min). Cyclic pentapeptide 19 was obtained after freeze-drying as TFA salt (2.4 mg, 6.5 μmol, 13%).

(125) HRMS (ESI.sup.+) calcd. for C.sub.26H.sub.37N.sub.6O.sub.5 [M+H].sup.+ 513.2820. found 513.2816.

(126) Solid-Phase Peptide Synthesis of Cyclo(Met-Ala-Trp-GOx-Gly-Leu) (20):

(127) ##STR00086##

(128) Cyclic peptide 20 was synthesised as described above starting from H-Leu-2-chlorotrityl resin (67.5 mg, 0.05 mmol). Tryptophan was incorporated without side chain protecting group. The crude cyclic peptide was purified by preparative HPLC (solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in MeCN; gradient: 0-3 min, 5% B; 3-28 min, 3-50% B; 28-32 min, 50-100% B; retention time: 25.3 min). Cyclic hexapeptide 20 was obtained after freeze-drying as TFA salt (2.5 mg, 6.5 μmol, 7%).

(129) HRMS (ESI.sup.+) calcd. for C.sub.31H.sub.46N.sub.7O.sub.6S* [M+H].sup.+ 644.3225. found 644.3220.

(130) Solid-Phase Peptide Synthesis of Cyclo(Ser-Met-Ala-Trp-GOx-Gly-Leu) (21):

(131) ##STR00087##

(132) Cyclic peptide 21 was synthesised as described above starting from H-Leu-2-chlorotrityl resin (67.5 mg, 0.05 mmol). Tryptophan was incorporated without side chain protecting group. The crude cyclic peptide was purified by preparative HPLC (solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in MeCN; gradient: 0-3 min, 5% B; 3-28 min, 3-50% B; 28-32 min, 50-100% B; retention time: 26.8 min). Cyclic heptapeptide 21 was obtained after freeze-drying as TFA salt (6.2 mg, 6.5 μmol, 13%).

(133) HRMS (ESI.sup.+) calcd. for C.sub.34H.sub.50N.sub.8NaO.sub.8S* [M+Na].sup.+ 753.3365. found 753.3358.

Example 6—Preparation of Cyclo(Trp-Leu-AOx-Gly) (12)

(134) ##STR00088##

(135) To a solution of H-Trp-Leu-AOx-Gly-OH (53) (47 mg, 0.10 mmol, 1.0 equiv) in anhydrous DMF (100 mL, 0.001 M) under an atmosphere of nitrogen was added DEPBT (60 mg, 0.20 mmol, 2.0 equiv) and DIPEA (35 μL, 0.20 mmol, 2.0 equiv) and the mixture was stirred for 48 h at room temperature. The solvent was removed under reduced pressure, and the residue was purified twice by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 9:1.fwdarw.85:15) to give cyclic tetrapeptide 12 as a white solid (1.sup.st run: 22.9 mg, 50 μmol, 50%; 2.sup.nd run (289 μmol scale): 64.4 mg, 142 μmol, 49%). R.sub.f (CH.sub.2Cl.sub.2/MeOH 9:1) 0.19; mp 220-223° C.; .sup.1H NMR (500 MHz, CD.sub.3OD) δ.sub.H 7.62 (d, J=7.9 Hz, 1H, ArH), 7.32 (d, J=8.1 Hz, 1H, ArH), 7.12 (s, 1H, ArH), 7.10 (t, J=7.7 Hz, 1H, ArH), 7.03 (t, J=7.4 Hz, 1H, ArH), 4.93 (dd, J=10.2, 7.2 Hz, 1H, CHα-Trp), 4.57 (q, J=7.2 Hz, 1H, CHα-AOx), 4.50 (d, J=6.8 Hz, 1H, OCHH-Ox), 4.40 (d, J=6.8 Hz, 1H, OCHH-Ox), 4.38 (d, J=7.8 Hz, 1H, OCHH-Ox), 4.20 (dd, J=10.9, 4.2 Hz, 1H, CHα-Leu), 4.04 (d, J=7.8 Hz, 1H, OCHH-Ox), 3.67 (d, J=16.6 Hz, 1H, CHHGly), 3.57 (d, J=16.6 Hz, 1H, CHHGly), 3.37-3.27 (m, 1H, CHHβ-Trp), 3.21 (dd, J=15.1, 7.2 Hz, 1H, CHHβ-Trp), 1.72-1.63 (m, 1H, CHHβ-Leu), 1.59-1.48 (m, 2H, CHHβ-Leu, CHγ-Leu), 1.12 (d, J=6.9 Hz, 3H, CH.sub.3β-AOx), 0.90 (d, J=6.0 Hz, 3H, CH.sub.3δ-Leu), 0.73 (d, J=6.0 Hz, 3H, CH.sub.3δ-Leu). N.B. CHHβ-Trp overlaps with solvent peak; .sup.13C NMR (126 MHz, CD.sub.3OD) δ.sub.C 176.4 (C═O), 175.3 (C═O), 173.7 (C═O), 138.0 (C), 128.5 (C), 123.8 (CH), 122.6 (CH), 119.9 (CH), 119.1 (CH), 112.3 (CH), 110.3 (C), 79.2 (OCH.sub.2), 77.3 (OCH.sub.2), 64.7 (C, Ox), 57.7 (CH, α-Trp), 55.3 (CH, α-Leu), 51.6 (CH, α-AOx), 48.8 (CH.sub.2, Gly), 40.6 (CH.sub.2, β-Leu), 27.6 (CH.sub.2, β-Trp), 26.2 (CH, γ-Leu), 23.3 (CH.sub.3, δ-Leu), 21.2 (CH.sub.3, δ-Leu), 13.5 (CH.sub.3, AOx). N.B. CH.sub.2, Gly signal overlaps with solvent peak; ν.sub.max (neat)=3256, 2956, 1659, 1532, 740 cm.sup.−1; MS (ESI.sup.+) m/z 478 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.24H.sub.33N.sub.5NaO.sub.4 [M+Na].sup.+ 478.2425. found 478.2423; [α].sub.D.sup.29 −108 (c 0.06, MeOH).

(136) The successful formation of 12 containing an alanine modified residue confirms that the methodology herein described has broader applicability and is not limited to glycine substitution.

Example 7—Kinetic Measurement of the Cyclization Reaction

(137) ##STR00089##

(138) To gain a deeper understanding of reaction rates and the products formed, time-course studies of an inherently difficult to cyclise tetrapeptide WLGG (6) and corresponding oxetane modified WLGOxG (7) (where GOx=oxetane modified glycine) were undertaken. For this investigation, substrates containing a tryptophan residue were used to allow quantitative monitoring by UV spectroscopy. Both substrates were subjected to the DEPBT method and conversions monitored over 74 h. From these data, it is clear that the initial rate of formation of oxetane containing cyclic peptide 9 (see structure above) is considerably faster than 8 (see structure above), even though both linear precursors 6 and 7 are consumed at similar rates (see FIG. 9). Appreciable quantities of the unwanted dimer and cyclodimer were produced in the cyclisation of 8, explaining the lower conversion and isolated yield. In contrast, for the oxetane-modified peptide 7 clean conversion to the cyclic product 9 was observed. Taken together, these studies establish that head-to-tail ring closures to form small cyclic peptides proceed more quickly, give higher yields and produce less side products when one of the backbone carbonyl groups is replaced by an oxetane ring.

(139) Method: HPLC measurements were conducted on an Agilent 1260 Infinity analytical HPLC system on an Agilent Eclipse Plus C18 column (5.0 μm, 4.6×150 mm) with a flow rate of 1.0 mL/min (solvent A: 0.1% TFA in water; solvent B: 0.1% TFA in MeCN; gradient: 0-3 min, 3% B; 3-14 min, 3-20% B; 14-20 min, 20% B; 20-41 min, 20-50% B; 41-43 min, 50-100% B; 43-45 min, 100% B). To separate 20 mL vials were added linear precursor 6 or 7 (10 μmol, 1.0 equiv) and anhydrous DMF (10 mL). At this time a 500 μL sample was withdrawn to determine the initial value by analytical HPLC before DEPBT (20 μmol, 2.0 equiv) and DIPEA (20 μmol, 2.0 equiv) were added to the solution. At designated time points, 500 μL of reaction mixture was taken and diluted with 200 μL distilled water. 10 μL of these samples were directly injected into the analytical HPLC. Further samples were taken after 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 26, 30, 34, 50 and 74 h and treated in the same manner. Signals for linear precursors 6 or 7 and cyclic peptides 8 or 9 as well as dimer 42 were integrated at 280 nm.

(140) To check the accuracy of the integration at 280 nm, calibration curves for the linear and cyclic oxetane modified peptide 7 and 9 were measured by injecting 10 μL of stock solutions of known concentration. UV signals at 280 nm were integrated and the resulting areas plotted against the amount of injected compound in nmol. Linear fitting gave equations shown below for each compound. The obtained data show that conversions obtained from sole integration of the peaks at 280 nm are in accordance with yields obtained from the calibrations curves. The same was assumed for the parent system (compound 6 and 8). Conversions were calculated from initial integral of linear precursor determined before addition of coupling reagent and led to 49% for cyclic peptide 8, 11% for the dimer 42 and 83% for cyclic oxetane modified peptide 9. Conversion obtained for the dimer was divided by two due to two Trp-residues in the structure. Retention times of linear precursors and formed products was confirmed by LC-MS (Bruker Amazon X) under the same HPLC conditions and injection of purified compounds.

Example 8—Comparison with Other Amino Acid Modifications

(141) To understand how significant the methodology herein disclosed is in the broader context of peptide macrocyclisation, a series of alternative modifications were made to the central G residue of LAGAY and the efficiencies of the ring closures compared. N-Methyl glycine, 2-methylalanine (Aib), ethylenediamine, dimethylethylenediamine and -alanine were all introduced in place of the glycine (see Table 3). These modifications could improve cyclisation efficiency by: (i) increasing the conformational flexibility of the peptide backbone by deletion of one of the amide bonds; (ii) enlarging the size of the macrocycle by introduction of an additional methylene group; (iii) bringing the reacting ends closer together by favouring the cis-amide conformation; or (iv) introducing a potentially beneficial Thorpe-Ingold effect. In fact, only the oxetane modification led to marked improvement in isolated yield of the derived cyclic peptides, suggesting that oxetane introduction is particularly beneficial.

(142) TABLE-US-00005 TABLE 3 Synthesis of cLAGAY and impact of modifications on cyclisation. 0 embedded image AA Yield Δ% 31% 36% +5% 29% −2% 28% −3% 51% +20% 32% +1% 39% +8%

Example 9—Solution Phase Synthesis of Cyclo-((D)Pro-Leu-GAz(H)-Gly) (GAz=Azetidine Modified Glycine)

(143) ##STR00099##

(144) In this example the tetrapeptide was made by solution phase synthesis and deprotection of a Boc group on the azetidine ring.

(145) Preparation of NO.sub.2-GAz(Boc)-Gly-OBn:

(146) ##STR00100##

(147) To a solution of H-Gly-OBn.Math.TsOH (3.49 g, 10.4 mmol, 2.0 equiv) in CH.sub.2Cl.sub.2 was added triethylamine (1.44 mL, 10.4 mmol, 2.0 equiv) and stirred at room temperature for 15 minutes. In a separate reaction vessel, N-Boc-3-azetidinone (0.89 g, 5.2 mmol, 1.0 equiv) in nitromethane (5.2 mL) was added triethylamine (144 μL, 1.0 mmol, 0.2 equiv) and stirred for 1 h at room temperature. The solvent was removed in vacuo and then resuspended in CH.sub.2Cl.sub.2 (21 mL), cooled to −78° C., and triethylamine (1.44 mL, 10.4 mmol, 2.0 equiv) was added followed by dropwise addition of methanesulfonyl chloride (0.40 mL, 5.2 mmol, 1.0 equiv). The reaction mixture was stirred at −78° C. for 1.5 h and the solution of the crude amine was added slowly via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. A saturated solution of NH.sub.4Cl (100 mL) was added and stirred for 10 min. The layers were separated and the aqueous one extracted with CH.sub.2Cl.sub.2 (2×60 mL) and EtOAc (2×60 mL). The combined organic phases were washed with saturated aqueous NaHCO.sub.3 solution (100 mL), brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, EtOAc/PE 3:7) to yield NO.sub.2-GAz(Boc)-Gly-OBn (1.66 g, 4.37 mmol, 84%) as a yellow oil. R.sub.f (EtOAc/PE 3:7) 0.34; .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.41-7.30 (m, 5H, ArH), 5.18 (s, 2H, CH.sub.2Ph), 4.67 (s, 2H, CH.sub.2NO.sub.2), 4.02-3.83 (m, 4H, 2×NCH.sub.2-Az), 3.51 (d, J=4.5 Hz, 2H, CH.sub.2Gly), 2.33 (s, 1H, NH), 1.44 (s, 9H, 3×CH.sub.3, Boc); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 171.4 (C═O), 156.2 (C═O, Boc), 135.2 (C), 128.9 (CH), 128.8 (CH), 128.7 (CH), 80.6 (C, Boc), 78.7 (CH.sub.2NO.sub.2), 67.4 (CH.sub.2, Bn), 54.3 (2×NCH.sub.2), 44.9 (CH.sub.2, Gly), 28.4 (CH.sub.3, Boc); ν.sub.max (neat)=2976, 1739, 1692, 1552, 1378, 1163 cm.sup.−1; MS (ESI.sup.+) m/z 402 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.18H.sub.25N.sub.3NaO.sub.6 [M+Na].sup.+ 402.1636. found 402.1636;

(148) Preparation of Fmoc-Leu-GAz(Boc)-Gly-OBn:

(149) ##STR00101##

(150) To a solution of NO.sub.2-GAz(Boc)-Gly-OBn (1.66 g, 4.4 mmol, 1.0 equiv) in THF (44 mL) was added Fmoc-Leu-OSu (3.89 g, 8.6 mmol, 2.0 equiv), NaHCO.sub.3 (1.47 g, 17.5 mmol, 4.0 equiv) and Raney Ni (slurry in H.sub.2O, 8 mL). The solution was placed under an atmosphere of nitrogen, evacuated and filled with hydrogen (balloon). The reaction mixture was stirred vigorously for 4.0 h at room temperature. Then, the mixture was filtered through a plug of Celite eluting with EtOAc, concentrated under reduced pressure, the filtrate was suspended in EtOAc (50 mL), washed with saturated Na.sub.2CO.sub.3 (3×50 mL), dried over Na.sub.2SO.sub.4 and concentrated in vacuo. Fmoc-Leu-GAz(Boc)-Gly-OBn was afforded after purification by column chromatography (SiO.sub.2, 9:1.fwdarw.3:2 CH.sub.2Cl.sub.2/EA) as a white foam (1.84 g, 2.7 mmol, 62%). R.sub.f (CH.sub.2Cl.sub.2/EtOAc 9:1) 0.26; mp 77-79° C.; .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.76 (d, J=7.5 Hz, 2H, ArH), 7.59 (d, J=7.2 Hz, 2H, ArH), 7.42-7.29 (m, 9H, ArH), 6.69 (s, 1H, NH), 5.20 (d, J=7.0 Hz, 1H, NH Fmoc), 5.11 (s, 2H, CH.sub.2Ph), 4.41 (d, J=7.1 Hz, 2H, CH.sub.2, Fmoc), 4.25-4.15 (m, 2H, CH, Fmoc, CHα-Leu), 3.69-3.60 (m, 4H, 2×NCH.sub.2-Az), 3.52-3.35 (m, 4H, CH.sub.2 Gly, CH.sub.2 GAz), 1.64-1.60 (m, 3H, CHγ-Leu, CH.sub.2β-Leu), 1.42 (s, 9H, 3×CH.sub.3, Boc), 0.98-0.91 (m, 6H, 2×CH.sub.3δ-Leu); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 172.9 (C═O), 172.6 (C═O), 156.3 (C═O, Boc), 144.0 (C), 143.9 (C), 141.5 (C), 135.2 (C), 132.1 (C), 128.82 (CH), 128.77 (CH), 128.6 (CH), 127.9 (CH), 127.2 (CH), 125.2 (CH), 120.2 (CH), 80.1 (C, Boc), 67.4 (CH.sub.2Ph, Bn), 67.1 (CH.sub.2, Fmoc), 55.0 (2×NCH.sub.2), 53.9 (CH, α-Leu), 47.3 (CH, Fmoc), 44.8 (CH.sub.2, GAz), 43.5 (CH.sub.2, Gly), 41.8 (CH.sub.2, 3-Leu), 28.5 (CH.sub.3, Boc), 24.9 (CH, γ-Leu), 23.1 (CH.sub.3, δ-Leu), 22.1 (CH.sub.3, δ-Leu). Note: Fmoc C═O overlaps with Boc C═O;

(151) ν.sub.max (neat)=2955, 1696, 1657, 1120, 757 cm.sup.−1; MS (ESI.sup.+) m/z 685 [M+H].sup.+, 707 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.39H.sub.49N.sub.4O.sub.7 [M+H].sup.+ 685.3596. found 685.3604.

(152) Preparation of Cbz-(D)Pro-Leu-GAz(Boc)-Gly-OBn:

(153) ##STR00102##

(154) To a solution of tripeptide Fmoc-Leu-GAz(Boc)-Gly-OBn (752 mg, 1.1 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (1 mL) was added diethylamine (1 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×25 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (11 mL), Cbz-D-Pro-OH (274 mg, 1.1 mmol, 1.0 equiv), EDC.Math.HCl (211 mg, 1.1 mmol, 1.0 equiv), HOBt-H.sub.2O (149 mg, 1.1 mmol, 1.0 equiv) and NMM (484 μL, 4.4 mmol, 4.0 equiv) were added subsequently, and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with EtOAc (50 mL) and washed with brine (50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The residue was purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 97:3) to give tetrapeptide Cbz-D-Pro-Leu-GAz(Boc)-Gly-OBn (471 mg, 0.68 mmol, 62%) as a white foam. R.sub.f (CH.sub.2Cl.sub.2/MeOH 97:3) 0.18; mp 65-67° C.; .sup.1H NMR (500 MHz, DMSO @ 373 K) δ 7.65 (s, 1H, NH), 7.46 (s, 1H, NH), 7.42-7.26 (m, 10H, ArH), 5.15 (s, 2H, CH.sub.2Ph Bn), 5.12-5.02 (m, 2H, CH.sub.2Ph Bn), 4.33-4.23 (m, 2H, CHα-Pro, CHα-Leu), 3.66-3.57 (m, 4H, 2×NCH.sub.2-Az), 3.50-3.42 (m, 4H, CH.sub.2 GAz, CH.sub.2δ-Pro), 3.38-3.28 (m, 2H, CH.sub.2 Gly), 2.17-2.09 (m, 1H, CHHγ-Pro), 1.93-1.78 (m, 3H, CHHγ-Pro, CH.sub.2β-Pro), 1.64-1.47 (m, 3H, CHγ-Leu, CH.sub.2β-Leu), 1.39 (s, 9H, 3×CH.sub.3, Boc), 0.88 (d, J=6.4 Hz, 3H, CH.sub.3δ-Leu), 0.85 (d, J=6.3 Hz, 3H, CH.sub.3δ-Leu); .sup.13C NMR (126 MHz, DMSO @ 373 K) δ 171.9 (C═O), 171.3 (C═O), 171.2 (C═O), 155.2 (C═O Cbz), 153.8 (C═O Boc), 136.4 (C), 135.5 (C), 127.7 (CH), 127.6 (CH), 127.3 (CH), 127.2 (CH), 127.0 (CH), 126.6 (CH), 77.9 (C, Boc), 65.6 (CH.sub.2Ph, Bn), 65.2 (CH.sub.2Ph, Bn), 59.7 (CH, α-Pro), 56.7 (2×NCH.sub.2), 54.6 (C, Az), 51.1 (CH, α-Leu), 46.3 (CH.sub.2, GAz), 44.1 (CH.sub.2, 6-Pro), 42.7 (CH.sub.2, Gly), 40.3 (CH.sub.2, β-Leu), 29.7 (CH.sub.2, γ-Pro), 27.6 (CH.sub.3, Boc), 23.8 (CH, γ-Leu), 23.0 (CH.sub.2, β-Pro), 22.2 (CH.sub.3, δ-Leu), 21.1 (CH.sub.3, δ-Leu); ν.sub.max (neat)=2955, 1740, 1667, 1407, 1120 cm.sup.−1; MS (ESI.sup.+) m/z [M+H].sup.+ 694, [M+Na].sup.+ 716; HRMS (ESI.sup.+) calcd. for C.sub.37H.sub.52N.sub.5O.sub.8 694.3810 [M+H].sup.+. found 694.3803.

(155) Preparation of H-(D)Pro-Leu-GAz(Boc)-Gly-OH:

(156) ##STR00103##

(157) To a solution of Cbz-D-Pro-Leu-GAz(Boc)-Gly-OBn (450 mg, 0.65 mmol, 1.0 equiv) in MeOH (6.5 mL) was added 10 wt % Pd/C (45 mg, 10 wt %) and the reaction flask was evacuated, filled with nitrogen, evacuated, and placed under an atmosphere of hydrogen (balloon). The mixture was stirred at room temperature for 16 h, placed under nitrogen and filtered through a plug of Celite, which was washed with MeOH (3×). The filtrate was concentrated in vacuo to give the title compound as a white solid (304 mg, 0.647 mmol, quant. yield), which required no further purification; mp 89-91° C.; H NMR (500 MHz, MeOD) δ 4.41-4.30 (m, 2H, CHα-Leu, CHα-Pro), 3.76-3.57 (m, 5H, 2×NCH.sub.2-Az, CHHGAz), 3.44-3.38 (m, 1H, CHHδ-Pro), 3.36-3.31 (m, 1H, CHHδ-Pro), 3.30-3.26 (m, 1H, CHHGAz), 3.17 (d, J=17.4 Hz, 1H, CHHGly), 3.06 (d, J=17.4 Hz, 1H, CHHGly), 2.39 (td, J=13.3, 7.8 Hz, 1H, CHHγ-Pro), 2.16-2.01 (m, 2H, CH.sub.2β-Pro), 1.96 (dq, J=15.6, 7.8 Hz, 1H, CHHγ-Pro), 1.79-1.62 (m, 2H, CHγ-Leu, CHHβ-Leu), 1.61-1.54 (m, 1H, CHHβ-Leu), 1.42 (s, 9H, CH.sub.3 Boc), 0.98 (d, J=6.5 Hz, 3H, CH.sub.3δ-Leu), 0.92 (d, J=6.4 Hz, 3H, CH.sub.3δ-Leu); .sup.13C NMR (126 MHz, MeOD) δ 178.5 (C═O), 173.0 (C═O), 169.2 (C═O), 156.9 (C═O Boc), 79.8 (C, Boc), 60.1 (CH, α-Pro), 55.5 (2×NCH.sub.2), 52.5 (CH, α-Leu), 46.2 (CH.sub.2, Gly), 45.6 (CH.sub.2, δ-Pro), 42.0 (CH.sub.2, GAz), 39.8 (CH.sub.2, β-Leu), 29.0 (CH.sub.2, γ-Pro), 27.2 (CH.sub.3, Boc), 24.8 (CH, γ-Leu), 23.8 (CH.sub.2, β-Pro), 22.2 (CH.sub.3, δ-Leu), 20.0 (CH.sub.3, δ-Leu); ν.sub.max (neat)=3262, 3052, 1651, 1556, 1387, 1118 cm.sup.−1; MS (ESI.sup.+) m/z 470 [M+H].sup.+, 492 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.22H.sub.40N.sub.5O.sub.6 470.2973 [M+H].sup.+. found 470.2971;

(158) Preparation of Cyclo-((D)Pro-Leu-GAz(Boc)-Gly):

(159) ##STR00104##

(160) To a solution of H-D-Pro-Leu-GAz(Boc)-Gly-OH (47 mg, 0.1 mmol, 1.0 equiv) in DMF (mL, 0.001 M) was added DEPBT (60 mg, 0.2 mmol, 2.0 equiv) and DIPEA (36 μL, 0.2 mmol, 2.0 equiv) and the mixture was stirred for 48 h at room temperature. The solvent was removed in vacuo and the residue was purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 40:1.fwdarw.19:1) to give the cyclic tetrapeptide as a glassy colourless solid (35.8 mg, 79 μmol, 79%); R.sub.f (CH.sub.2Cl.sub.2/MeOH 19:1) 0.40; mp 129-131° C.; .sup.1H NMR (500 MHz, MeOD) δ 4.49-4.40 (m, 2H, CHα-Pro, CHα-Leu), 3.95 (s, 1H, NCHH-Az), 3.84-3.78 (m, 1H, NCHH-Az), 3.74-3.55 (m, 5H, NCHH-Az, NCHH-Az, CHHδ-Pro, CH.sub.2Gly), 3.37-3.35 (m, 3H, CHHδ-Pro, CH.sub.2GAz), 2.38-2.29 (m, 1H, CHHγ-Pro), 2.05-1.89 (m, 3H, CHHγ-Pro, CH.sub.2β-Pro), 1.73-1.65 (m, 2H, CHγ-Leu, CHHβ-Leu), 1.61-1.56 (m, 1H, CHHβ-Leu), 1.45 (s, 9H, 3×CH.sub.3Boc), 1.00 (d, J=6.1 Hz, 3H, CH.sub.3δ-Leu), 0.95 (d, J=6.0 Hz, 3H, CH.sub.3δ-Leu), Note: CHHδ-Pro, CH.sub.2GAz overlaps with solvent signal; .sup.13C NMR (126 MHz, MeOD) δ 176.3 (C═O), 175.7 (C═O), 173.0 (C═O), 158.2 (C═O, Boc), 81.2 (C, Boc), 62.1 (CH, α-Leu), 57.0 (2×NCH.sub.2), 54.0 (CH, α-Pro), 48.4 (CH.sub.2, GAz), 47.8 (CH.sub.2, Gly), 38.3 (CH.sub.2, β-Leu), 33.0 (CH.sub.2, γ-Pro), 28.6 (CH.sub.3, Boc), 26.1 (CH, γ-Leu), 23.5 (CH.sub.2, β-Pro), 23.0 (CH.sub.3, δ-Leu), 22.5 (CH.sub.3, δ-Leu), Note: CH.sub.2, δ-Pro overlaps with solvent signal;

(161) ν.sub.max (neat)=2954, 1662, 1623, 1404, 1164 cm.sup.−1; MS (ESI.sup.+) m/z 474 [M+Na].sup.+, 925 [2M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.22H.sub.37N.sub.5NaO.sub.5 [M+Na].sup.+474.2687. found 474.2684.

(162) Preparation of Cyclo-((D)Pro-Leu-GAz(H)-Gly):

(163) To a solution of Cyclo-((D)Pro-Leu-GAz(Boc)-Gly) (17.5 mg, 38.7 μmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (1 mL) was added TFA (1 mL). The solution was stirred for 1 h at room temperature. The solvent was removed in vacuo to reveal the title compound as a pale yellow glassy solid as the TFA salt in quantitative yield, which required no further purification. .sup.1H and .sup.19F NMR using TFE as an internal standard confirmed 1 eq. TFA salt; mp 123-125° C. (decomp.); .sup.1H NMR (500 MHz, MeOD) δ 4.49 (dd, J=8.1, 4.3 Hz, 1H, CHα-Pro), 4.27 (t, J=7.7 Hz, 1H, CHα-Leu), 4.01 (d, J=11.2 Hz, 1H, NCHH-Az), 3.98 (d, J=11.4 Hz, 1H, NCHH-Az), 3.81 (d, J=10.9 Hz, 1H, NCHH-Az), 3.75 (d, J=11.1 Hz, 1H, NCHH-Az), 3.70 (d, J=14.4 Hz, 1H, CHHGAz), 3.65-3.60 (m, 1H, CHHδ-Pro), 3.59-3.54 (m, 1H, CHHδ-Pro), 3.41 (d, J=8.5 Hz, 1H, CHHGly), 3.38 (d, J=8.3 Hz, 1H, CHHGly), 3.23 (d, J=14.2 Hz, 1H, CHHGAz), 2.36 (dt, J=19.7, 7.6 Hz, 1H, CHHγ-Pro), 2.04-1.88 (m, 3H, CHHγ-Pro, CH.sub.2β-Pro), 1.71-1.60 (m, 3H, CH.sub.2β-Leu, CHγ-Leu), 0.99 (d, J=6.4 Hz, 3H, CH.sub.3δ-Leu), 0.94 (d, J=6.4 Hz, 3H, CH.sub.3δ-Leu); .sup.13C NMR (126 MHz, MeOD) δ 176.2 (C═O), 176.0 (C═O), 173.1 (C═O), 62.0 (CH, α-Pro), 59.8 (C, Az), 57.2 (NCH.sub.2), 55.9 (CH, α-Leu), 54.2 (NCH.sub.2), 47.5 (CH.sub.2, GAz), 46.5 (CH.sub.2, Gly), 39.2 (CH, 3-Leu), 33.0 (CH.sub.2, γ-Pro), 26.1 (CH, γ-Leu), 23.7 (CH.sub.2, β-Pro), 22.9 (CH.sub.3, δ-Leu), 22.5 (CH.sub.3, δ-Leu). Note: CH.sub.2, 6-Pro overlaps with solvent signal; ν.sub.max (neat)=2956, 1665, 1199, 1177, 1128 cm.sup.−1; MS (ESI.sup.+) m/z 352 [M+H].sup.+, 374 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.17H.sub.30N.sub.5O.sub.3 352.2343 [M+H].sup.+. found 352.2338.

Example 10—Solution Phase Synthesis of Cyclo-(Leu-Tyr(.SUP.t.Bu)-Val-GAz(Boc)-Thr(.SUP.t.Bu)-Phe) (GAz=Azetidine Modified Glycine)

(164) ##STR00105##

(165) In this example the hexapeptide was made by solution phase synthesis. The example illustrates access to different ring sizes using an azetidine turn-inducing element and the tolerance of azetidine to side-chain deprotections (e.g. tyrosine, threonine).

(166) Preparation of NO.sub.2-GAz(Boc)-Thr(.sup.tBu)-OCumyl:

(167) ##STR00106##

(168) To a solution of Fmoc-Thr(.sup.tBu)-OCumyl (1.91 g, 3.71 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (3.7 mL) was added diethylamine (3.7 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×15 mL) and concentrated under reduced pressure to give the crude amine. In a second reaction vessel, N-Boc-3-azetidinone (0.63 g, 3.71 mmol, 1.0 equiv), nitromethane (3.7 mL) and triethylamine (103 μL, 0.74 mmol, 0.2 equiv) were combined and stirred for 1 h at room temperature. The solvent was removed in vacuo and resuspended in CH.sub.2Cl.sub.2 (15 mL), cooled to −78° C., and triethylamine (1.03 mL, 7.40 mmol, 2.0 equiv) was added followed by dropwise addition of a solution of methanesulfonyl chloride (287 μL, 3.71 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (3.7 mL). The reaction mixture was stirred at −78° C. for 1.5 h and the solution of the crude amine in anhydrous CH.sub.2Cl.sub.2 (30 mL) was added slowly via syringe. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. A saturated solution of NH.sub.4Cl (50 mL) was added and stirred for 10 min. The layers were separated and the aqueous one extracted with CH.sub.2Cl.sub.2 (2×30 mL) and EtOAc (2×30 mL). The combined organic phases were washed with saturated aqueous NaHCO.sub.3 solution (50 mL), brine (50 mL), dried over MgSO.sub.4, filtered, and concentrated in vacuo. The residue was purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/EtOAc 4:1) to give the title compound (1.62 g, 3.18 mmol, 86%) as a pale-yellow oil. R.sub.f (CH.sub.2Cl.sub.2/EtOAc 4:1) 0.30; .sup.1H NMR (500 MHz, CDCl.sub.3) 7.46-7.42 (m, 2H, ArH), 7.36-7.31 (m, 3H, ArH), 4.62 (d, J=13.1 Hz, 1H, CHHNO.sub.2), 4.53 (d, J=13.1 Hz, 1H, CHHNO.sub.2), 3.93 (d, J=9.4 Hz, 1H, NCHH-Az), 3.82-3.72 (m, 4H, NCHH-Az, NCH.sub.2-Az, CHα-Thr), 3.20 (d, J=2.9 Hz, 1H, CHβ-Thr), 2.61-2.25 (m, 1H, NH), 1.83 (s, 3H, CH.sub.3-Cumyl), 1.80 (s, 3H, CH.sub.3-Cumyl), 1.43 (s, 9H, 3×CH.sub.3 Boc), 1.20 (s, 9H, 3×CH.sub.3 .sup.tBu), 1.08 (d, J=6.2 Hz, 3H, CH.sub.3γ-Thr); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 172.7 (C═O), 156.2 (C═O Boc), 145.2 (C), 128.4 (CH), 127.5 (CH), 124.8 (CH), 83.3 (C, Cumyl), 80.3 (C, Boc), 79.0 (CH.sub.2NO.sub.2), 74.2 (C, .sup.tBu), 69.2 (α-CH, Thr), 61.8 (β-CH, Thr), 54.2 (2×NCH.sub.2), 28.6 (CH.sub.3, .sup.tBu), 28.4 (CH.sub.3, Boc), 27.9 (2×CH.sub.3, Cumyl), 19.4 (CH.sub.3, γ-Thr); ν.sub.max (neat)=2975, 1700, 1555, 1377, 1194, 699 cm.sup.−1; MS (ESI.sup.+) m/z 530 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.26H.sub.41N.sub.3NaO.sub.7 530.2837 [M+Na].sup.+. found 530.2833.

(169) Preparation of Fmoc-Val-GAz(Boc)-Thr(.sup.tBu)-OCumyl:

(170) ##STR00107##

(171) To a solution of NO.sub.2-GAz(Boc)-Thr(.sup.tBu)-OCumyl (1.61 g, 3.18 mmol, 1.0 equiv) in THF (32 mL) was added Fmoc-Val-OSu (2.77 g, 6.35 mmol, 2.0 equiv) and Raney Ni (slurry in H.sub.2O, 6 mL). The solution was placed under an atmosphere of nitrogen, evacuated and filled with hydrogen (balloon). The reaction mixture was stirred vigorously for 4.0 h at room temperature. Then, the mixture was filtered through a plug of Celite eluting with EtOAc, concentrated in vacuo, the filtrate was diluted with EtOAc (50 mL), washed with saturated Na.sub.2CO.sub.3 (3×50 mL), dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. Fmoc-Val-GAz(Boc)-Thr(.sup.tBu)-OCumyl was afforded after purification by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/EtOAc 19:1) as a white foam (1.53 g, 1.91 mmol, 60%); mp 73-75° C.; R.sub.f (CH.sub.2Cl.sub.2/EtOAc 19:1) 0.19; .sup.1H NMR (500 MHz, CDCl.sub.3) 7.76 (d, J=7.5 Hz, 2H, ArH), 7.59 (d, J=7.2 Hz, 2H, ArH), 7.43-7.37 (m, 4H, ArH), 7.34-7.28 (m, 4H, ArH), 7.25-7.20 (m, 1H, ArH), 6.50 (s, 1H, NH), 5.44 (d, J=8.6 Hz, 1H, NH Fmoc), 4.42 (dd, J=10.3, 7.5 Hz, 1H, CHH Fmoc), 4.34-4.28 (m, 1H, CHH Fmoc), 4.21 (t, J=7.0 Hz, 1H, CHFmoc), 4.00-3.92 (m, 1H, CHα-Thr), 3.90-3.82 (m, 1H, CHα-Val), 3.81-3.70 (m, 1H, CHHGAz), 3.62 (d, J=8.7 Hz, 1H, NCHH-Az), 3.60-3.45 (m, 3H, NCHH-Az, NCH.sub.2-Az), 3.23-2.70 (m, 2H, CHHGAz, CHβ-Thr), 2.00-1.92 (m, 1H, CHβ-Val) 1.83 (s, 3H, CH.sub.3-Cumyl), 1.75 (s, 3H, CH.sub.3-Cumyl), 1.42 (s, 9H, 3×CH.sub.3 Boc), 1.19 (s, 9H, 3×CH.sub.3 .sup.tBu), 1.17 (d, J=6.1 Hz, 3H, CH.sub.3γ-Thr), 0.89 (d, J=6.5 Hz, 3H, CH.sub.3γ-Val), 0.85 (d, J=6.6 Hz, 1H, CH.sub.3γ-Val); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 174.0 (C═O), 171.6 (C═O), 156.4 (C═O, Boc, C═O Fmoc) 145.0 (C), 144.1 (C), 144.0 (2×C), 141.4 (C), 128.4 (CH), 127.8 (CH), 127.6 (CH), 127.2 (CH), 125.27 (CH), 124.7 (CH), 120.1 (CH), 83.4 (C, Cumyl), 79.8 (C, Boc), 74.2 (C, .sup.tBu), 69.2 (CHα-Thr), 67.1 (CH.sub.2, Fmoc), 61.8 (CH, β-Thr), 60.4 (CHα-Val), 54.7 (2×NCH.sub.2), 47.3 (CH, Fmoc), 44.0 (CH.sub.2, GAz), 31.5 (CH, β-Val), 28.8 (CH.sub.3, Cumyl), 28.7 (CH.sub.3, Boc), 28.5 (CH.sub.3, .sup.tBu), 27.3 (2×CH.sub.3, Cumyl), 20.2 (CH.sub.3, γ-Thr), 19.3 (CH.sub.3, γ-Val), 17.8 (CH.sub.3, γ-Val); ν.sub.max (neat)=2968, 1702, 1658, 1399, 1365 cm.sup.−1; MS (ESI.sup.+) m/z 799 [M+H].sup.+, 821 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.46H.sub.62N.sub.4NaO.sub.8 821.4460 [M+Na].sup.+. found 821.4467.

(172) Preparation of Fmoc-Val-GAz(Boc)-Thr(.sup.tBu)-Phe-OBn:

(173) ##STR00108##

(174) Fmoc-Val-GAz(Boc)-Thr(Bu)-OCumyl (1.47 g, 1.84 mmol, 1.0 equiv) was dissolved in 2% TFA/CH.sub.2Cl.sub.2 and stirred at room temperature until completion, monitored by ESI-MS. The mixture was concentrated under reduced pressure, the resulting residue was repeatedly re-dissolved in CH.sub.2Cl.sub.2 (20 mL) and the solvent was removed under reduced pressure. To the crude acid in CH.sub.2Cl.sub.2 (20 mL) was added H-Phe-OBn-HCl (0.65 g, 2.22 mmol, 1.2 equiv), NMM (1.01 mL, 9.22 mmol, 5.0 equiv), HOBt-H.sub.2O (0.25 g, 1.84 mmol, 1.0 equiv) and EDC.Math.HCl (0.35 g, 1.84 mmol, 1.0 equiv). The reaction mixture was allowed to stir for 18 h at room temperature under an atmosphere of nitrogen. The mixture was diluted with EtOAc (50 mL) and washed with brine (3×50 mL), dried (Na.sub.2SO.sub.4) and concentrated in vacuo to afford a yellow oil which was purified by flash column chromatography (SiO.sub.2, CH.sub.2C.sub.2/MeOH 49:1). Fmoc-Val-GAz(Boc)-Thr(.sup.tBu)-Phe-OBn was obtained as a yellow solid (1.26 g, 1.37 mmol, 74%). R.sub.f (CH.sub.2Cl.sub.2/MeOH 49:1) 0.44; mp 56-58° C.; .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.80 (d, J=8.1 Hz, 1H, NH), 7.76 (d, J=7.5 Hz, 2H, ArH), 7.63-7.57 (m, 2H, ArH), 7.42-7.27 (m, 10H, ArH), 7.22-7.18 (m, 3H, ArH), 7.08-7.02 (m, 2H, 1×NH, 1×ArH), 5.49 (d, J=8.8 Hz, 1H, NH Fmoc), 5.18 (d, J=12.1 Hz, 1H, CHHPh Bn), 5.10 (d, J=12.1 Hz, 1H. CHHPh Bn), 4.90 (dt, J=7.9, 5.9 Hz, 1H, CHα-Phe), 4.43 (dd, J=10.2, 7.5 Hz, 1H, CHHFmoc), 4.33 (dd, J=10.1, 7.5 Hz, 1H, CHHFmoc), 4.23 (t, J=7.1 Hz, 1H, CHFmoc), 4.12-4.03 (m, 1H, CHα-Val), 3.78 (d, J=8.6 Hz, 2H, NCHH-Az, CHHGAz), 3.59-3.48 (m, 4H, CHα-Thr, NCHH-Az, NCH.sub.2-Az), 3.17 (dd, J=13.9, 5.5 Hz, 1H, CHHβ-Phe), 3.00 (dd, J=13.8, 7.9 Hz, 1H, CHHβ-Phe), 2.95-2.70 (m, 2H, CHα-Thr, CHHGAz), 2.40 (s, 1H, NH), 2.15-2.08 (m, 1H, CHβ-Val), 1.42 (s, 9H, 3×CH.sub.3 Boc), 1.08 (s, 9H, 3×CH.sub.3 .sup.tBu), 1.01-0.95 (m, 6H, CH.sub.3γ-Thr, CH.sub.3γ-Val), 0.93 (d, J=6.6 Hz, 3H, CH.sub.3γ-Val); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 173.1 (C═O), 172.1 (C═O), 156.5 (C═O Fmoc), 156.4 (C═O Boc), 144.04 (C), 143.97 (C), 141.4 (C), 136.1 (2×C), 135.1 (C), 129.3 (CH), 128.75 (CH), 128.74 (CH), 128.69 (CH), 128.66 (CH), 127.9 (CH), 127.3 (CH), 127.2 (CH), 125.3 (CH), 120.1 (CH), 79.8 (C, Boc), 74.9 (C, .sup.tBu), 67.5 (CH.sub.2Ph, Bn), 67.2 (CH.sub.2, Fmoc), 60.6 (CH, α-Val, CH, β-Thr), 55.6 (2×NCH.sub.2), 53.3 (CH, α-Phe), 47.3 (CH, Fmoc), 38.2 (CH.sub.2, β-Phe), 31.3 (CH, β-Val), 28.5 (CH.sub.3, Boc or .sup.tBu), 28.4 (CH.sub.3, Boc or .sup.tBu), 19.4 (CH.sub.3, γ-Thr), 18.0 (2×CH.sub.3, γ-Val), Note: CH.sub.2, GAz, CHα-Thr missing; ν.sub.max (neat)=2970, 1734, 1698, 1667, 1390, 1106 cm.sup.−1; MS (ESI.sup.+) m/z 918 [M+H].sup.+, 940 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.53H.sub.68N.sub.5O.sub.9 918.5012 [M+H].sup.+. found 918.5010.

(175) Preparation of Fmoc-Tyr(.sup.tBu)-Val-GAz(Boc)-Thr(.sup.tBu)-Phe-OBn:

(176) ##STR00109##

(177) To a solution of tetrapeptide Fmoc-Val-GAz(Boc)-Thr(.sup.tBu)-Phe-OBn (994 mg, 1.08 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (2 mL) was added diethylamine (2 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (11 mL), Fmoc-Tyr(tBu)-OH (496 mg, 1.08 mmol, 1.0 equiv), EDC.Math.HCl (207 mg, 1.08 mmol, 1.0 equiv), HOBt-H.sub.2O (146 mg, 1.08 mmol, 1.0 equiv) and NMM (475 μL, 4.32 mmol, 4.0 equiv) were added subsequently, and the mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with EtOAc (50 mL) and washed with brine (3×50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 49:1) to give pentapeptide Fmoc-Tyr(.sup.tBu)-Val-GAz(Boc)-Thr(.sup.tBu)-Phe-OBn (898 mg, 0.79 mmol, 73%) as a white solid. R.sub.f (CH.sub.2Cl.sub.2/MeOH 97:3) 0.27; mp 103-105° C.; .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.82 (d, J=8.0 Hz, 1H, NH), 7.76 (d, J=7.5 Hz, 2H, ArH), 7.54 (dd, J=7.3, 2.9 Hz, 2H, ArH), 7.44-7.27 (m, 9H, ArH), 7.25-7.19 (m, 3H, ArH), 7.11-7.04 (m, 4H, ArH), 6.90 (d, J=8.1 Hz, 2H, ArH), 6.53 (d, J=6.6 Hz, 1H, NH), 5.35-5.28 (m, 1H, Fmoc NH), 5.18 (d, J=12.1 Hz, 1H, CHHPh Bn), 5.12 (d, J=12.2 Hz, 1H, CHHPh Bn), 4.91 (dd, J=13.8, 7.6 Hz, 1H, CHα-Phe), 4.55-4.36 (m, 2H, CHH Fmoc, CHα-Tyr), 4.36-4.21 (m, 2H, CHH Fmoc, CHα-Val), 4.18 (t, J=6.9 Hz, 1H, CH Fmoc), 3.86-3.41 (m, 6H, 2×NCH.sub.2-Az, CHHGAz, CHα-Thr), 3.16 (dd, J=13.9, 5.7 Hz, 1H, CHHβ-Phe), 3.12-2.76 (m, 5H, CHβ-Thr, CHHGAz, CHHβ-Phe, CH.sub.2-Tyr), 2.12-2.05 (m, 1H, m, 1H, CHβ-Val), 1.80 (s, 1H, NH), 1.41 (s, 9H, 3×CH.sub.3 Boc), 1.31 (s, 9H, 3×CH.sub.3 .sup.tBu), 1.12 (s, 9H, 3×CH.sub.3 .sup.tBu), 1.02 (d, J=6.2 Hz, 3H, CH.sub.3γ-Val), 0.88 (d, J=6.7 Hz, 3H, CH.sub.3γ-Thr), 0.81 (d, J=6.6 Hz, 3H, CH.sub.3γ-Val), Note: 1 NH missing; .sup.13C NMR (126 MHz, CDCl.sub.3) δ 173.1 (C═O), 171.8 (C═O), 171.4 (C═O), 171.1 (C═O), 156.4 (C═O Boc), 156.1 (C═O Fmoc), 154.6 (C), 143.9 (C), 143.8 (C), 141.4 (C), 136.1 (C), 135.1 (C), 131.2 (C), 129.3 (CH), 128.8 (CH), 128.74 (CH), 128.67 (CH), 128.65 (CH), 127.9 (CH), 127.3 (CH), 127.2 (CH), 125.20 (CH), 125.16 (CH), 124.5 (CH), 120.1 (CH), 79.8 (C, Boc), 78.6 (C, tBu), 74.9 (C, tBu), 67.5 (CH.sub.2Ph Bn), 67.2 (CH.sub.2Ph Fmoc), 58.9 (CH, α-Val), 56.3 (CH, α-Tyr), 55.6 (2×NCH.sub.2), 53.4 (CH, α-Phe), 47.2 (CH, Fmoc), 38.2 (CH.sub.2, β-Tyr), 37.4 (CH.sub.2, β-Phe), 30.9 (CH, β-Val), 29.0 (CH.sub.3, .sup.tBu), 28.5 (CH.sub.3, .sup.tBu), 28.45 (CH.sub.3, Boc), 19.4 (CH.sub.3, γ-Val), 18.0 (CH.sub.3, γ-Thr, CH.sub.3, γ-Val), Note: Two aromatic quaternary C, CH.sub.2 GAz, CH β-Thr and CH α-Thr missing; ν.sub.max (neat)=2970, 1698, 1643, 1529, 1232 cm.sup.−1; MS (ESI.sup.+) m/z 1137 [M+H].sup.+, 1160 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.66H.sub.84N.sub.6NaO.sub.11 1159.6090 [M+Na].sup.+. found 1159.6101.

(178) Preparation of Cbz-Leu-Tyr(.sup.tBu)-Val-GAz(Boc)-Thr(.sup.tBu)-Phe-OBn:

(179) ##STR00110##

(180) To a solution of pentapeptide Fmoc-Tyr(.sup.tBu)-Val-GAz(Boc)-Thr(.sup.tBu)-Phe-OBn (845 mg, 0.74 mmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (2 mL) was added diethylamine (2 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the resulting residue repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated under reduced pressure to give the crude amine. The residue was dissolved in CH.sub.2Cl.sub.2 (8 mL), Cbz-Leu-OH (197 mg, 0.74 mmol, 1.0 equiv), EDC.Math.HCl (143 mg, 0.74 mmol, 1.0 equiv), HOBt-H.sub.2O (101 mg, 0.74 mmol, 1.0 equiv) and NMM (327 μL, 2.98 mmol, 4.0 equiv) were added subsequently, and the mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with EtOAc (50 mL) and washed with brine (3×50 mL), dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 97:3) to give hexapeptide Cbz-Leu-Tyr(.sup.tBu)-Val-GAz(Boc)-Thr(.sup.tBu)-Phe-OBn (708 mg, 0.61 mmol, 82%) as a white foam. R.sub.f (CH.sub.2Cl.sub.2/MeOH 97:3) 0.23; mp 126-128° C.; .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.79 (d, J=8.1 Hz, 1H, NH), 7.43-7.15 (m, 14H, ArH, NH), 7.10-7.04 (m, 4H, ArH), 6.88 (d, J=8.1 Hz, 2H, ArH), 6.77-6.60 (m, 2H, 2×NH), 5.18-4.96 (m, 5H, 2×CH.sub.2Ph, Cbz NH), 4.88 (dd, J=13.9, 7.3 Hz, 1H, CHα-Phe), 4.58-4.49 (m, 1H, CHα-Tyr), 4.34-4.25 (m, 1H, CHα-Val), 4.07-4.00 (m, 1H, CHα-Leu), 3.87-3.48 (m, 6H, CHHGAz, 2×NCH.sub.2-Az, CHα-Thr), 3.21-2.80 (m, 6H, CHHGAz, CH.sub.2β-Phe, CH.sub.2β-Tyr, CH.sub.3β-Thr), 2.57 (s, 1H, NH), 2.28-2.14 (m, 1H, CHβ-Val), 1.59-1.48 (m, 2H, CHHβ-Leu, CHγ-Leu), 1.39 (s, 10H, CHHβ-Leu, 3×CH.sub.3 .sup.tBu), 1.30 (s, 9H, 3×CH.sub.3 .sup.tBu), 1.11 (s, 9H, 3×CH.sub.3 .sup.tBu), 1.00 (d, J=6.2 Hz, 3H, CH.sub.3γ-Thr), 0.91 (d, J=6.4 Hz, 3H, CH.sub.3γ-Val), 0.87-0.81 (m, 9H, CH.sub.3γ-Val, 2×CH.sub.3δ-Leu); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 173.3 (C═O), 172.9 (C═O), 171.6 (C═O), 171.5 (C═O), 171.1 (C═O), 156.5 (C═O Boc/Cbz), 156.4 (C═O Boc/Cbz), 154.5 (C), 136.1 (C), 136.0 (C), 135.1 (C), 131.5 (C), 129.8 (CH), 129.3 (CH), 128.8 (CH), 128.73 (CH), 128.69 (CH), 128.64 (CH), 128.63 (CH), 128.4 (CH), 128.3 (CH), 127.3 (CH), 124.5 (CH), 79.7 (C, .sup.tBu), 78.5 (C, .sup.tBu), 74.9 (C, Boc), 67.40 (CH.sub.2Ph Bn), 67.37 (CH.sub.2Ph Bn), 61.1 (CH, α-Thr), 59.0 (CH, α-Val), 55.8 (CH, α-Tyr), 55.5 (2×NCH.sub.2), 54.1 (CH, α-Leu), 53.5 (CH, α-Phe), 41.0 (CH.sub.2, β-Leu), 38.2 (CH.sub.2, β-Phe), 36.4 (CH.sub.2, β-Tyr), 30.3 (CH, γ-Val), 29.0 (CH.sub.3, .sup.tBu), 28.5 (CH.sub.3, .sup.tBu), 28.4 (CH.sub.3, Boc), 24.8 (CH, γ-Leu), 23.0 (CH.sub.3, δ-Leu), 21.9 (CH.sub.3, γ-Val), 19.5 (CH.sub.3, δ-Leu), 17.7 (CH.sub.3, γ-Val, CH.sub.3, γ-Thr). Note: CH.sub.2 GAz and CH α-Thr missing; ν.sub.max (neat)=2971, 1697, 1637, 1365, 696 cm.sup.−1; MS (ESI.sup.+) m/z 1184 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.65H.sub.91N.sub.7NaO.sub.12 1184.6618 [M+Na].sup.+. found 1184.6623.

(181) Preparation of H-Leu-Tyr(.sup.tBu)-Val-GAz(Boc)-Thr(.sup.tBu)-Phe-OH:

(182) ##STR00111##

(183) To a solution of Cbz-Leu-Tyr(Bu)-Val-GAz(Boc)-Thr(Bu)-Phe-OBn (590 mg, 0.518 mmol, 1.0 equiv) in MeOH (5 mL) was added 10 wt % Pd/C (59 mg, 10 wt %) and the reaction flask was evacuated, filled with nitrogen, evacuated, and placed under an atmosphere of hydrogen (balloon). The reaction mixture was stirred at room temperature for 16 h, placed under nitrogen and filtered through a plug of Celite, which was washed with MeOH (3×). The filtrate was concentrated in vacuo to give the title compound as a white solid (491 mg, 0.517 mmol, quant. yield). mp 126-128° C.; .sup.1H NMR (500 MHz, MeOD) δ 7.31-7.21 (m, 7H, ArH), 6.91 (d, J=8.3 Hz, 2H, ArH), 4.76-4.70 (m, 2H, CHα-Tyr, CHα-Phe), 4.21 (d, J=6.9 Hz, 1H, CHα-Val), 3.87-3.81 (m, 1H, CHα-Leu), 3.71-3.44 (m, 6H, CHHGAz, 2×NCH.sub.2-Az, CHα-Thr), 3.26-3.21 (m, 2H, CHHβ-Phe, CHHβ-Tyr), 3.07-2.82 (m, 5H, CHHGAz, CHHβ-Phe, CHHβ-Tyr, CHβ-Thr), 2.10 (td, J=13.5, 6.8 Hz, 1H, CHβ-Val), 1.72-1.62 (m, 3H, CH.sub.2β-Leu, CHγ-Leu), 1.42 (s, 9H, CH.sub.3 Boc), 1.32 (s, 9H, CH.sub.3 .sup.tBu), 1.14 (s, 9H, CH.sub.3 .sup.tBu), 1.06 (d, J=5.8 Hz, 3H, CH.sub.3γ-Thr), 0.97 (d, J=3.2 Hz, 3H, CH.sub.3δ-Leu), 0.96 (d, J=3.0 Hz, 3H, CH.sub.3δ-Leu), 0.94 (d, J=3.3 Hz, 3H, CH.sub.3γ-Val), 0.93 (d, J=3.2 Hz, 3H, CH.sub.3γ-Val); .sup.13C NMR (126 MHz, MeOD) δ 175.4 (C═O), 174.9 (C═O), 173.8 (C═O), 173.4 (C═O), 170.8 (C═O), 158.1 (C═O Boc), 155.4 (C), 138.4 (C), 133.4 (C), 130.8 (CH), 130.4 (CH), 129.7 (CH), 128.0 (CH), 125.3 (CH), 81.2 (C, .sup.tBu), 79.5 (C, .sup.tBu), 75.6 (C, Boc), 70.7 (CH, α-Thr), 63.7 (CH, β-Thr), 60.5 (CH, α-Val), 57.1 (2×NCH.sub.2), 56.6 (CH, α-Tyr/Phe), 54.9 (CH, α-Tyr/Phe), 52.8 (CH, α-Leu), 44.9 (CH.sub.2, GAz), 41.8 (CH, β-Leu), 38.7 (CH.sub.2, β-Tyr/Phe), 37.7 (CH.sub.2, β-Tyr/Phe), 32.0 (CH, β-Val), 29.2 (CH.sub.3, .sup.tBu), 28.9 (CH.sub.3, .sup.tBu), 28.6 (CH.sub.3, Boc), 25.3 (CH, γ-Leu), 23.3 (CH.sub.3, δ-Leu), 21.8 (CH.sub.3, δ-Leu), 20.3 (CH.sub.3, γ-Thr), 19.9 (CH.sub.3, γ-Val), 18.6 (CH.sub.3, γ-Val). ν.sub.max (neat)=3287, 2967, 1641, 1608, 1506, 1160 cm.sup.−1; MS (ESI.sup.+) m/z 938 [M+H].sup.+, 960 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.50H.sub.30N.sub.7NaO.sub.10 938.5961 [M+H].sup.+. found 938.5966.

(184) Preparation of Cyclo-(Leu-Tyr(.sup.tBu)-Val-GAz(Boc)-Thr(.sup.tBu)-Phe):

(185) ##STR00112##

(186) To a solution of H-Leu-Tyr(.sup.tBu)-Val-GAz(Boc)-Thr(.sup.tBu)-Phe-OH (94 mg, 0.1 mmol, 1.0 equiv) in DMF (20 mL, 0.005 M) was added DEPBT (60 mg, 0.2 mmol, 2.0 equiv) and DIPEA (36 μL, 0.2 mmol, 2.0 equiv) and the reaction mixture was stirred for 48 h at room temperature. The solvent was removed under reduced pressure at 60° C. over 30 min, and the residue was dried in vacuo. The residue was analysed by LCMS and purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 49:1.fwdarw.19:1) to give the cyclic hexapeptide as a white glassy solid (52.1 mg, 57 μmol, 57%). R.sub.f (CH.sub.2Cl.sub.2/MeOH 19:1) 0.35; mp 156-158° C.; .sup.1H NMR (500 MHz, MeOD) δ 7.38-7.31 (m, 2H, ArH), 7.29-7.24 (m, 3H, ArH), 7.15 (d, J=8.3 Hz, 2H, ArH), 6.93 (d, J=8.3 Hz, 2H, ArH), 4.62 (dd, J=9.9, 5.6 Hz, 1H, CHα-Phe), 4.36 (d, J=9.0 Hz, 1H, CHα-Val), 4.08 (dd, J=11.5, 3.3 Hz, 1H, NCHH GAz), 3.98-3.83 (m, 2H, NCHH GAz, CHH GAz), 3.81-3.71 (m, 2H, NCHH GAz, CHα-Leu), 3.66-3.49 (m, 3H, NCHH GAz, CHα-Tyr, CHHβ-Tyr), 3.37 (m, 2H, CHHβ-Tyr, CHα-Thr), 3.02-2.94 (m, 1H, CHH GAz), 2.90-2.82 (m, 1H, CHβ-Thr), 2.74 (dd, J=13.4, 10.8 Hz, 1H, CHHβ-Phe), 2.18-1.88 (m, 1H, CHβ-Val), 1.58-1.51 (m, 1H, CHHβ-Leu), 1.42 (s, 9H, CH.sub.3 Boc), 1.35-1.30 (m, 10H, CHHβ-Leu, CH.sub.3 .sup.tBu), 1.26-1.22 (m, 1H, CHγ-Leu), 1.09-0.97 (m, 18H, CH.sub.3 .sup.tBu, 2×CH.sub.3γ-Val, CH.sub.3γ-Thr), 0.81 (d, J=6.5 Hz, 3H, CH.sub.3δ-Leu), 0.77 (d, J=6.4 Hz, 3H, CH.sub.3δ-Leu). Note: CHHβ-Phe overlaps with solvent signal; .sup.13C NMR (126 MHz, MeOD) δ 176.2 (C═O), 174.9 (C═O), 174.7 (C═O), 174.5 (C═O), 172.8 (C═O), 158.2 (C═O Boc), 155.3 (C), 137.7 (C), 134.9 (C), 130.6 (CH), 130.2 (CH), 130.0 (CH), 128.3 (CH), 125.3 (CH), 81.3 (C, .sup.tBu), 79.4 (C, .sup.tBu), 75.5 (C, Boc), 69.5 (CH, α-Thr), 65.1 (CH, 3-Thr), 62.6 (CH, α-Val), 59.5 (CH, α-Tyr), 57.7 (2×NCH.sub.2), 55.8 (CH, α-Leu), 54.7 (CH, α-Phe), 40.3 (CH.sub.2, β-Leu), 39.3 (CH.sub.2, β-Phe), 35.1 (CH.sub.2, β-Tyr), 33.6 (CH.sub.2, β-Val), 29.2 (CH.sub.3, .sup.tBu), 28.9 (CH.sub.3, .sup.tBu), 28.6 (CH.sub.3, Boc), 25.2 (CH, γ-Leu), 23.1 (CH.sub.3, δ-Leu), 22.3 (CH.sub.3, 6-Leu), 21.8 (CH.sub.3, γ-Thr), 19.9 (CH.sub.3, γ-Val), 19.5 (CH.sub.3, γ-Val). Note: GAz CH.sub.2 missing; ν.sub.max (neat)=2973, 1642, 1505, 1161 cm.sup.−1; MS (ESI.sup.+) m/z 920 [M+H].sup.+, 942 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.2H.sub.76N.sub.7O.sub.9 942.5699 [M+H].sup.+. found 942.5692.

(187) Preparation of Cyclo-(Leu-Tyr-Val-GAz-Thr-Phe):

(188) To a solution of Cyclo-(Leu-Tyr(.sup.tBu)-Val-GAz(Boc)-Thr(.sup.tBu)-Phe) (15.9 mg, 16.3 μmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (1 mL) was added TFA (1 mL). The solution was stirred for 1.5 h at room temperature. The solvent was removed in vacuo to reveal the title compound as a white glassy solid as the TFA salt in quantitative yield, which required no further purification. 1H and .sup.19F NMR using TFE as an internal standard confirmed 1 eq. TFA salt; mp 186-187° C. (decomp.); .sup.1H NMR (500 MHz, MeOD) δ 7.37-7.25 (m, 5H, ArH), 7.05 (d, J=7.8 Hz, 2H, ArH), 6.73 (d, J=7.6 Hz, 2H, ArH), 4.64 (dd, J=9.8, 4.9 Hz, 1H, CHα-Phe), 4.33 (d, J=9.2 Hz, 1H, CHα-Val), 4.18 (d, J=11.0 Hz, 1H, NCHH GAz), 4.10 (dd, J=11.3, 3.5 Hz, 1H, CHα-Tyr), 4.05-3.98 (m, 2H, CHH GAz, NCHH GAz), 3.98-3.88 (m, 2H, NCH.sub.2 GAz), 3.75 (t, J=7.2 Hz, 1H, CHα-Leu), 3.46 (m, 1H, CHHβ-Tyr), 3.03 (d, J=16.2 Hz, 1H, CHH GAz), 2.88-2.76 (m, 2H, CHHβ-Phe, CHβ-Thr), 2.05-1.92 (m, 1H, CHβ-Val), 1.54-1.47 (m, 1H, CHHβ-Leu), 1.43-1.32 (m, 2H, CHHβ-Leu, CHγ-Leu), 1.06 (d, J=6.5 Hz, 3H, CH.sub.3γ-Thr), 1.03 (d, J=6.9 Hz, 3H, CH.sub.3γ-Val), 1.00 (d, J=5.8 Hz, 3H, CH.sub.3γ-Val), 0.82 (d, J=6.0 Hz, 3H, CH.sub.3δ-Leu), 0.79 (d, J=6.1 Hz, 3H, CH.sub.3δ-Leu) Note: CHH-Tyr, CHα-Thr and CHHβ-Phe overlaps with solvent signal; .sup.13C NMR (126 MHz, MeOD) δ 175.4 (C═O), 174.9 (C═O), 174.7 (C═O), 174.6 (C═O), 173.0 (C═O), 157.3 (C), 137.6 (C), 131.0 (CH), 130.2 (C), 130.1 (CH), 129.8 (CH), 128.2 (CH), 116.3 (CH), 69.2 (CH, α-Thr), 66.0 (CH, 3-Thr), 62.8 (CH, α-Val), 60.6 (C, GAz), 59.4 (CH, α-Tyr), 55.9 (2×NCH.sub.2), 55.8 (CH, α-Leu), 54.8 (CH, α-Phe), 44.2 (CH.sub.2, GAz), 40.3 (CH.sub.2, β-Leu), 38.9 (CH.sub.2, β-Phe), 35.1 (CH.sub.2, β-Tyr), 33.4 (CH, β-Val), 25.1 (CH, γ-Leu), 22.8 (CH.sub.3, β-Leu), 22.6 (CH.sub.3, δ-Leu), 20.8 (CH.sub.3, γ-Thr), 19.9 (CH.sub.3, γ-Val), 19.5 (CH.sub.3, γ-Val). ν.sub.max (neat)=3302, 3029, 1639, 1515, 1436, 1198, 1132 cm.sup.−1; MS (ESI.sup.+) m/z 708 [M+H].sup.+, 730 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.37H.sub.54N.sub.7O.sub.7 708.4079 [M+Na].sup.+. found 708.4082.

Example 12—Solid Phase Synthesis of Cyclo-(Phe-Glu-GAz-Thr-Gly) (GAz=Azetidine Modified Glycine)

(189) ##STR00113##

(190) In this example the pentapeptide is made by solid phase peptide synthesis with side-chain and Boc deprotection exemplified.

(191) Preparation of Fmoc-GAz(Boc)-Thr(.sup.tBu)-OCumyl:

(192) ##STR00114##

(193) To a solution of NO.sub.2-GAz(Boc)-Thr(.sup.tBu)-OCumyl (2.00 g, 3.94 mmol, 1.0 equiv) in THF (40 mL) was added Fmoc-OSu (2.67 g, 7.88 mmol, 2.0 equiv), NaHCO.sub.3 (1.32 g, 15.76 mmol, 4.0 equiv) and Raney Ni (slurry in H.sub.2O, 8 mL). The solution was placed under an atmosphere of nitrogen, evacuated and filled with hydrogen (balloon). The reaction mixture was stirred vigorously for 4.0 h at room temperature. Then, the mixture was filtered through a plug of Celite eluting with EtOAc, concentrated in vacuo, the filtrate was diluted with EtOAc (50 mL), washed with saturated Na.sub.2CO.sub.3 (3×50 mL), dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. Fmoc-GAz(Boc)-Thr(.sup.tBu)-OCumyl was afforded after purification by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/EtOAc 19:1) as a white foam (1.43 g, 2.04 mmol, 52%); R.sub.f (CH.sub.2Cl.sub.2/EtOAc 19:1) 0.23; mp 67-69° C.; .sup.1H NMR (500 MHz, CDCl.sub.3) δ 7.76 (d, J=7.5 Hz, 2H, ArH), 7.59 (d, J=4.5 Hz, 1H, ArH), 7.58 (d, J=4.4 Hz, 1H, ArH), 7.45-7.38 (m, 4H, ArH), 7.37-7.29 (m, 4H, ArH), 7.28-7.20 (m, 1H, ArH), 5.42 (t, J=5.0 Hz, 1H, Fmoc NH), 4.48-4.29 (m, 2H, CH.sub.2 Fmoc), 4.19 (t, J=7.0 Hz, 1H, CH Fmoc), 3.82-3.73 (m, 1H, CHα-Thr), 3.68-3.51 (m, 2H, 2×NCH.sub.2-Az, CHHGAz), 3.31-2.98 (m, 2H, CHβ-Thr, CHHGAz), 2.08 (s, 1H, NH), 1.84 (s, 3H, CH.sub.3-Cumyl), 1.80 (s, 3H, CH.sub.3-Cumyl), 1.43 (s, 9H, CH.sub.3 Boc), 1.20 (s, 9H, CH.sub.3 .sup.tBu), 1.16 (d, J=6.1 Hz, 3H, CH.sub.3γ-Thr); .sup.13C NMR (126 MHz, CDCl.sub.3) δ 173.6 (C═O), 156.8 (C═O, Boc), 156.3 (C═O, Fmoc), 144.8 (C), 144.0 (C), 141.3 (C), 128.3 (CH), 127.7 (CH), 127.4 (CH), 127.0 (CH), 125.2 (CH), 124.7 (CH), 120.0 (CH), 83.2 (C, Cumyl), 79.6 (C, Boc), 74.3 (C, tBu), 69.1 (CH, α-Thr), 66.8 (CH.sub.2, Fmoc) 61.8 (CH, 3-Thr), 54.6 (2×NCH.sub.2), 47.3 (CH Fmoc), 45.5 (CH.sub.2, GAz), 28.6 (CH.sub.3, Boc), 28.4 (CH.sub.3, tBu), 27.5 (CH.sub.3, Cumyl), 20.0 (CH.sub.3, γ-Thr). Note: One CH.sub.3, Cumyl overlapping; ν.sub.max (neat)=2974, 1699, 1364, 1100, 758 cm.sup.−1; MS (ESI.sup.+) m/z 700 [M+H].sup.+, 722 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.41H.sub.53N.sub.3NaO.sub.7722.3776 [M+Na].sup.+. found 722.3782.

(194) Preparation of H-Phe-Glu(.sup.tBu)-GAz(Boc)-Thr(.sup.tBu)-Gly-OH:

(195) ##STR00115##

(196) Fmoc-GAz(Boc)-Thr(.sup.tBu)-OCumyl (420 mg, 0.60 mmol, 4.0 equiv) was stirred at room temperature in 2% TFA in CH.sub.2Cl.sub.2 (12 mL) until complete deprotection of the cumyl ester was observed by ESI-MS. The solvent was removed under reduced pressure and the resulting residue was repeatedly dissolved in CH.sub.2Cl.sub.2 (3×20 mL) and concentrated under reduced pressure. The crude Fmoc-GAz(Boc)-Thr(.sup.tBu)-OH was used for coupling without further purification. H-Gly-2-chlorotrityl resin (resin loading 0.52 mmol/g, 288 mg, 0.15 mmol, 1.0 equiv) was placed in a 10 mL reaction vessel and the resin was pre-swollen in DMF (2.0 mL) for 30 min. Fmoc-GAz(Boc)-Thr(.sup.tBu)-OH was dissolved in DMF (3.0 mL). HATU (228 mg, 0.6 mmol, 4.0 equiv) and DIPEA (209 μL, 1.2 mmol, 8.0 equiv) were added and this solution was added directly to the resin. The coupling reaction was allowed to proceed for 2 h at room temperature under agitation. The resin was filtered, washed with DMF (5×2.0 mL) and in case of a positive TNBS test, the coupling step was repeated. The Fmoc group was removed with 20% piperidine in DMF (4.0 mL) for 20 min at room temperature. After washing the resin with DMF (5×2.0 mL), Fmoc-Glu(tBu)-OH (319 mg, 0.75 mmol, 5.0 equiv) was coupled with HATU (285 mg, 0.75 mmol, 5.0 equiv), DIPEA (261 μL, 1.50 mmol, 10 equiv) in DMF (3.0 mL) for 1 h at room temperature. The resin was washed with DMF (5×2.0 mL) before the Fmoc group was removed and coupled with Fmoc-Phe-OH (290 mg, 0.75 mmol, 5.0 equiv.) followed by Fmoc deprotection as described before. The pentapeptide was then cleaved from the resin with TFE in CH.sub.2Cl.sub.2 (1:4, 3.0 mL) for 1 h at room temperature. This was repeated twice and the combined cleavage solutions were evaporated to dryness under reduced pressure to reveal the title compound as a glassy white solid (74.3 mg, 0.10 mmol, 68%) which required no further purification; mp 118-120° C.; .sup.1H NMR (500 MHz, MeOD) δ 7.42-7.26 (m, 5H, ArH), 4.31 (dd, J=8.6, 5.5 Hz, 1H, CHα-Glu), 4.19 (t, J=7.4 Hz, 1H, CHα-Phe), 3.98-3.57 (m, 8H, CHα-Thr, 2×NCH.sub.2 GAz, CH.sub.2 Gly, CHH GAz), 3.20 (dd, J=13.6, 7.4 Hz, 1H, CHHβ-Phe), 3.12-2.96 (m, 3H, CHHβ-Phe, CHH GAz, CHβ-Thr), 2.34-2.15 (m, 2H, CH.sub.2γ-Glu), 2.11-2.04 (m, 1H, CHHβ-Glu), 1.99-1.87 (m, 1H, CHHβ-Glu), 1.48-1.43 (m, 18H, CH.sub.3 Boc, CH.sub.3 CO.sub.2 .sup.tBu), 1.25-1.17 (m, 12H, CH.sub.3 .sup.tBu, CH.sub.3γ-Thr); .sup.13C NMR (126 MHz, MeOD) δ 175.9 (C═O), 175.9 (C═O), 174.0 (C═O), 173.7 (C═O), 170.1 (C═O), 158.3 (C═O Boc), 136.0 (C), 130.6 (CH), 130.1 (CH), 128.7 (CH), 81.7 (C, tBu), 81.2 (C, tBu), 75.4 (C, Boc), 69.9 (CH, α-Thr), 64.3 (CH, 3-Thr), 57.4 (2×NCH.sub.2), 56.0 (CH, α-Phe), 55.0 (CH, α-Glu), 45.7 (CH.sub.2, Gly), 44.1 (CH.sub.2, GAz), 38.7 (CH.sub.2, β-Phe), 32.6 (CH.sub.2, γ-Glu), 29.0 (CH.sub.3, CO.sub.2 .sup.tBu), 28.6 (CH.sub.3, Boc), 28.4 (CH.sub.3, tBu), 28.1 (CH.sub.2, 3-Glu), 21.4 (CH.sub.3, γ-Thr); ν.sub.max (neat)=2971, 1653, 1391, 1151 cm.sup.−1; MS (ESI.sup.+) m/z 749 [M+H].sup.+, 771 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.37H.sub.61N.sub.6O.sub.10 749.4444 [M+Na].sup.+. found 749.4438.

(197) Preparation of Cyclo-(Phe-Glu(.sup.tBu)-GAz(Boc)-Thr(.sup.tBu)-Gly):

(198) ##STR00116##

(199) To a solution of H-Phe-Glu(.sup.tBu)-GAz(Boc)-Thr(.sup.tBu)-Gly-OH (60.6 mg, 81 μmol, 1.0 equiv) in DMF (16.2 mL, 0.005 M) was added DEPBT (48.4 mg, 0.16 mmol, 2.0 equiv) and DIPEA (28 μL, 0.16 mmol, 2.0 equiv) and the reaction mixture was stirred for 68 h at room temperature. The solvent was removed under reduced pressure at 60° C. over 30 min, and the residue was dried in vacuo. The residue was purified by column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 49:1.fwdarw.19:1) to give the cyclic hexapeptide as a white glassy solid (43.2 mg, 59 μmol, 73%). R.sub.f (CH.sub.2Cl.sub.2/MeOH 19:1) 0.29; mp 125-127° C.; .sup.1H NMR (500 MHz, MeOD) δ 7.36-7.20 (m, 5H, ArH), 4.49 (dd, J=8.9, 5.2 Hz, 1H, CHα-Glu), 4.45 (dd, J=10.0, 3.9 Hz, 1H, CHα-Phe), 4.00-3.90 (m, 2H, CHHGly, NCHH GAz), 3.83-3.74 (m, 3H, NCHH GAz, CHH GAz, CHα-Thr), 3.66 (d, J=8.3 Hz, 1H, NCHHGAz), 3.60 (d, J=8.9 Hz, 1H, NCHH GAz), 3.52 (d, J=14.6 Hz, 1H, CHHGly), 3.29 (dd, J=14.4, 4.1 Hz, 1H, CHHβ-Phe), 3.15 (d, J=4.3 Hz, 1H, CHβ-Thr), 3.02-2.94 (m, 2H, CHH GAz, CHHβ-Phe), 2.34 (t, J=7.5 Hz, 2H, CH.sub.2γ-Glu), 2.25-2.14 (m, 1H, CHHβ-Glu), 2.00-1.91 (m, 1H, CHHβ-Glu), 1.51-1.44 (m, 18H, CH.sub.3 Boc, CH.sub.3 CO.sub.2 .sup.tBu), 1.23 (s, 9H, CH.sub.3 .sup.tBu), 1.10 (d, J=6.2 Hz, 3H, CH.sub.3γ-Thr); .sup.13C NMR (126 MHz, MeOD) δ 177.0 (C═O), 174.1 (C═O), 173.82 (C═O), 173.79 (C═O), 158.3 (C═O Boc), 138.4 (C), 130.0 (CH), 129.7 (CH), 128.0 (CH), 81.8 (C, .sup.tBu), 81.1 (C, .sup.tBu), 75.4 (C, Boc), 71.2 (CH, α-Thr), 62.5 (CH, β-Thr), 58.5 (2×NCH.sub.2), 57.1 (CH, α-Phe), 54.0 (CH, α-Glu), 46.3 (CH.sub.2 GAz), 44.8 (CH.sub.2 Gly), 37.7 (CH.sub.2, β-Phe), 32.8 (CH.sub.2, γ-Glu), 28.64 (CH.sub.3, CO.sub.2 .sup.tBu), 28.58 (CH.sub.3, Boc), 28.4 (CH.sub.3, .sup.tBu), 28.2 (CH.sub.2, β-Glu), 19.1 (CH.sub.3, γ-Thr). Note: One C═O overlapping; ν.sub.max (neat)=2974, 1649, 1532, 1366, 1152 cm.sup.−1; MS (ESI.sup.+) m/z 731 [M+H].sup.+, 753 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.37H.sub.58N.sub.6NaO.sub.9 753.4157 [M+Na].sup.+. found 753.4162.

(200) Preparation of Cyclo-(Phe-Glu-GAz-Thr-Gly):

(201) To a solution of Cyclo-(Phe-Glu(.sup.tBu)-GAz(Boc)-Thr(.sup.tBu)-Gly) (29 mg, 40 μmol, 1.0 equiv) in CH.sub.2Cl.sub.2 (1 mL) was added TFA (1 mL). The solution was stirred for 1 h at room temperature. The solvent was removed in vacuo to reveal the title compound as a white glassy solid as the TFA salt in quantitative yield, which required no further purification. .sup.1H and .sup.19F NMR using TFE as an internal standard confirmed 1 eq. TFA salt; mp 81-83° C.; .sup.1H NMR (500 MHz, MeOD) δ 7.37-7.29 (m, 5H, ArH), 4.50 (dd, J=9.7, 4.3 Hz, 1H, CHα-Glu), 4.39 (dd, J=10.0, 4.4 Hz, 1H, CHα-Phe), 4.26 (d, J=10.9 Hz, 1H, NCHH GAz), 4.03 (d, J=14.9 Hz, 1H, CHH Gly), 3.91 (d, J=11.0 Hz, 1H, NCHH GAz), 3.78 (d, J=10.3 Hz, 1H, NCHH GAz), 3.74-3.63 (m, 3H, CHH GAz, NCHH GAz, CHα-Thr), 3.54 (d, J=14.9 Hz, 1H, CHH Gly), 3.25 (dd, J=14.4, 4.4 Hz, 1H, CHHβ-Phe), 3.10-2.98 (m, 3H, CHH GAz, CHHβ-Phe, CHβ-Thr), 2.43-2.26 (m, 3H, CH.sub.2γ-Glu, CHHβ-Glu), 2.05-1.92 (m, 1H, CHHβ-Glu), 1.20 (d, J=6.3 Hz, 3H, CH.sub.3γ-Thr); .sup.13C NMR (126 MHz, MeOD) δ 176.5 (C═O), 176.4 (C═O), 175.1 (C═O), 174.4 (C═O), 173.9 (C═O), 138.1 (C), 130.0 (CH), 129.7 (CH), 128.1 (CH), 70.4 (CH, α-Thr), 63.3 (CH, β-Thr), 60.3 (C, Az), 60.0 (NCH.sub.2), 59.1 (CH, α-Phe), 53.5 (NCH.sub.2), 53.5 (CH, α-Glu), 45.9 (CH.sub.2 GAz), 44.4 (CH.sub.2 Gly), 37.4 (CH.sub.2, β-Phe), 31.3 (CH.sub.2, γ-Glu), 27.8 (CH.sub.2, β-Glu), 19.2 (CH.sub.3, γ-Thr); ν.sub.max (neat)=3273, 3033, 2922, 1649, 1538, 1177 cm.sup.−1; MS (ESI.sup.+) m/z 519 [M+H].sup.+, 541 [M+Na].sup.+; HRMS (ESI.sup.+) calcd. for C.sub.24H.sub.35N.sub.6O.sub.7 519.2562 [M+H].sup.+. found 519.2555.

Example 13—Solution Phase Synthesis of Other Azetidine-Containing Peptide Macrocycles

(202) The following protected cyclic peptides were prepared by solution phase peptide synthesis using methods analogous to those in Examples 10 and 11 (the figures below the structures give the yield). Deprotection using 50% TFA/DCM for 1 hour provided the corresponding deprotected analogues.

(203) ##STR00117##

Example 14—Solid Phase Synthesis of Other Azetidine-Containing Macrocycles

(204) The following cyclic peptides were prepared by SPPS using methods analogous to that in Example 12:

(205) ##STR00118## ##STR00119##
LP=yield of linear precursor from resin
CP=yield of cyclic peptide after cyclisation

Example 15—Relative Stability of Cyclic Peptides Including the Oxetane and Azetidine Turn-Inducing Elements

(206) An experiment was conducted to determine the relative stability of the following oxetane and azetidine-containing compounds in acid (TFA):

(207) ##STR00120##

(208) Each compound was added to 70% TFA with stirring. After 24 hours the resulting products were analysed by .sup.1H NMR and LC-MS.

(209) The LC-MS traces are provided in FIG. 10. The ring-opened azetidine product is visible by LC-MS, but is not present in a high quantity and was not observed in .sup.1H NMR. This is in contrast with the ring-opened oxetane which was visible by LC-MS and clearly present in a higher concentration. It was also observed in .sup.1H NMR.

(210) This demonstrates that the macrocycle containing the azetidine ring is more stable than that containing the oxetane ring under the forcing conditions used.

Example 16—Comparison of Yield of Macrocyclisation for Oxetane and Azetidine-Containing Peptides

(211) TABLE-US-00006 embedded image Concentration Yield Substrate.sup.a (M) (%).sup.b 1 0.001 20.sup.c 2 0.001 54 3 0.001 79 3.sup.d 0.005 82 .sup.aReactions were run at 0.1 mmol scale .sup.bIsolated yield of macrocycle after column chromatography .sup.cProduct isolated as the dimeric octapeptide .sup.dReaction carried out on 0.44 mmol scale

(212) The macrocyclisation yield is lowest for the non-modified compound 1. Higher yields are obtained for both the oxetane and azetidine-containing compounds 2 and 3. In this example, the highest yield of the macrocycle is obtained for the peptide containing the azetidine turn-inducing element.

SUMMARY

(213) We have established that the introduction of a carbonyl bioisosteric turn-inducing residue into the peptide backbone offers a new way to improve difficult peptide macrocyclizations. The substrates are easily made and the cyclization works across a range of ring sizes. It is beneficial for head-to-tail, head-to-side-chain, side-chain-to-tail, and side-chain-to-side-chain cyclizations, and tolerates variation in location of the turn-inducing element with respect to the bond being formed. The products are compatible with the harsh acidic conditions needed to deprotect amino acid side chains, and CBMCPs have clear potential as bioisosteres for conventional cyclic peptides.

REFERENCES

(214) 1. Zorzi, A.; Deyle, K.; Heinis, C. Curr. Opin. Chem. Biol. 2017, 38, 24-29. 2. (a) Nielsen, D. S.; Shepherd, N. E.; Xu, W.; Lucke, A. J.; Stoermer, M. J.; Fairlie, D. P. Chem. Rev. 2017, 117, 8094-8128. (b) Bockus, A. T.; McEwen, C. M.; Lokey, R. S. Curr. Top. Med. Chem. 2013, 13, 821-836. 3. (a) Dougherty, P. G.; Qian, Z.; Pei, D. Biochem. J. 2017, 474, 1109-1125. (b) Cardote, T. A. F.; Ciulli, A. ChemMedChem 2016, 11, 787-794. 4. For reviews, see: (a) Passioura, T.; Suga, H. Chem. Commun. 2017, 53, 1931. (b) De Leon Rodriguez, L. M.; Weidkamp, A. J.; Brimble, M. A. Org. Biomol. Chem. 2015, 13, 6906-6921. (c) Mar-tí-Centelles, V.; Pandey, M. D.; Burguete, M. I.; Luis, S. V. Chem. Rev. 2015, 115, 8736-8834. (d) Yudin, A. K. Chem. Sci. 2015, 6, 30-49. (e) White, C. J.; Yudin, A. K. Nature Chem. 2011, 3, 509-524. 5. (a) Hubert, J. G.; Stepek, I. A.; Noda, H.; Bode, J. W. Chem. Sci. 2018, 9, 2159-2167. (b) Malins, L. R.; deGruyter, J. N.; Rob-bins, K. J.; Scola, P. M.; Eastgate, M. D.; Ghadiri, M. R.; Baran, P. S. J. Am. Chem. Soc. 2017, 139, 5233-5241. (c) Johnson, A. M.; An-slyn, E. V. Org. Lett. 2017, 19, 1654-1657. (d) Le, D. N.; Riedel, J.; Kozlyuk, N.; Martin, R. W.; Dong, V. M. Org. Lett. 2017, 19, 114-117. (e) McCarver, S. J.; Qiao, J. X.; Carpenter, J.; Borzilleri, R. M.; Poss, M. A.; Eastgate, M. D.; Miller, M. M.; MacMillan, D. W. C. Angew. Chem. Int. Ed. 2017, 56, 728-732. (f) Puentes, A. R.; More-jón, M. C.; Rivera, D. G.; Wessjohann, L. A. Org. Lett. 2017, 19, 4022-4025. (g) Tang, J.; He, Y.; Chen, H.; Sheng, W.; Wang, H. Chem. Sci. 2017, 8, 4565-4570. (h) Rojas, A. J.; Zhang, C.; Vinogradova, E. V.; Buchwald, N. H.; Reilly, J.; Pentelute, B. L.; Buchwald, S. L. Chem. Sci. 2017, 8, 4257-4563. (i) Stephens, T. C.; Lodi, M.; Steer, A. M.; Lin, Y.; Gill, M. T.; Unsworth, W. P. Chem. Eur. J. 2017, 23, 13314-13318. (j) Frost, J. R.; Scully, C. C. G.; Yudin, A. K. Nature Chem. 2016, 8, 1105-1111. 6. (a) Skropeta, D.; Jolliffe, K. A.; Turner, P. J. Org. Chem. 2004, 69, 8804-8809. (b) Fairweather, K. A.; Sayyadi, N.; Luck, I. J.; Clegg, J. K.; Jolliffe, K. A. Org. Lett. 2010, 12, 3136-3139. 7. Powell, N. H.; Clarkson, G. J.; Notman, R.; Raubo, P.; Martin, N. G.; Shipman, M. Chem. Commun. 2014, 50, 8797-8800. 8. (a) Bull, J. A.; Croft, R. A.; Davis, O. A.; Doran, R.; Morgan, K. F. Chem. Rev. 2016, 116, 12150-12233. (b) Wuitschik, G.; Car-reira, E. M.; Wagner, B.; Fischer, H.; Parrilla, I.; Schuler, F.; Roger-Evans, M.; Müller, K. J. Med. Chem. 2010, 53, 3227-3246. 9. (a) Boutureira, O.; Martínez-Sáez, N.; Brindle, K. M.; Neves, A. A.; Corzana, F.; Bernardes, G. J. L. Chem. Eur. J. 2017, 23, 6483-6489. (b) Martínez-Sáez, N.; Sun, S.; Oldrini, D.; Sormanni, P.; Boutureira, O.; Carboni, F.; Compañón, I.; Deery, M. J.; Vendruscolo, M.; Corzana, F.; Adamo, R.; Bernardes, G. J. L. An-gew. Chem. Int. Ed. 2017, 56, 14963-14967. 10. Möller, G. P.; Müller, S.; Wolfstädter, B. T.; Wolfrum, S.; Schepmann, D.; Wünsch, B.; Carreira, E. M. Org. Lett. 2017, 19, 2510-2513. 11. (a) Beadle, J. D.; Knuhtsen, A.; Hoose, A.; Raubo, P.; Jamieson, A. G.; Shipman, M. Org. Lett. 2017, 19, 3303-3306. (b) McLaughlin, M.; Yazaki, R.; Fessard, T. C.; Carreira, E. M. Org. Lett. 2014, 16, 4070-4073. 12. Tang, Y.; Xie, H.; Tian, G.; Ye, Y. J. Peptide Res. 2002, 60, 95-103. 13. Wong, M. S. Y.; Taleski, D.; Jolliffe, K. A. Aust. J. Chem. 2015, 68, 627-634. 14. Arap, W.; Pasqualini, R.; Ruoslahti, E. Science 1998, 279, 377-380. 15. Graziadio, A.; Zanda, M.; Frau, S.; Fleming, I. N.; Musolino, M.; Dall'Angelo, S.; Baldassarre, M.; Piras, M. Bioconjugate Chem. 2016, 27, 1332-1340.

Macrocyclization of peptidomimetics

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G01N33/6803

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Abstract

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