Discovery of Subnanomolar Arginine-Glycine-Aspartate-Based Î±VÎ²3

SUPPORTING INFORMATION Discovery of Sub-Nanomolar Arginine-Glycine-Aspartate-Based V 3/ V 5

Integrin Binders Embedding 4-Aminoproline Residues

Franca Zanardi,*,‡ Paola Burreddu,† Gloria Rassu,† Luciana Auzzas,† Lucia Battistini,‡ Claudio Curti,‡ Andrea Sartori,‡ Giuseppe Nicastro,*,# Gloria Menchi,§ Nicoletta Cini,§,$ Anna Bottonocetti,$ Silvia Raspanti,$ and Giovanni Casiraghi*,‡ ‡

Dipartimento Farmaceutico, Università di Parma, Viale G. P. Usberti 27A, I-43100 Parma, Italy, †Istituto di Chimica Biomolecolare del CNR, Traversa La Crucca 3, I-07040 Li Punti, Sassari, Italy, #Centro Interdipartimentale Misure “G. Casnati”, Università di Parma, Viale G. P. Usberti 23A, I-43100 Parma, Italy, §Dipartimento di Chimica Organica “Ugo Shiff”, Università degli Studi di Firenze, Polo Scientifico di Sesto e Agraria, Via della Lastruccia 13, I-50019 Sesto Fiorentino, Italy, and $Dipartimento di Fisiopatologia Clinica, Unità di Medicina Nucleare, Università degli Studi di Firenze, Viale Pieraccini 6, I-50134 Firenze, Italy

[email protected]

[email protected]

[email protected]

N

O

NH

HN HN

O NH HO2C

HN

N H

NH2

O

O IC50h: 0.08 nM 5 IC50h: 0.88 nM

V 3 V

Contents •

General methods

S2

•

Experimental procedures and spectroscopic data for compounds 15-28

S3

•

Elemental analyses of intermediary and target compounds (Table S1)

S15

•

Copies of 1H NMR spectra (600 MHz, D2O) of cyclopeptides 5-12 (Figures S1-S8)

S16

•

Concentration-response curves of cyclopeptides 5-12 to

(Figures S9-S16)

S20

•

Schematic view of observed interactions between ligand 9 and the

receptor (Fig. S17)

S28

•

Surface representations of the

•

Superposition of docked 5, 9, and 10 to the X-ray

•

References

V 3

V 3

and

V 5 V 3

binding site with ligands 5, 9, and 10 (Figure S18) V 3-bound

S29

conformation of 1 (Figure S19) S30 S31 S1

General. All solid-phase synthesis chemicals purchased from commercial sources were dried in dessicator before use; all other chemicals were used as supplied without further purification. Apart from NMP, all organic solvents were dried and freshly distilled before use according to literature procedures. cTrt resin (2-chlorotrityl chloride polymer bound, 100-300 mesh, nominal loading ~1.6 mmol/g, calculated loading 1.55 mmol/g) was from Fluka; coupling reagents were from either Bachem or Aldrich. All moisture sensitive reactions were carried out under a positive pressure of nitrogen or argon. TNBS test was performed according to the following procedure. A few resin beads were sampled and accurately washed with ethanol. The sample was then placed in a vial and 1 drop of a 10% solution of DIEA in DMF and 1 drop of 1% TNBS in DMF were added. The sample was then observed under a microscope and colour changes noted. The TNBS test is considered positive (presence of free amino groups) when the resin beads turn orange or red within 1 min and negative (no free amino groups) when the beads remain colourless. Thin layer chromatography (TLC) was performed on silica gel 60 F254 precoated plates (Merck) with visualization under short-wavelength UV light or by dipping the plates with molybdate reagent (H2O/concentrated H2SO4/(NH4)6Mo7O24·4H2O/Ce2(SO4)3 90/10/25/1 v/v/w/w) followed by heating. Flash chromatography was performed on 40-63 µm silica gel (Merck) using the indicated solvent mixtures. Analytical reversed-phase HPLC was performed on a SpectraSystem P2000 apparatus (Thermo Separation Products, UV detection 220 or 254 nm) equipped with a C18-10µm column (Discovery BIO Wide Pore, 250 × 4.6 mm). Semipreparative reversed-phase HPLC was performed on a Prostar 210 apparatus (Varian, UV detection at 220 or 254 nm) equipped with a C18-10µm column (Discovery BIO Wide Pore, 250 × 10 mm). Direct infusion ESI-MS spectra were recorded on API 150EX apparatus (Applied Biosystems). High-resolution mass spectrometry measurements (HRMS) were performed on a APEX IIIQ Fourier transform mass spectrometer (Bruker) equipped with an

S2

external electrospray ion source. Melting points were determined with an optical thermomicroscope Optiphot2-Pol (Nikon) and are uncorrected. Optical rotations were measured using a Perkin-Elmer model 341 polarimeter at ambient temperature using a 100-mm cell with a 1-mL capacity and are given in units of 10-1 deg cm2 g-1. Elemental analyses were performed by the Microanalytical Laboratory of the University of Parma. Routine NMR spectra were recorded on Avance 300 (Bruker) or Mercury Plus MP-400 (Varian) NMR spectrometers. Chemical shifts ( ) are reported in parts per million (ppm) with TMS (CDCl3), CD2HOD, and HOD resonance peaks set at 0, 3.31, and 4.80 ppm, respectively. For sample preparation and NMR experiments of target cyclopeptides 5-12, see the Experimental Section in the main text. Fmoc-Asp(But)-OH, Fmoc-Arg(Pmc)-OH, and Fmoc-Gly-OH were purchased from Fluka or Bachem. Trans-4-hydroxy-L-proline (13) was purchased from Fluka. Cis-4-hydroxy-D-proline (14) was prepared from 13 in a 65% yield according to a known three-step procedure (Ac2O, AcOH; then 2N HCl; then Ag2CO3).1 Compound 14 is also commercially available from Fluka. (2S,4R)-1-(tert-Butoxycarbonyl)-4-hydroxyproline Benzyl Ester (15). Typical Procedure. To a

NH

HO

Boc2O, Et3N CO2H

13

N

HO

Boc

CO2H 13a

BnBr, Cs2CO3

N

Boc

solution of triethylamine (4 mL) and methanol (36 mL) was added

HO

CO2Bn 15

trans-4-hydroxy-L-proline

(13)

(2.0 g, 15.3 mmol) and di-tert-butyl dicarbonate (6.66 g, 30.5 mmol). After being refluxed for 2 h, the reaction mixture was allowed to cool to room temperature and the solvent removed in vacuo. The residue was cooled to 0 °C, followed by addition of NaH2PO4 (150 mg), and the solution was acidified with diluted HCl to pH 2. The mixture was stirred at 0 °C for 30 min. Then it was extracted with EtOAc (4 ×), and the combined organic layers were collected, dried (MgSO4), and filtered. Evaporating off the solvent under reduced pressure afforded trans-N-(tert-butoxycarbonyl)-4-hydroxy-L-proline (13a) (3.0 g, 85%) as a colourless foam and used directly for the next step: [ ]25D –77 (c 0.8, H2O); S3

1

H NMR (300 MHz, CD3OD)

4.40 (m, 1H), 4.32 (t, J = 8.0 Hz, 1H), 3.56 (dd, J = 11.5, 4.1 Hz, 1H),

3.45 (m, 1H), 2.28 (m, 1H), 2.07 (m, 1H), 1.48 and 1.45 (2s, 9H);

13

C NMR (75.4 MHz, CD3OD)

176.0 (Cq), 147.9 (Cq), 81.7 (Cq), 70.1 (CH), 59.4 (CH), 55.6 (CH2), 40.1 (CH2), 28.5 (3C, CH3). The spectral and chiro-optical characteristics of this compound fully matched those reported for the commercially available material (Fluka). Proline 13a (3.0, 13.0 mmol) was dissolved in methanol (50 mL) and the solution was cooled to 0 °C. Aqueous caesium carbonate (2.12 g in 32 mL H2O) was added. The solution was concentrated and sufficient DMF was added to azeotrope the water, leaving a white solid which was dissolved in DMF (60 mL). Benzyl bromide (1.5 mL, 13.0 mmol) was added at 0 °C and the mixture was stirred vigorously at room temperature for 20 h. The reaction mixture was concentrated in vacuo, dissolved in EtOAc (40 mL), and washed with water (2 ×) and saturated aqueous sodium chloride (2 ×). The organic layer was dried (MgSO4), filtered, and concentrated in vacuo to produce a residue which was purified by silica gel flash chromatography (hexanes/EtOAc 50:50) affording benzylproline 15 as a colorless oil (3.97 g, 95%): [ ]25D –61.3 (c 4.0, CHCl3); [lit.2 [ ]25D –63.34 (c 1, CHCl3)]; 1H NMR (300 MHz, CDCl3, mixture of rotamers)

7.30 (m, 5H, Ph), 5.15 (m, 2H, CH2Ph), 4.45 (dd, J = 8.8, 1.7 Hz, 1H,

H2), 4.37 (m, 1H, H4), 3.74 (bs, 1H, OH), 3.55 (dd, J = 11.6, 4.3 Hz, 1H, H5A), 3.42 (m, 1H, H5B), 2.24 (m, 1H, H3A), 1.97 (m, 1H, H3B), 1.40 and 1.24 (2s, 9H, But); mixture of rotamers, major isomer)

13

C NMR (75.4 MHz, CDCl3,

173.03 (Cq, CO2R), 154.1 (Cq, Boc), 135.5 (Cq, Ph), 128.8 (2C,

CH, Ph), 128.4 (2C, CH, Ph), 128.1 (CH, Ph), 80.4 (Cq, Boc), 69.1 (CH, C4), 66.8 (CH2, CH2Ph), 58.1 (CH, C2), 54.7 (CH2, C5), 39.1 (CH2, C3), 28.4 (3C, CH3, Boc). Anal. (C17H23NO5): C, H, N. (2R,4R)-1-(tert-Butoxycarbonyl)-4-hydroxyproline Benzyl Ester (16). The title compound was prepared from hydroxyproline 14 NH HO

Boc2O, Et3N CO2H

14

N HO

Boc CO2H

14a

BnBr, Cs2CO3

N HO

Boc

(1.0 g, 7.63 mmol) according to CO2Bn

16

the two-step procedure utilized to S4

convert 13 into 15. After the first step, cis-N-(tert-butoxycarbonyl)-4-hydroxy-D-proline (14a) (85%) was obtained as a white powder and used directly for the following step: [ ]25D +46.1 (c 5.0, MeOH); [lit.3 [ ]22D +47.1 (c 2.29, EtOH)]; 1H NMR (300 MHz, CDCl3, mixture of rotamers)

7.60 (bs, 2H),

4.31 (m, 1H), 4.20 (bd, J = 8.2 Hz, 1H), 3.42 (m, 2H), 2.0-2.3 (m, 2H), 1.34 and 1.31 (2s, 9H);

13

C

NMR (75.4 MHz, CDCl3, mixture of rotamers, major isomer) 175.7 (Cq), 154.2 (Cq), 80.7 (Cq), 69.3 (CH), 57.8 (CH), 54.2 (CH2), 38.0 (CH2), 27.9 (3C). Proline 14a is also commercially available from Flamma Spa, Italy. After the second step, compound 16 (1.96 g, 80% yield from 14) was obtained as a colorless oil: [ ]25D +10.38 (c 2.0, CHCl3); 1H NMR (300 MHz, CDCl3, mixture of rotamers)

7.39

(m, 5H, Ph), 5.1-5.4 (m, 2H, CH2Ph), 4.3-4.5 (m, 2H, H2, H4), 3.5-3.8 (m, 2H, H5A, H5B), 3.29 and 3.19 (2d, J = 10.0 and 9.9 Hz, 1H, OH), 2.35 (m, 1H, H3A), 2.12 (bd, J = 12.5 Hz, 1H, H3B), 1.49 and 1.36 (2s, 9H, But);

13

C NMR (75.4 MHz, CDCl3, mixture of rotamers, major isomer)

173.9 (Cq,

CO2R), 153.5 (Cq, Boc), 135.1 (Cq, Ph), 128.4 (2C, CH, Ph), 128.2 (2C, CH, Ph), 128.1 (CH, Ph), 80.1 (Cq, Boc), 69.6 (CH, C4), 67.0 (CH2, CH2Ph), 57.8 (CH, C2), 54.9 (CH2, C5), 38.4 (CH2, C3), 28.2 (3C, CH3, Boc). Anal. (C17H23NO5): C, H, N. (2S,4S)-1-(tert-Butoxycarbonyl)-4-azidoproline Benzyl Ester (17). Typical Procedure. A solution of DEAD (7.9 mL of a 40% solution in toluene, N HO

Boc CO2Bn

15

DPPA, PPh3, DEAD

N N3 17

Boc

18.20 mmol) and DPPA (3.9 mL, 18.20 mmol) in

CO2Bn

THF (15 mL) was added dropwise over 30 min to a solution of proline 15 (1.95 g, 6.07 mmol) and PPh3

(4.77 g, 18.20 mmol) in THF (50 mL) at 0 °C under argon. The mixture was stirred for 24 h at room temperature. After addition of EtOH (20 mL), the solvent was concentrated to dryness in vacuo. The residue was purified by silica gel flash chromatography (hexanes/EtOAc 80:20 to 70:30) to afford azidoproline 17 (2.06 g, 98%) as a yellowish oil: [ ]25D –40.1 (c 1.0, CHCl3); [lit.4 [ ]D –32.2 (c 1.5, CH2Cl2)]; 1H NMR (300 MHz, CDCl3, mixture of rotamers)

7.31 (m, 5H, Ph), 5.21 (m, 2H, CH2Ph),

4.50 (dd, J = 8.9, 3.5 Hz, 0.4H, H2), 4.38 (dd, J = 8.9, 3.9 Hz, 0.6H, H2), 4.16 (m, 1H, H4), 3.73 S5

(ddd, J = 18.0, 11.7, 6.1 Hz, 1H, H5A), 3.50 (ddd, J = 17.8, 11.7, 3.7 Hz, 1H, H5B), 2.46 (m, 1H, H3A), 2.20 (dt, J = 13.5, 3.8 Hz, 1H, H3B), 1.48 and 1.36 (2s, 9H, But); 13C NMR (75.4 MHz, CDCl3, mixture of rotamers, major isomer, DEPT assignment)

171.4 (Cq), 153.7 (Cq), 135.6 (Cq), 128.6 (2C, CH),

128.5 (2C, CH), 128.2 (CH), 80.6 (Cq), 67.1 (CH2), 58.3 (CH), 57.8 (CH), 51.4 (CH2), 36.1 (CH2), 28.1 (3C, CH3). Anal. (C17H22N4O4): C, H, N. (2S,4R)-1-(tert-Butoxycarbonyl)-4-azidoproline Benzyl Ester (18). Typical Procedure. Proline 15 (2.0 g, 6.22 mmol) and CBr4 (2.13 N HO

Boc

CBr4, Diphos

CO2Bn 15

Boc N Br

Boc

NaN3, DMF

N N3

CO2Bn 15a

g, 6.41 mmol) were dissolved in dry CO2Bn

18

THF (18 mL). Then, Diphos5 (1.49

g, 3.73 mmol) was added. The reaction was allowed to stir at room temperature for 20 h, at which time the suspension was filtered through a Celite pad. The pad was rinsed with THF and the filtrate was evaporated to give a yellow oil. The crude mixture was purified by silica gel flash chromatography (hexanes/EtOAc 50:50) to afford a bromoproline intermediate 15a (2.0 g, 84%) as a pale yellow oil: [ ]25D –40.8 (c 1.0, CHCl3); [lit.4 [ ]D –41.3 (c 1.5, CH2Cl2)]; 1H NMR (300 MHz, CDCl3, mixture of rotamers)

7.20 (m, 5H), 4.9-5.1 (m, 2H), 4.10-4.30 (m, 2H), 3.85 (m, 1H), 3.55 (m, 1H), 2.5-3.7 (m,

1H), 2.28 (m, 1H), 1.35 and 1.21 (2s, 9H); isomer, DEPT assignment)

13

C NMR (75.4 MHz, CDCl3, mixture of rotamers, major

171.6 (Cq), 153.8 (Cq), 135.8 (Cq), 128.8 (2C, CH), 128.7 (2C, CH),

128.3 (CH), 80.8 (Cq), 67.2 (CH2), 58.5 (CH), 55.9 (CH), 42.8 (CH2), 41.1 (CH2), 28.5 (3C, CH3). Bromoproline intermediate 15a (2.0 g, 5.22 mmol) and NaN3 (1.70 g, 26.1 mmol) were suspended in 75 mL of dry DMF. This mixture was heated at 55 °C in an oil bath for 24 h. The mixture was allowed to cool to room temperature, whereupon it was poured into an ice cold water. The mixture was extracted with EtOAc (3 ×), and the combined organic layers were washed with H2O and brine, dried (MgSO4), filtered, and concentrated to give azidoproline 18 (1.75 g, 97%) as a colorless oil: [ ]25D – 46.1 (c 1.0, CHCl3); [lit.4 [ ]D –49.3 (c 1.0, CH2Cl2)]; 1H NMR (300 MHz, CDCl3, mixture of S6

rotamers) 7.27 (m, 5H), 5.18 (m, 2H), 4.45 (dd, J = 8.5, 6.6 Hz, 0.4H), 4.28 (t, J = 7.5 Hz, 0.6H), 4.05 (m, 1H), 3.3-3.6 (m, 2H), 2.20 (m, 1H), 2.05 (m, 1H), 1.40 and 1.30 (2s, 9H); CDCl3, mixture of rotamers, major isomer, DEPT assignment)

13

C NMR (75.4 MHz,

171.9 (Cq), 153.9 (Cq), 135.6 (Cq),

128.6 (2C, CH), 128.5 (2C, CH), 128.2 (CH), 80.6 (Cq), 67.0 (CH2), 58.9 (CH), 57.8 (CH), 51.4 (CH2), 36.5 (CH2), 28.3 (3C, CH3). Anal. (C17H22N4O4): C, H, N. (2R,4S)-1-(tert-Butoxycarbonyl)-4-azidoproline Benzyl Ester (19). The title compound was prepared from hydroxyproline 16 (1.0 g, 3.11 N HO

Boc

DPPA, PPh3, DEAD

N N3

CO2Bn

Boc

mmol) according to the procedure utilized to

CO2Bn

convert 15 into 17. After purification by silica gel

19

16

flash chromatography (hexanes/EtOAc 80:20 to 70:30), the title compound 19 (1.04 g, 96%) was obtained as a colorless oil: [ ]25D +48.2 (c 1.1, CHCl3). The 1H and

13

C NMR data fully matched those reported for the enantiomeric counterpart 18.

Anal. (C17H22N4O4): C, H, N. (2R,4R)-1-(tert-Butoxycarbonyl)-4-azidoproline Benzyl Ester (20). The title compound was prepared from hydroxyproline 16 N HO

Boc CO2Bn

16

CBr4, Diphos

Boc N Br

CO2Bn 16a

Boc

NaN3, DMF

N N3

(1.0 g, 3.11 mmol) according to the CO2Bn

20

two-step

procedure

utilized

to

convert 15 into 18. After the first step, bromoproline intermediate 16a4 (1.02 g, 85%) was obtained as a white powder: [ ]25D +51.55 (c 2.0, CHCl3); 1H NMR (300 MHz, CDCl3, mixture of rotamers)

7.35

(m, 5H), 5.1-5.4 (m, 2H), 4.4-4.7 (m, 2H), 3.7-4.0 (m, 2H), 2.60 (m, 1H), 2.40 (m, 1H), 1.48 and 1.37 (2s, 9H); 13C NMR (75.4 MHz, CDCl3, mixture of rotamers, major isomer, DEPT assignment)

172.1

(Cq), 153.4 (Cq), 135.3 (Cq), 128.6 (2C, CH), 128.5 (2C, CH), 128.1 (CH), 80.7 (Cq), 67.0 (CH2), 58.2 (CH), 55.9 (CH), 44.3 (CH2), 41.4 (CH2), 28.2 (3C, CH3). After the second step, the title compound 204 (883 mg, 82% from 16) was obtained as a colorless oil: [ ]25D +45.8 (c 2.0, CHCl3). The 1H and 13C S7

NMR data fully matched those reported for the enantiomeric counterpart 17. Anal. (C17H22N4O4): C, H, N. (2S,4S)-1-(tert-Butoxycarbonyl)-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (21). Typical Procedure. Azidoproline 17 (0.5 g, N N3

Boc H2, Pd/C CO2Bn

17

N

H2N

Boc FmocOSu, aq Na2CO3 CO2H

17a

Boc N HN Fmoc 21

1.44 mmol) was dissolved in CO2H

MeOH (30 mL) and palladium on carbon (10%, 50 mg) was added.

The reaction vessel was evacuated by aspirator and thoroughly purged with hydrogen (three times), and the resulting heterogeneous mixture was stirred under a hydrogen balloon for 24 h at room temperature. The mixture was filtered through a pad of Celite and the pad was washed with MeOH. The filtrate was concentrated in vacuo to give crude N-Boc-4-aminoproline intermediate 17a (332 mg, 100%) which was used as such in the following step: a white powder; [ ]25D +14.3 (c 1.0, H2O); [lit.6 [ ]D +21 (c 0.24, H2O)]; 1H NMR (300 MHz, D2O, mixture of rotamers)

4.07 (dd, J = 9.4, 2.8 Hz, 1H), 3.90 (m,

1H), 3.62 (m, 2H), 2.57 (ddd, J = 14.9, 9.3, 5.8 Hz, 1H), 2.02 (dt, J = 14.5, 2.8 Hz, 1H), 1.36 and 1.32 (2s, 9H); 13C NMR (75.4 MHz, CDCl3, mixture of rotamers, major isomer, DEPT assignment)

177.1

(Cq), 154.9 (Cq), 83.1 (Cq), 58.2 (CH), 49.3 (CH2), 48.7 (CH), 33.5 (CH2), 27.5 (3C, CH3). N-Boc-4-aminoproline intermediate 17a (322 mg, 1.44 mmol) was dissolved in THF (3 mL), and 10% aqueous Na2CO3 solution (4 mL) was added. FmocOSu (486 mg, 1.44 mmol) dissolved in THF (10 mL) was then added to the solution pre-cooled to 0 °C. The reaction mixture was stirred for 2 h at room temperature and concentrated in vacuo to leave a residue which was dissolved in EtOAc (10 mL) and treated with saturated aq NH4Cl solution. The mixture was extracted with EtOAc (3 ×) and the organic layers were collected, dried over MgSO4, filtered, and concentrated to afford a crude residue which was purified by silica gel flash chromatography (EtOAc/MeOH 70:30) to afford aminoproline 21 (586 mg, 90%) as a white solid: mp 168 °C; [ ]25D –17.2 (c 0.87, MeOH); 1H NMR (300 MHz, CDCl3, S8

mixture of rotamers)

7.78 (d, J = 7.4 Hz, 2H), 7.61 (d, J = 7.5 Hz, 2H), 7.42 (t, J = 7.3 Hz, 2H), 7.34

(t, J = 7.3 Hz, 2H), 5.77 (bd, J = 5.9 Hz, 1H), 4.51 (d, J = 8.3 Hz, 1H), 4.25-4.45 (m, 3H), 4.22 (t, J = 7.1 Hz, 1H), 3.58 (m, 2H), 2.49 (bd, J = 13.6 Hz, 1H), 2.37 (m, 1H), 1.52 and 1.46 (2s, 9H); 13C NMR (75.4 MHz, CDCl3, HSQC assignment)

180.9 (Cq), 155.7 (2C, Cq), 143.5 (2C, Cq), 141.0 (2C, Cq),

127.5 (2C, CH), 126.9 (2C, CH), 124.9 (2C, CH), 119.8 (2C, CH), 82.9 (Cq), 66.9 (CH2), 58.7 (CH), 53.9 (CH2), 50.5 (CH), 46.9 (CH), 32.4 (CH2), 28.1 (3C, CH3). Anal. (C25H28N2O6): C, H, N. The title amino acid is also commercially available from Sigma-Aldrich or NeoMPS. (2S,4S)-1-Benzoyl-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (22). Typical Procedure. A O N N3

Boc CO2Bn

17

NH

TFA N3

PhCO2H, DIC, HOBt CO2Bn

17b

O H2, Ph Pd/C

N

CO2Bn

N3 17c

N

O FmocOSu, Ph aq Na2CO3

CO2H

H2N 17d

HN Fmoc

N

Ph CO2H

22

solution of azidoproline 17 (0.5 g, 1.44 mmol) in dry DCM (10 mL) was cooled to 0 °C and treated with anhydrous TFA (2 mL). After the mixture was stirred at room temperature for 2 h, saturated aq NaHCO3 solution was added until neutral pH. The mixture was extracted with EtOAc (4 ×) and the organic layers were collected, dried over MgSO4, filtered, and concentrated to afford a crude residue which was purified by silica gel flash chromatography (EtOAc/MeOH 96:4) to afford the N deprotected azidoproline intermediate 17b (294 mg, 83%) as a colorless oil: [ ]25D –42.5 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3)

7.34 (m, 5H, Ph), 5.21 (1/2 ABq, J = 12.2 Hz, 1H, CH2Ph),

5.16 (1/2 ABq, J = 12.2 Hz, 1H, CH2Ph), 4.05 (ddt, J = 6.0, 4.8, 2.5 Hz, 1H, H4), 3.83 (dd, J = 9.5, 4.3 Hz, 1H, H2), 3.13 (ddd, J = 12.0, 2.3, 1.4 Hz, 1H, H5A), 2.96 (dd, J = 12.0, 4.8 Hz, 1H, H5B), 2.64 (bs, 1H, NH), 2.33 (ddd, J = 14.0, 9.5, 6.0 Hz, 1H, H3A), 2.11 (dddd, J = 14.0, 4.2, 2.7, 1.4 Hz, 1H, H3B); 13

C NMR (75.4 MHz, CDCl3, HSQC assignment)

173.9 (Cq), 135.5 (Cq), 128.6 (2C, CH), 128.5

(CH), 128.3 (2C, CH), 67.1 (CH2), 61.3 (CH), 58.9 (CH), 52.6 (CH2), 36.0 (CH2). Azidoproline intermediate 17b (294 mg, 1.20 mmol) was dissolved in DCM (10 mL) and the S9

resulting solution was cooled to 0 °C and treated with benzoic acid (176 mg, 1.44 mmol), HOBt (195 mg, 1.44 mmol), and DIC (0.26 mL, 1.68 mmol). After stirring at room temperature for 2 h, the reaction mixture was filtered to take off the solid precipitate. The organic layer was washed with aq NaHCO3 solution (3 ×), dried over MgSO4, filtered, and concentrated to afford a crude residue which was purified by silica gel flash chromatography (hexanes/EtOAc 40:60). N -Benzoyl azidoproline intermediate 17c (370 mg, 88%) was obtained as an oil. Compound 17c (370 mg, 1.06 mmol) was transformed into the title amino acid 22 via the two-step sequence previously described to convert 17 into 21. Compound 22 (425 mg, 88%, over two steps, corresponding to a 64% yield from 17): a white solid; mp 165 °C; [ ]25D –47.0 (c 1.0, MeOH); 1H NMR (300 MHz, CDCl3, mixture of rotamers) 7.76 (d, J = 7.4 Hz, 2H), 7.4-7.6 (m, 8H), 7.30 (m, 3H), 5.85 (bd, J = 6.7 Hz, 1H), 4.93 (t, J = 5.5 Hz, 1H), 4.3-4.6 (m, 3H), 4.19 (t, J = 6.8 Hz, 1H), 3.90 (dd, J = 11.8, 6.0 Hz, 1H), 3.60 (bd, J = 10.1 Hz, 1H), 2.51 (m, 2H); 13C NMR (75.4 MHz, CDCl3, HSQC assignment)

185.8 (Cq), 172.8 (Cq), 155.7

(Cq), 143.7 (2C, Cq), 141.3 (2C, Cq), 131.3 (Cq), 128.7 (2C, CH), 127.8 (2C, CH), 127.3 (CH), 127.1 (4C, CH), 125.1 (2C, CH), 120.0 (2C, CH), 67.1 (CH2), 59.8 (CH), 56.7 (CH2), 50.8 (CH), 47.1 (CH), 32.4 (CH2). Anal. (C27H24N2O5): C, H, N. The title amino acid is commercially available from NeoMPS. (2S,4S)-1-Propanoyl-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (23). The title compound O N

Boc

N3

CO2Bn 17

NH

TFA N3

EtCO2H, DIC, HOBt CO2Bn

17b

N

O H2, Pd/C

CO2Bn

N3 17e

FmocOSu, aq Na2CO3

N CO2H

H2N 17f

O

HN Fmoc

N CO2H 23

was prepared from azidoproline 17 (0.5 g, 1.44 mmol) according to the procedure followed for the transformation of 17 into 22 and using propanoic acid instead of benzoic acid. Compound 23 (365 mg, 62% yield from 17): a white foam; [ ]25D –9.1 (c 4.1, MeOH); 1H NMR (400 MHz, CD3OD, mixture of rotamers)

7.81 (d, J = 7.2 Hz, 2H), 7.65 (bd, J = 6.0 Hz, 2H), 7.41 (t, J = 7.2 Hz, 2H), 7.33 (t, J = 7.2

Hz, 2H), 4.3-4.5 (m, 3H), 4.24 (m, 2H), 3.91 (m, 1H), 3.40 (m, 1H), 2.57 (m, 1H), 2.38 (m, 2H), 1.95 S10

(m, 1H), 1.13 (m, 3H); 13C NMR (100 MHz, CD3OD) 176.0 (Cq), 160.7 (Cq), 159.0 (Cq), 146.1 (2C, Cq), 143.4 (2C, Cq), 129.6 (2C, CH), 129.0 (2C, CH), 127.0 (2C, CH), 121.8 (2C, CH), 68.7 (CH2), 60.1 (CH), 53.9 (CH2), 53.5 (CH), 52.4 (CH), 36.5 (CH2), 29.3 (CH2), 9.9 (CH3). Anal. (C23H24N2O5): C, H, N. (2S,4S)-1-Heptyl-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (24). A solution of azidoproline N

Boc TFA

NH

CO2Bn N3

N3 17

heptanal, NaBH(OAc)3 CO2Bn

17b

N

N3

17g

6

H2, Pd/C

CO2Bn H N 2

N

6

FmocOSu, aq Na2CO3

CO2H

17h

N

6

HN CO2H Fmoc 24

17 (0.5 g, 1.44 mmol) in dry DCM (10 mL) was cooled to 0 °C and treated with anhydrous TFA (2 mL). After the mixture was stirred at room temperature for 2 h, saturated aq NaHCO3 solution was added until neutral pH. The mixture was extracted with EtOAc (4 ×) and the organic layers were collected, dried over MgSO4, filtered, and concentrated to afford a crude residue which was purified by silica gel flash chromatography (EtOAc/MeOH 96:4) to afford N -deprotected azidoproline intermediate 17b (294 mg, 83%), whose optical and spectral characteristics are reported above (see transformation of compound 17 into 22, first step). To an ice-cooled solution7 of azidoproline intermediate 17b (294 mg, 1.19 mmol) in DCE (10 mL) were added freshly distilled heptanal (0.20 mL, 1.43 mmol) and sodium triacetoxyborohydride [NaBH(OAc)3] (353 mg, 1.67 mmol). The resulting mixture was stirred at room temperature for 1 h, and quenched with saturated aq NaHCO3 solution until neutral pH. Extraction with EtOAc (4 ×), drying of the organic layers over MgSO4, filtration, and evaporation of the solvent in vacuo gave a crude residue, which was purified by silica gel flash chromatography (hexanes/EtOAc 80:20) to yield Nheptyl azidoproline 17g (348 mg, 85%) as an oil; 1H NMR (300 MHz, CDCl3) 7.30 (m, 5H), 5.21 (m, 2H), 4.01 (m, 1H), 3.28 (dd, J = 8.9, 6.3 Hz, 1H), 3.23 (dd, J = 10.3, 1.8 Hz, 1H), 2.73 (dt, J = 12.3, 8.0 Hz, 1H), 2.62 (dd, J = 9.9, 5.7 Hz, 1H), 2.51 (dt, J = 13.3, 8.7 Hz, 1H), 2.39 (dt, J = 11.6, 7.0 Hz, 1H), 2.12 (ddd, J = 13.9, 5.4, 2.5 Hz, 1H), 1.45 (m, 2H), 1.2-1.4 (m, 8H), 0.75 (m, 3H);

13

C NMR (75.4 S11

MHz, CDCl3)

172.9 (Cq), 136.0 (Cq), 128.3 (3C, CH), 128.2 (2C, CH), 66.8 (CH2), 65.0 (CH), 58.9

(CH), 58.6 (CH2), 54.3 (CH2), 35.8 (CH2), 28.3 (CH2), 25.1 (CH2), 24.6 (CH2), 23.8 (CH2), 13.5 (CH3). Anal. (C19H28N4O2): C, H, N. Azidoproline intermediate 17g (348 mg, 1.01 mmol) was converted into the title amino acid 24 via the two-step sequence previously described to transform 17 into 21. Compound 24 (396 mg, 87%, over two steps, corresponding to a 61% yield from 17): a white foam; [ ]25D –12.8 (c 4.75, MeOH); 1H NMR (400 MHz, CD3OD, mixture of rotamers)

7.79 (d, J = 7.6 Hz, 2H), 7.64 (dd, J = 7.6, 2.4 Hz,

2H), 7.40 (t, J = 7.2 Hz, 2H), 7.32 (t, J = 7.2 Hz, 2H), 4.38 (m, 2H), 4.30 (m, 1H), 4.19 (t, J = 6.4 Hz, 1H), 3.80 (bt, J = 8.0 Hz, 1H), 3.60 (bd, J = 10.4 Hz, 1H), 3.29 (m, 1H), 3.22 (m, 1H), 3.01 (m, 1H), 2.75 (dt, J = 13.6, 8.0 Hz, 1H), 2.08 (m, 1H), 1.69 (m, 2H), 1.34 (m, 8H), 0.92 (m, 3H); 13C NMR (75.4 MHz, CDCl3, HSQC assignment)

181.3 (Cq), 162.4 (Cq), 156.0 (Cq), 143.7 (2C, Cq), 141.3 (2C,

Cq), 131.8 (Cq), 128.7 (2C, CH), 127.8 (2C, CH), 127.3 (CH), 127.1 (4C, CH), 125.1 (2C, CH), 120.0 (2C, CH), 67.1 (CH2), 59.8 (CH), 56.7 (CH2), 50.8 (CH), 47.1 (CH), 32.4 (CH2). Anal. (C27H34N2O4): C, H, N. (2S,4R)-1-(tert-Butoxycarbonyl)-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (25). The title compound N

N3

Boc

N

H2, Pd/C CO2Bn

18

H2N

Boc FmocOSu, aq Na2CO3

CO2H 18a

was

obtained

from

Boc

azidoproline 18 (0.5 g, 1.44 mmol)

N HN Fmoc 25

CO2H

via the two-step sequence utilized to convert 17 into 21. Compound 25

(573 mg, 88% from 18), a white solid: mp 190 °C; [ ]25D –11.58 (c 0.95, MeOH); 1H NMR (300 MHz, CDCl3, mixture of rotamers)

7.79 (d, J = 7.4 Hz, 2H), 7.59 (d, J = 7.3 Hz, 2H), 7.43 (t, J = 7.4 Hz,

2H), 7.34 (t, J = 7.4 Hz, 2H), 4.95 and 4.88 (2bs, 1H), 4.4-4.6 (m, 2H), 4.2-4.4 (m, 3H), 3.78 (m, 1H), 3.3-3.5 (m, 1H), 2.53 (m, 1H), 2.12 (m, 1H), 1.50 and 1.45 (2s, 9H); HSQC assignment)

13

C NMR (75.4 MHz, CDCl3,

179.2 (Cq), 156.3 (2C, Cq), 143.7 (2C, Cq), 141.4 (2C, Cq), 127.8 (2C, CH), S12

127.1 (2C, CH), 124.9 (2C, CH), 120.0 (2C, CH), 81.1 (Cq), 66.6 (CH2), 57.9 (CH), 51.9 (CH2), 49.95 (CH), 47.2 (CH), 34.0 (CH2), 28.3 (3C, CH3). Anal. (C25H28N2O6): C, H, N. The title amino acid is also commercially available from NeoMPS. (2S,4R)-1-Benzoyl-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (26). The title compound was O N

Boc

N3

NH

TFA CO2Bn

N3

PhCO2H, DIC, HOBt CO2Bn

H2, Ph Pd/C

N

CO2Bn

N3

18a

18

O N

FmocOSu, Ph aq Na CO 2 3 CO2H

H2N

18b

O

18c

HN Fmoc

N

Ph CO2H

26

obtained from azidoproline 18 (1.0 g, 2.88 mmol) via the four-step sequence utilized to convert 17 into 22. Compound 26 (881 mg, 67% yield from 17): a glassy solid: [ ]25D –14.0 (c 1.5, MeOH); 1H NMR (400 MHz, CD3OD, mixture of rotamers)

7.88 (m, 2H), 7.2-7.7 (m, 11H), 4.68 (t, J = 7.2 Hz, 1H),

4.25-4.45 (m, 3H), 4.10-4.25 (m, 1H), 3.98 and 3.87 (2m, 1H), 3.63 and 3.44 (2m, 1H), 2.0-2.4 (m, 2H);

13

C NMR (100 MHz, CD3OD, HSQC assignment)

185.8 (Cq), 173.0 (Cq), 163.9 (Cq), 157.2

(Cq), 146.0 (2C, Cq), 143.4 (2C, Cq), 132.4 (Cq), 130.3 (2C, CH), 129.6 (2C, CH), 129.2 (2C, CH), 129.0 (2C, CH), 127.0 (3C, CH), 121.8 (2C, CH), 68.4 (CH2), 61.5 (CH), 57.3 (CH2), 52.8 (CH), 48.1 (CH), 37.0 (CH2). Anal. (C27H24N2O5): C, H, N. (2R,4S)-1-(tert-Butoxycarbonyl)-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (27). The title compound was obtained from azidoproline 19 (1.0 g, 2.88 mmol) via the two-step sequence utilized to convert 17 into 21. Compound 27 N N3

Boc H2, Pd/C CO2Bn

19

of rotamers)

N

H2N

Boc FmocOSu, aq Na2CO3 CO2H

19a

Boc

N HN Fmoc 27

CO2H

(1.1 g, 85% from 19), a white foam: [ ]25D +9.2 (c 4.2, MeOH);

1

H

NMR (400 MHz, CD3OD, mixture

7.78 (d, J = 7.2 Hz, 2H), 7.63 (d, J = 7.2 Hz, 2H), 7.39 (t, J = 7.2 Hz, 2H), 7.31 (t, J =

7.2 Hz, 2H), 4.35 (m, 2H), 4.26 (m, 2H), 4.18 (t, J = 6.4 Hz, 1H), 3.73 (dd, J = 10.4, 6.0 Hz, 1H), 3.38 (m, 1H), 2.53 (m, 1H), 2.0 and 2.2 (2m, 1H), 1.47 and 1.46 (2s, 9H);

13

C NMR (100 MHz, CD3OD, S13

mixture of rotamers, major isomer, HSQC assignment)

179.0 (Cq), 159.1 (Cq), 156.9 (Cq), 146.1

(2C, Cq), 143.4 (2C, Cq), 129.6 (2C, CH), 129.0 (2C, CH), 127.0 (2C, CH), 121.8 (2C, CH), 82.3 (Cq), 68.5 (CH2), 61.3 (CH), 53.3 (CH2), 51.4 (CH), 50.7 (CH), 38.3 (CH2), 29.5 (3C, CH3). Anal. (C25H28N2O6): C, H, N. (2R,4R)-1-(tert-Butoxycarbonyl)-4-N-(9-fluorenylmethoxycarbonyl)aminoproline (28). The title

N N3

Boc

H2, Pd/C CO2Bn

20

Boc

Boc FmocOSu, N aq Na2CO3

H2N

CO2H 20a

compound

was

obtained

from

N HN Fmoc 28

azidoproline 20 (0.5 g, 1.44 mmol) CO2H

via the two-step sequence utilized to

convert 17 into 21. Compound 28 (573 mg, 88% from 20), a glassy solid: [ ]25D +17.5 (c 0.8, MeOH); 1

H NMR (400 MHz, CDCl3, mixture of rotamers)

7.71 (d, J = 7.4 Hz, 2H), 7.57 (m, 2H), 7.35 (t, J =

7.3 Hz, 2H), 7.27 (t, J = 7.3 Hz, 2H), 6.0 and 6.4 (2bs, 1H), 4.0-4.5 (m, 5H), 3.3-3.7 (m, 2H), 1.9-2.4 (m, 2H), 1.43 and 1.37 (2s, 9H); 13C NMR (100 MHz, CDCl3, HSQC assignment)

176.4 (Cq), 156.0

(2C, Cq), 143.9 (2C, Cq), 141.2 (2C, Cq), 127.6 (2C, CH), 127.0 (2C, CH), 125.3 (2C, CH), 119.9 (2C, CH), 81.8 (Cq), 66.9 (CH2), 59.7 (CH), 54.1 (CH2), 50.5 (CH), 47.1 (CH), 33.8 (CH2), 28.3 (3C, CH3). Anal. (C25H28N2O6): C, H, N.

S14

Table S1. Elemental Analyses of Intermediary and Target Compounds

Elemental Analyses Calcd (%)

Found (%)

Compound

Formula

C

H

N

C

H

N

5

C17H28N8O6 HCl

42.81

6.13

23.50

42.59

6.40

23.25

6

C17H28N8O6 HCl

42.81

6.13

23.50

42.70

6.31

23.36

7

C17H28N8O6 HCl

42.81

6.13

23.50

42.74

6.44

23.19

8

C17H28N8O6 HCl

42.81

6.13

23.50

42.98

6.29

23.28

9

C24H42N8O6 HCl

50.12

7.54

19.48

49.97

7.81

19.37

10

C20H32N8O7 HCl

45.07

6.24

21.02

45.33

6.20

20.90

11

C24H32N8O7 HCl

49.61

5.72

19.29

49.76

5.98

19.14

12

C24H32N8O7 HCl

49.61

5.72

19.29

49.33

5.94

19.35

15

C17H23NO5

63.54

7.21

4.36

63.28

7.05

4.50

16

C17H23NO5

63.54

7.21

4.36

63.37

7.52

4.20

17

C17H22N4O4

58.95

6.40

16.17

58.68

6.78

16.24

18

C17H22N4O4

58.95

6.40

16.17

59.12

6.59

16.01

19

C17H22N4O4

58.95

6.40

16.17

59.09

6.74

16.07

20

C17H22N4O4

58.95

6.40

16.17

58.69

6.61

16.35

21

C25H28N2O6

66.36

6.24

6.19

66.08

6.45

6.00

22

C27H24N2O5

71.04

5.30

6.14

70.87

5.63

5.97

23

C23H24N2O5

67.63

5.92

6.86

67.73

6.04

6.63

24

C27H34N2O4

71.97

7.61

6.22

71.78

7.50

6.35

25

C25H28N2O6

66.36

6.24

6.19

66.18

6.47

5.96

26

C27H24N2O5

71.04

5.30

6.14

71.22

5.54

6.02

27

C25H28N2O6

66.36

6.24

6.19

66.13

6.29

6.07

28

C25H28N2O6

66.36

6.24

6.19

66.48

6.50

5.97

S15

Figure S1. 1H NMR spectrum (600 MHz, D2O, 298 K) of cyclopeptide 5.

Figure S2. 1H NMR spectrum (600 MHz, D2O, 298 K) of cyclopeptide 6. S16







S19

V 3

V 5

Figure S9. Compound 5: Inhibition of [125I]-echistatin specific binding to purified human integrin protein

V 3

(up) and

V 5

(down). Each point represents the mean value ±SEM of triplicate

determinations.

S20

V 3

V 5


V 3

(up) and

V 5


determinations.

S21

V 3

V 5


V 3

(up) and

V 5


determinations.

S22

V 3

V 5


V 3

(up) and

V 5


determinations.

S23

V 3

V 5


V 3

(up) and

V 5


determinations.

S24

V 3

V 5


V 3

(up) and

V 5


determinations.

S25

V 3

V 5


V 3

(up) and

V 5


determinations.

S26

V 3

V 5


V 3

(up) and

V 5


determinations.

S27

Tyr166 Asp150 Tyr178

O

Arg214

O

N NH2

NH2

NH NH

HN O

O

HN H

O

NH

O

O HN O

Asn215 HN

O O

H O Ser123

Mn2+

Asp218

Figure S17. Schematic 2D representation of ligand 9 bound to the

V 3

receptor highlighting the

protein residues that form the main interactions with the different structural units of the inhibitor. The dashed lines represent H-bonding and salt bridges.

S28

Figure S18. Best-scored docking solution of ligands 5 (up), 9 (middle), and 10 (down) into the

V 3

binding site (as Connolly surface) highlighting the complementarity between the ligands and the receptor. An evident hydrophobic hollow hosting the N -n-heptyl and N -propanoyl chains of ligands 9 and 10 is visible. The protein residues involved in the ligand-receptor binding are colored in red; the manganese ion sites are colored in magenta. Nonpolar hydrogen atoms are omitted for clarity.

S29

Figure S19. Superposition of docked structure 5 (red), 9 (blue), and 10 (green) to the X-ray

V 3-

bound conformation of 1 (yellow). The highly active, aminoproline-based ligands 5, 9, and 10 share a common cyclopeptide backbone arrangement, as well as similar C Arg-Asp distances. Hydrogen atoms are omitted for clarity.

S30

References 1. Baker, G. L.; Fritschel, S. J.; Stille, J. R.; Stille, J. K. J. Org. Chem. 1981, 46, 2954-2960. 2. Barraclough, P.; Hudhomme, P.; Spray, C. A.; Young, D. W. Tetrahedron 1995, 51, 4195-4212. 3. Nakamura, T.; Matsuyama, M.; Kamigata, N.; Iyoda, M. J. Org. Chem. 1992, 57, 3783-3789. 4. Fisher, A.; Mann, A.; Verma, V.; Thomas, N.; Mishra, R. K.; Johnson, R. L. J. Med. Chem. 2006, 49, 307-317. 5. Pollastri, M. P.; Sagal, J. F.; Chang, G. Tetrahedron Lett. 2001, 42, 2459-2460. 6. Webb, T. R.; Eigenbrot, C. J. Org. Chem. 1991, 56, 3009-3016. 7. Levatala, M. K.; Banerjee, S. R.; Maresca, K. P.; Babich, J. W.; Zubieta, J. Synthesis 2004, 1759-1766.

S31

Discovery of Subnanomolar Arginine-Glycine-Aspartate-Based Î±VÎ²3

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