Patent Application
20080039376
This Example describes the isolation of Compounds 1 to 10.
Water was Milli-Q filtered, while all other solvents used were Omnisolv. A YMC basic C18 5 uM, 21.2 mm×150 mm, column and Hypersil BDS C18 5 uM, 21.2×150 mm column were used for preparative HPLC. NMR spectra were recorded on a Varian Inova 600 or 500 MHz NMR spectrometer. Samples were dissolved in d6-DMSO and chemical shifts were calculated relative to the solvent peak (DMSO 1H □ 2.49 and 13C 39.5 ppm). Mass spectra were measured on a Fisons VG Platform II, using positive electrospray ionisation mode. The elution solvent was a mixture acetonitrile/water 50% at 0.1 ml/min.
The sponge (Melophlus sp.) was collected by SCUBA diving off Ribbon Reef No. 5, Australia and a voucher sample (G319104) is lodged at the Queensland Museum, Brisbane, Australia.
A freeze dried ground sample of the sponge Melophlus sp (128 g) collected from Ribbon Reef No. 5 in far North Queensland, Australia was exhaustively extracted with methanol (2 l). The solvent was evaporated to yield a dark brown residue (28 g). The residue was redissolved in a mixture of EtOAc (20 mL) and water (60 mL) and separated by droplet countercurrent chromatography with water as the stationary phase and a gradient from EtOAc to butanol as the mobile phase at 5 mL/min. Two minute fractions were collected and every second fraction analysed by electrospray mass spectrometry. Like fractions were combined yielding 5 fractions. Fraction 2 (320 mg) was separated by centrifugal partition chromatography (Sanki CPC, ascending mode) using a trisolvent mixture CHCl3/MeOH/H2O (7:13:8) with the lower phase as stationary phase. A flow rate of 2 mL/min was used and two minute fractions were collected for 360 min. Every second fraction was analyzed by positive electrospray mass spectrometry and like fractions combined. Fractions 91-101 were combined to yield impure Compound 2 (10.8 mg) and fractions 107-120 were combined to yield impure Compound 1 (12.4 mg). The impure peptide fractions of Compounds 1 and 2 were each partitioned between aqueous TFA (1%) and hexane. The aqueous layers from each partition contained pure Compound 2 (9.5 mg) and Compound 1 (11.5 mg). Fractions 1, 3 and 4 from the original DCCC separation were combined with the remaining fractions from the CPC separation and preabsorbed onto C18 (3 g). The preabsorbed fractions were further separated by C18 HPLC hypersil BDS C18 (5 uM, 20 mm×150 mm) using a water/methanol gradient from water containing 1% TFA to methanol containing 1% TFA at 10 mL/min over 60 min. One minute fractions were collected and all fractions analyzed by electrospray mass spectrometry. Like fractions were combined. Fractions 51-58 contained peptides related to Compounds 1 and 2, and were combined (fraction A; 65 mg). This peptide fraction A was further purified by RP HPLC on YMC basic C18 5 uM, 20 mm×150 mm elution with 65% water (containing 1% TFA) and 35% MeCN (containing 1% TFA) at a flow rate of 10 mL/min. Twelve second fractions were collected for 36 minutes. Fractions 58-60 was pure Compound 2 (11 mg), fractions 67-69 was pure Compound 1 (11 mg), fractions 70-72 was pure Compound 3 (2 mg), fractions 73-77 was pure Compound 7 (11.2 mg), fractions 79-82 was pure Compound 4 (7.29 mg), fractions 91-96 was pure Compound 8 (8.75 mg), fractions 101-106 was pure Compound 9 (6.02 mg), fractions 118-125 was pure Compound 5 (2.08 mg), fractions 128-138 was pure Compound 10 (5.73 mg) and fractions 140-150 was pure Compound 6 (5.94 mg).
After extensive studies including 1H, gHSQC, gHMBC, and gCOSY experiments, Compounds 1-10 were identified as cyclic peptides. The absolute stereochemistry of Compound 1 was confirmed by single crystal X-ray diffraction analysis.
1H (600 MHz), 13C (125 MHz), HMBC and COSY
13C (mult)a
1H (mult, J Hz)
2,3JCH correlations
aChemical shifts determined from 2D heteronuclear experiments
1H (600 MHz), 13C (125 MHz), HMBC and COSY
13C (mult)a
1H (mult, J Hz)
2,3JCH correlations
aChemical shifts determined from 2D heteronuclear experiments
1H (600 MHz), 13C (125 MHz), HMBC and COSY
13C (mult)a
1H (mult, J Hz)
2,3JCH correlations
aChemical shifts determined from 2D heteronuclear experiments
1H (600 MHz), 13C (125 MHz), and COSY
13C (mult)a
1H (mult, J Hz)
aChemical shifts determined from 2D heteronuclear experiments
1H (600 MHz), 13C (125 MHz), HMBC and COSY
13C (mult)a
1H (mult, J Hz)
2,3JCH correlations
aChemical shifts determined from 2D heteronuclear experiments
1H (600 MHz), 13C (125 MHz), HMBC and COSY
13C (mult)a
1H (mult, J Hz)
2,3JCH correlations
aChemical shifts determined from 2D heteronuclear experiments
1H (600 MHz), 13C (125 MHz), HMBC and COSY
13C (mult)a
1H (mult, J Hz)
2,3JCH correlations
aChemical shifts determined from 2D heteronuclear experiments
1H (600 MHz), 13C (125 MHz) and COSY
13C (mult)a
1H (mult, J Hz)
aChemical shifts determined from 2D heteronuclear experiments
1H (600 MHz), 13C (125 MHz), HMBC and COSY
13C (mult)a
1H (mult, J Hz)
2,3HCH correlations
aChemical shifts determined from 2D heteronuclear experiments
1H (600 MHz), 13C (125 MHz), HMBC and COSY
13C (mult)a
1H (mult, J Hz)
2,3JCH correlations
aChemical shifts determined from 2D heteronuclear experiments
This Example describes the isolation of Compound 11.
Water was Milli-Q filtered, while all other solvents used were Omnisolv. A Hypersil BDS basic C18 5 uM, 21.2 mm×150 mm, column were used for preparative HPLC. NMR spectra were recorded on a Varian Inova 600 or 500 MHz NMR spectrometer. Samples were dissolved in d6-DMSO and chemical shifts were calculated relative to the solvent peak (DMSO 1H □ 2.50 and 13C 39.5 ppm). Mass spectra were measured on a Fisons VG Platform II, using positive electrospray ionisation mode. The elution solvent was a mixture acetonitrile/water 50% at 0.1 ml/min.
Six sponge samples of Candidaspongia flabellata were collected by SCUBA diving at Outer Gneering, Sunshine Coast, Old Reef, Fairfax Is and Chauvel Reef, Queensland, Australia and voucher samples (G315106, G314580, G314025, G315402, G318260, G317513) were lodged at the Queensland Museum, Brisbane, Australia.
The freeze-dried sponge materials (529 g) were ground and exhaustively extracted with methanol to afford six methanol extracts. The methanol crude extracts underwent a series of partitions: MeOH/n-hexane, H2O:MeOH (4:1)/DCM, H2O:MeOH (4:1)/EtOAc. Bioactivity was spread in the H2O:MeOH (4:1) and EtOAc layers. The H2O:MeOH (4:1) and EtOAc layers were combined for all six biota and then partitioned with H2O/butanol. The activity was in the butanol layer (900 mg), which then underwent countercurrent chromatography {H2O/MeOH/EtOAc (4:1:5)}, upper layer mobile phase. The very early eluting fractions, 13-24, were combined (325 mg) and partitioned n-hexane:EtOAc:MeOH:H2O (1:1:1:1). The bioactive aqueous layer (150 mg) was then chromatographed further by counter current chromatography {(CHCl3:MeOH:H2O (7:13:8)}, lower layer mobile phase. The early eluting active fractions, 25-32, were combined to give 85 mg of material. This underwent a final purification step by HPLC (Hypersil BDS C18) using a 30 min H2O/MeCN gradient from H2O (containing 1% TFA) to MeCN (containing 1% TFA). This yielded 0.4 mg of Compound 11 eluting after 18.2 mins.
Compound 11 was also identified as a cyclic peptide after detailed studies, including 1H, 13C, gHSQC, gHMBC, and gCOSY experiments.
1H (600 MHz), 13C (125 MHz), HMBC and COSY
13C (mult)a
1H (mult, J Hz)
2,3JCH correlations
a1.42
a1.44
a3.23
aChemical shift estimated from 2D NMR experiments
This Example describes the synthesis of Compound 12.
High resolution mass spectra were recorded on a Micromass LCT mass spectrometer equipped with an electrospray interface (LC-HRMS). 1H NMR measurements were performed on Varian UNITY plus 400, 500 and 600 spectrometers, operating at 1H frequencies of 400, 500 and 600 MHz respectively. NMR spectra were recorded in d6-DMSO with chemical shifts given in ppm with the solvent as internal standard.
Compound 12 was prepared according to a literature procedure (Marsh and Bradley, J. Org. Chem., 1997, 62, 6199-6203) with the following modifications: Fmoc-L-Arg-Nω,ω′-(Boc)2-OH was first coupled to the resin/linker. After removal of the Fmoc group, the free amine was coupled with Nα-(4-nitrophenyloxycarbonyl)-Nε-(9-fluorenylmethoxycarbonyl)-D-lysine allyl ester. Fmoc peptide synthesis continued on the side chain of the lysine residue using Fmoc-L-Ala followed by Fmoc-L-N-MeAla, Fmoc-L-Leu and Fmoc-L-Ala. Allyl ester and Fmoc removal was followed by cyclization and finally cleavage from the resin/linker. Purification of the residue by reversed-phase HPLC (Ace C8 column, linear gradient 5%→95% MeCN in 0.1 M aqueous NH4OAc) gave Compound 12 (1.8 mg, 1.3%).
1H NMR (500 MHz, d6-DMSO): □ 9.2 (broad s, 1H), 8.66 (d, 1H), 8.52 (d, 1H), 7.4-8.0 (broad signal, 4H), 7.47 (dd, 1H), 7.10 (d, 1H), 6.56 (d, 1H), 6.08 (d, 1H), 4.77-4.83 (m, 1H), 4.70-4.77 (m, 1H), 4.23 (qd, 1H), 4.07 (qd, 1H), 3.88-3.98 (m, 1H), 3.65-3.75 (m, 1H), 3.47-3.52 (m, 1H), 3.03 (broad t, 2H), 2.71-2.78 (m, 1H), 2.52 (s, 3H), 1.78-1.84 (m, 1H), 1.68-1.79 (m, 1H), 1.30-1.65 (m, 12H), 1.15-1.23 (m, 2H), 1.18 (two d, 6H), 0.94 (d, 3H), 0.93 (d, 3H), 0.89 (d, 3H), 0.88 (d, 3H).
HRMS (ESI) calculated for C32H59N10O8 711.4517 (M+H)+, found 711.4525.
This Example describes the synthesis of Compounds 1 and 13 to 16.
a) Synthesis of Intermediate A
TFA (2 mL) was added to Boc-D-Lys(Fmoc)-OAllyl (2.86 g, 5.6 mmol) and left to stand for 5 min. The TFA was then removed by a stream of dry nitrogen to afford H-D-Lys(Fmoc)-OAllyl which was dried on a high vacuum line for 2 h to remove all traces of TFA. 2-Chlorotrityl resin (1 g, 1.4 mmol) was pre-swelled in DCM (10 mL) for 1 h. The resin was drained and a solution of H-D-Lys(Fmoc)-OAllyl (2.30 g, 5.64 mmol) and DIEA (729 mg, 982 μL, 5.64 mmol) in DCM (10 mL) was added and the reaction mixture shaken for 1 h. Further DIEA (1.46 g, 1.95 mL, 11.3 mmol) was added to the resin and the reaction mixture shaken for a further 1 h. Methanol (1 mL) was added to end-cap any unreacted resin and the reaction mixture shaken for a further 1 h. The resin was filtered and washed with DMF (2×5 mL), DCM (2×5 mL) and DMF (2×5 mL). The resin was subjected to Fmoc-solid phase peptide synthesis (SPPS) using the following conditions:
A solution of Pd(PPh3)4 (1.62 g, 1.4 mmol) and dimedone (1.96 g, 14 mmol) in THF:DCM (1:1, 50 mL) was sparged with nitrogen gas for 10 min., added to the resin and the mixture shaken for 16 h. The reaction mixture was filtered and washed with DCM (3×5 mL), DMF (3×5 mL) a solution of 0.5% DIEA and 0.5% diethyldithiocarbamic acid sodium salt in DMF (3×5 mL) and DMF (3×5mL). The resin was treated with 20% piperidine in DMF (2×10 mL) for 2 min. followed by washing with DMF (4×5 mL), DCM (4×5 mL), 10% pyridinium hydrochloride in DCM:DMF (1:1, 4×5 mL) and DMF (4×5 mL). A solution of PyBroP (718 mg, 1.54 mmol) and DIEA (1 mL, 5.74 mmol) in DCM:DMF (1:1, 10 mL) was added to the resin and the mixture shaken for 3 h after which a ninhydrin test was negative. The cyclic peptide was cleaved from the resin by treatment with 50% TFA in DCM (20 mL) for 1 h. The resin was filtered, washed with TFA (2×5 mL) and DCM (2×5 mL), concentrated to dryness, re-dissolved in MeCN:H2O (0.1% TFA) and lyophilised to afford crude Intermediate A (435 mg, 50% based on the 2-chlorotrityl resin). Purification by RPHPLC (95:5 H2O (1% TFA):MeCN (1% TFA) to 2:3 H2O 1% TFA):MeCN (1% TFA)) over 60 min afforded Intermediate A (0.417 g, 3.6%).
b) Allyl-N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-N5-{imino[(2, 2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5-yl)amino]methyl}ornithinate
N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-N5-{imino[(2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5-yl)amino]methyl}ornithine (1.0 g, 1.54 mmol) was dissolved in DMF (5 mL). Caesium carbonate (377 mg, 1.16 mmol) was added and the reaction mixture stirred for 1 h. Allyl bromide (0.913 mL, 10.8 mmol) was then added and stirring was continued for a further 1 h resulting in a milky white solution. Water (25 mL) was added and the reaction mixture acidified with 2M KHSO4. DCM (50 mL) was added and the phases separated. The aqueous phase was washed with DCM (2×50 mL) and the combined organics washed with brine (50 mL), dried (MgSO4), filtered and concentrated to dryness to afford allyl-N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-N5-{imino[(2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5-yl)amino]methyl}ornithinate as colourless foam (857 mg, 81%).
1HNMR (CDCl3, 500 MHz): □ 1.43 (s, 6H), 1.59 (m, 2H), 1.73 (m, 1H), 1.86 (m, 1H), 2.09 (s, 3H), 2.52 (s, 3H), 2.61 (s, 3H), 2.91 (s, 2H), 3.22 (m, 2H), 4.17 (t, J 7 Hz, 1H), 4.32 (m, 1H), 4.37 (m, 1H), 4.59 (br d, J 4.5 Hz, 2H), 5.21 (d, J 10.5 Hz, 1H), 5.30 (d, J 17 Hz, 1H), 5.83 (m, 1H), 5.88 (m, 1H), 6.26 (br s, 1H), 6.35 (br s, 2H), 7.26 (t, J 7.5 Hz, 2H), 7.37 (t, J 7.5 Hz, 2H), 7.57 (m, 2H), 7.74 (d, J 7.5 Hz, 2H).
13CNMR (CDCl3, 125 MHz): □ 12.68, 18.22, 19.54, 25.69, 28.78, 29.93, 40.96, 43.43, 47.36, 53.72, 54.10, 66.23, 67.39, 86.63, 117.78, 119.12, 120.19, 124.93, 125.40, 127.34, 127.96, 131.79, 132.47, 133.17, 138.54, 141.49, 143.97, 144.08, 156.63, 159.03, 171.42). MS: (positive ESI) [M+H]+ m/z 689.
c) Allyl-N5-[[(4-ethyl-2,2,6,7-tetramethyl-2,3-dihydro-1-benzofuran-5-yl)amino](imino)methyl]-N2-[(4-nitrophenoxycarbonyl]ornithinate
Allyl-N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-N5-{imino[(2,2,4,6,7-pentamethyl-2,3-dihydro-1-benzofuran-5-yl)amino]methyl}ornithinate (800 mg, 1.16 mmol) was dissolved in DMF (4 mL). Piperidine (1 mL) was added, and the reaction mixture was stirred at room temperature for 30 min and then concentrated. The resulting residue was dissolved in DCM (9 mL) and added to a suspension of 4-nitrophenylchloroformate (370 mg, 1.85 mmol) and pyridine (750 uL, 9.3 μmol) in DCM (6 mL) with cooling in an ice-salt bath. After stirring for 2.5 h, 1M KHSO4 (20 mL) was added, the organic layer separated and the aqueous phase extracted with DCM (4×20 mL). The combined organic extracts were dried (MgSO4), filtered, concentrated and the resulting residue purified by flash chromatography on silica gel (100% Hexane to 7:3 EtOAc:hexane) to afford allyl-N5-[[(4-ethyl-2,2,6,7-tetramethyl-2,3-dihydro-1-benzofuran-5-yl)amino](imino)methyl]-N2-[(4-nitrophenoxy)carbonyl]ornithinate (138 mg, 18%).
1HNMR (CDCl3, 500 MHz): □ 1.42 (s, 6H), 1.62 (m, 2H), 1.79 (m, 1H), 1.89 (m, 1H), 2.04 (s, 3H), 2.48 (s, 3H), 2.55 (s, 3H), 2.90 (s, 2H), 3.20 (m, 2H), 4.30 (m, 1H), 4.60 (br d, J 4.5 Hz, 2H), 5.22 (d, J 10.5 Hz, 1H), 5.29 (d, J 17 Hz, 1H), 5.86 (m, 1H), 6.25 (br s, 1H), 6.33 (br s, 1H), 6.50 (br d, J 6.5 Hz, 1H), 6.90 (d, J 7.5 Hz, 1H), 7.25 (d, J 8 Hz, 2H), 8.05 (d, J 7.5 Hz, 1H), 8.15 (d, J 8 Hz, 2H).
13CNMR (CDCl3, 125 MHz): □ 12.63, 18.16, 19.45, 25.74, 28.76, 29.44, 40.8, 43.41, 54.41, 66.39, 86.71, 115.99, 117.78, 119.21, 122.22, 124.97, 125.23, 126.22, 131.66, 132.40, 133.02, 138.43, 140.75, 144.97, 153.45, 156.06, 156.67, 159.04, 163.07, 163.80, 171.6. MS: (positive ESI) [M+H]+ m/z 632.
d) Compound 1
Intermediate A (49.9 mg, 0.08 mmol) was dissolved in DMF (8 mL). Allyl-N5-[[(4-ethyl-2,2,6,7-tetramethyl-2,3-dihydro-1-benzofuran-5-yl)amino](imino)methyl]-A2-[(4-nitrophenoxy)carbonyl]ornithinate (60.6 mg, 0.096 mmol) was added, followed by DIEA (17 uL, 0.096 mmol) and the reaction mixture stirred at room temperature for 16 h. The reaction mixture was concentrated to give the crude urea. A solution of palladium(tetrakis)triphenylphosphine (8 mg, 0.0072 mmol) and dimedone (25 mg, 0.18 mmol) in TBF:DCM (1:1, 5 mL) was sparged with dry nitrogen and then added via canula to the urea and stirred at room temperature overnight to afford the crude carboxylic acid. The carboxylic acid was dissolved in DCM (1 mL), and p-Cresol (340 μL) and TFA (250 μL) were added and the reaction mixture stirred at room temperature for 20 h to afford crude Compound 1. The reaction mixture was purified by reverse phase HPLC (YMC basic semi prep column, linear gradient 65% Water (1% TFA) 35% MeCN (1% TFA)→100% MeCN (1% TFA)) to afford Compound 1 (11.3 mg, 17%). NMR and MS data were found to be identical with an authentic sample.
The Intermediate of formula A was also prepared by the following route.
a) Synthesis of Intermediate C
2-Chlorotrityl resin (300 mg, 0.42 mmol) was pre-swelled in DCM (2 mL) for 1 h. The resin was drained and a solution of Boc-D-Lysine(Fmoc)-OH (394 mg, 0.84 mmol) and DIEA (0.586 mL, 3.36 mmol) in DCM (2 mL) was added and the reaction mixture shaken for 1 h. A further aliquot of DIEA (0.293 mL, 1.68 mmol) was then added and the resin shaken for another 1 hr. Methanol (1 mL) was added to end-cap any unreacted resin and the reaction mixture shaken for a further 1 h. The resin was filtered and washed with DMF (2×5 mL), DCM (2×5 mL) and DMF (2×5 mL). The resin was then subjected to Fmoc-solid phase peptide synthesis (SPPS) using the following conditions:
Following Fmoc deprotection and thorough washing with DMF (4×5 ml), DCM (4×5 mL) and DMF (4×5 mL), the linear peptide was cleaved from resin with 2% TFA in DCM (150 mL) by rapid flow-wash into 250 mL of water. The DCM was removed in vacuo and the resulting solution frozen and freeze dried. The resulting gum was resuspended in 1:1 MeCN:H2O (100 mL), frozen and freeze-dried to afford crude Intermediate C (265 mg, 0.276 mmol, 65.9% based on the 2-chlorotrityl resin).
b) Synthesis of Intermediate A
Crude Intermediate C (0.401 g, 0.419 mmol) and DIEA (0.438 mL, 1.26 mmol) in DMF (208 mL) were added dropwise with stirring to a solution of PyBOP (1.09 g, 2.10 mmol) and DIEA (0.146 mL, 0.838 mmol) in DMF (208 mL). The resulting solution was stirred at room temperature for 18 h then concentrated to dryness and partitioned between EtOAc (100 mL) and water (100 mL). The organic phase was washed several times with water (3×100 mL), dried (MgSO4), filtered and concentrated to dryness. The crude product was treated with a solution of 90:9:1 (TFA:TIS[b1]:DCM) for 2 h, concentrated to dryness and purified using reverse phase HPLC (95:5 H2O (1% TFA):MeCN (1% TFA) to 3:2 H2O (1% TFA):MeCN (1% TFA) over 60 min to afford Intermediate A (0.167 g, 0.226 mmol, 53.9%).
Compound 13 was synthesised using a procedure similar to the procedure for Compound 1, starting from Intermediate A and N2-[(benzyloxy)carbonyl]-N5-(tert-butoxycarbonyl)ornithine. HRMS C39H61N9O8 822.4280 (M+H)+, found 822.4262.
Compound 14 was synthesised using a procedure similar to the procedure for Compound 1, starting from Intermediate A and tert-butyl N6-(tert-butoxycarbonyl)-L-lysinate.
1H NMR (500 MHz, CD3OD): □ 8.98 (d, 1H), 8.71 (d, 1H), 7.95 (dd, 1H), 7.79 (d, 1H), 7.64 (d, 1H), 7.31 (d, 1H), 7.08 (t, 1H), 7.01 (t, 1H), 6.78 (s, 1H), 5.00-4.88 (m, 2H), 4.78-4.70 (m, 1H), 4.36-4.23 (m, 2H), 4.19-4.13 (m, 1H), 3.88-3.77 (m, 1H), 3.55 (dd, 1H), 3.04-2.86 (m, 4H), 2.03-1.88 (m, 3H), 1.85 (s, 3H), 1.84-1.66 (m, 6H), 1.66-1.57 (m, 3H), 1.52 (d, 3H), 1.56-1.44 (m, 3H), 1.42-1.30 (m, 3H), 1.04 (two d, 6H), 0.95 (two d, 6H). HRMS (ESI) calculated for C40H64N9O8 798.4878 (M+H)+, found 798.4858.
Compound 15 was synthesised using a procedure similar to the procedure for Compound 1, starting from Intermediate A and 3-{6-[(tert-butoxycarbonyl)amino]pyridin-3-yl}alanine (WO 01/02364). HRMS C42H61N10O8 833.4674 (M+H)+, found 833.4678.
a) Synthesis of Intermediate B
Intermediate B was synthesised using a procedure similar to the procedure for Intermediate A.
b) Synthesis of Compound 16
Compound 16 was synthesised according to the procedure for Compound 1, starting from Intermediate B.
1H NMR (500 MHz, d6-DMSO): □ 12.70 (broad s 1H), 10.83 (s, 1H), 8.86 (d, 1H), 8.47 (d, 1H), 7.70-7.79 (m, 3H), 7.57 (t, 1H), 7.46 (d, 1H), 7.45 (dd, 1H), 7.35 (d, 1H), 7.28 (d, 1H), 7.02 (dd, 1H), 6.96 (dd, 1H), 6.81 (broad s, 1H), 6.47 (d, 1H), 6.46 (d, 1H), 4.82 (m, 1H), 4.74-4.75 (ddd, 1H), 4.43 (ddd, 1H), 4.22-4.24 (m, 1H), 4.13 (ddd, 1H), 4.02 (ddd, 1H), 3.78 (dd, 1H), 3.71 (dd, 1H), 3.60 (m, 1H), 3.35 (m, 1H), 3.11 (dt, 2H), 2.86-2.92 (m, 1H), 2.78-2.80 (m, 1H), 1.83 (s, 3H), 1.79-1.83 (m, 1H), 1.52-1.56 (m, 1H), 1.57-1.60 (m, 1H), 1.60-1.64 (m, 3H), 1.69-1.70 (m, 1H), 1.42-1.48 (m, 5H), 1.33-1.36 (m, 1H), 1.22-1.25 (m, 2H), 1.18-1.20 (m, 1H), 0.95 (d, 3H), 0.91 (d, 3H), 0.89 (d, 3H), 0.85 (d, 3H). HRMS C40H64N11O9 842.4888 (M+H)+, found 842.4885.
The Intermediate of formula B was also prepared by the following route.
2-Chlorotrityl resin (1 g, 1.4 mmol) was pre-swelled in DCM (5 mL) for 1 h. The resin was drained and a solution of Boc-D-Lysine(Fmoc)-OH (1.31 g, 2.8 mmol) and DIEA (1.45 g, 1.98 mL, 11.2 mmol) in DCM (4 mL) was added and the reaction mixture shaken for 2 h. Methanol (1 mL) was added to end-cap any unreacted resin and the reaction mixture shaken for a further 1 h. The resin was filtered and washed with DMF (2×5 mL), DCM (2×5 mL) and DMF (2×5 mL). The resin was then subjected to Fmoc-solid phase peptide synthesis (SPPS) using the following conditions:
Following Fmoc deprotection and thorough washing with DMF (4×5 ml), DCM (4×5 mL) and DMF (4×5 mL), the linear peptide was cleaved from resin with 2% TFA in DCM (400 mL) by rapid flow-wash into 500 mL of water. The DCM was removed in vacuo and the resulting solution frozen and freeze dried. The resulting gum was resuspended in 1:1 MeCN:H2O (100 mL), frozen and freeze-dried to afford a crude Intermediate D (994.6 mg, 0.88 mmol, 63% based on the 2-chlorotrityl resin).
Crude Intermediate D (905 mg, 0.88 mmol) and DIEA (0.304 mL, 1.74 mmol) were dissolved in DMF (440 mL) and added dropwise with stirring to a solution of PyBOP (2.13 g, 4.1 mmol) and DIEA (0.918 mL, 5.3 mmol) in DMF (440 mL). Once addition was complete the resulting solution was stirred at room temperature for 20 h then concentrated to dryness to afford an orange gum, which was purified using Sephadex LH-20 (MeOH) to give the protected cyclic peptide (551 mg, 70%). The protected crude cyclic peptide was then treated with a solution of 95:2.5:2.5 (TFA:TIS:DCM) for 20 h. The reaction mixture was concentrated to dryness and purified using reverse phase HPLC (95:5 H2O (1% TFA):MeCN (1% TFA) to 3:2 H2O (1% TFA):MeCN (1% TFA) over 60 min to afford Intermediate B (214 mg, 32% from Intermediate D).
The activities of certain Examples in the assay described in: Dirk Hendriks, Simon Sharpé and Marc van Sande, Clinical Chemistry, 31, 1936-1939 (1985), using a substrate concentration of 4 mM, are presented in Table I below.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0302853-7 | Oct 2003 | SE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/SE04/01568 | 10/28/2004 | WO | 00 | 3/6/2007 |
