Supporting Information for Synthesis of ... - Beilstein Journal

Supporting Information for Synthesis of acylhydrazino-peptomers, a new class of peptidomimetics, by consecutive Ugi and hydrazino-Ugi reactions Angélica de Fátima S. Barreto,* Veronica Alves dos Santos and Carlos Kleber Z. Andrade*

Address: Laboratório de Química Metodológica e Orgânica Sintética, Instituto de Química, Universidade de Brasília, CP 4478, 70910-970 Brasília-DF, Brazil.

Email: Carlos Kleber Z. Andrade* - [email protected] *Corresponding author

Detailed experimental procedures, NMR and mass spectra of all compounds General information

S2

General procedure for the preparation of hydrazides

S2

General procedure for the preparation of Boc-protected amido acids

S3

References

S14

Spectra of compounds

S15–S57

S1

Experimental General information NMR spectra were recorded on a Bruker Ascend instrument using a 5-mm internal diameter probe operating at 600 MHz for 1H and at 150 MHz for

13

C or

in a Varian Mercury Plus 300 spectrometer at 300 MHz for 1H and 75.46 MHz for

13

C both in the presence of TMS as internal standard. High resolution

ESI(+)-MS analyses were carried out on a triple TOF 5600+ (AB Sciex) with internal calibration and direct solution (1 ppm) infusion. Reactions under microwave were performed on a CEM Co. Discover microwave reactor using sealed vessels, dynamic program, and temperature detection by internal fiber optic probe and media stirring. TLC plates were revealed by treatment with a 10% solution of phosphomolybdic acid in ethanol, followed by heating. Melting points were recorded on a Marconi melting point and are uncorrected. Commercially available reagents and solvents were analytical grade or were purified by standard procedures prior to use. Compounds were analyzed by 1H NMR,

13

C NMR and high resolution ESI mass spectra giving data consistent

with the proposed structures.

General procedure for the preparation of hydrazides [1] The ester (10.0 mmol) was added in small portions to a solution of hydrazine hydrate (40.0 mmol) in 3.0 mL of ethanol. After refluxing for 3–6.5 h, the residue was concentrated in vacuum and purified by column chromatography.

S2

General procedure for the preparation of Boc-protected amino acids [2] The amino acid (10.0 mmol) was dissolved in dioxane/water (2:1, 30 mL), which was made alkaline with NaOH (1 M, 10 mL) and cooled in an ice-bath, and (Boc)2O (3.27 g, 15.0 mmol) and NaHCO3 (0.84 g, 10.0 mmol) were added. The reaction mixture was stirred overnight at room temperature and was then evaporated to half the volume. The residue was diluted with EtOAc (40 mL), cooled in an ice-bath and acidified to pH = 2.5–3.0 with KHSO4 (1 M). The layers were separated, the aqueous fraction was extracted with EtOAc (2 × 20 mL), and the combined organic extracts were washed with water, dried and evaporated to yield the respective Boc-Amino acid, which was used in the next step without further purification.

Cbz-glycine hydrazide (3a) [3] To a stirred solution of Cbz-glycine (1, 2.56 mmol, 0.54 g) in DMF (3.0 mL) was added sodium bicarbonate (3.38 mmol, 0,28 g) followed by methyl iodide (9.64 mmol, 0.60 mL). The mixture was stirred under nitrogen atmosphere for 46 h at room temperature. After this time, 30 mL of ethyl acetate was added and the mixture was washed with distilled water (three times). The organic phase was separated, dried with sodium sulfate and concentrated to yield product 2 (2.38 mmol, 0.53 g, 93% yield), which was used without further purification. Compound 3a was prepared following the general procedure for the preparation of hydrazide (refluxing for 3 h) using product 2 (4.93 mmol, 1.10 g), hydrazine hydrate (17.2 mmol, 0.86 g) and 1.4 mL of ethanol. Purification by column chromatography (CH2Cl2 → 10% MeOH/CH2Cl2) furnished 3a in 69% yield (0.76 g, 3.42 mmol) as a white solid. Rf (CH2Cl2/MeOH 15%) = 0.51. m.p = 112S3

114 ºC. 1H NMR (300 MHz, CD3OD) δ 7.37-7.27 (m, 5H), 5.09 (s, 2H), 3.76 (s, 2H).

13

C NMR (75.46 MHz, CDCl3) δ 171.4, 158.9, 138.0, 129.4, 129.0, 128.9,

67.8, 43.6.

Boc-glycine hydrazide (3b) [4] Compound 5 was prepared following the general procedure for the preparation of Boc-amino acids using glycine methyl ester hydrochloride (1.25 g, 10.0 mmol) in quantitative yields as a colorless oil, which was used without further purification. Compound 3b was then prepared following the general procedure for the preparation of hydrazide (refluxing for 3 h) using ester 5 (9.90 mmol, 1.87 g), hydrazine hydrate (39.6 mmol, 1.98 g) and 3.0 mL of ethanol. Purification by column chromatography (CH2Cl2 → 10% MeOH/CH2Cl2) furnished Boc-glycine hydrazide (3b) in 78% yield (1.45 g, 7.67 mmol) as a white solid. Rf (CH2Cl2/MeOH 15%) = 0.43. m.p = 105-107 ºC. 1H NMR (300 MHz, CD3OD) δ 3.68 (s, 2H), 1.44 (s, 9H).

13

C NMR (75.46 MHz, CD3OD) δ

171.9, 158.6, 80.9, 43.5, 28.8.

Hydrazide 3c Compound 7 was prepared in 73% yield as a viscous colorless oil following the general procedure for the preparation of Boc-Amino acids using amine 6 (1.46 g, 10.0 mmol) and was used without further purification. Compound 3c was then prepared following the general procedure for the preparation of hydrazide (refluxing for 3 h) using ester 7 (4.35 mmol, 1.07 g), hydrazine hydrate (17.4 mmol, 0.87 g) and 2.3 mL of ethanol. Purification by column chromatography (CH2Cl2 → 15% MeOH/CH2Cl2) furnished hydrazide 3c in 47% yield (0.51 g, S4

2.05 mmol) as a white solid. Rf (CH2Cl2/MeOH 15%) = 0.24. m.p = 44-46 ºC. 1H NMR (300 MHz, CD3OD) δ 3.87 (s, 2H), 3.73 (s, 2H), 1.46 (s, 9H).

13

C NMR

(75.46 MHz, CDCl3) δ 173.3, 170.9, 158.8, 81.0, 44.9, 42.4, 28.8. HRMS (ESI) m/z: calcd. for [M+Na]+ C9H18N4O4Na: 269.1226; found: 269.1217.

Compound 11a To a stirred solution of Cbz-glycine hydrazide (3a, 2.13 mmol, 0.476 g) in methanol (4.0 mL) was added isobutyraldehyde (8a, 2.13 mmol, 0.19 mL) and stirring was continued for 0.5 h at room temperature. After the solvent was evaporated and the imine was dissolved in trifluoroethanol (4.5 mL), acetic acid (10b, 2.13 mmol, 0.122 mL) and methyl isocyanoacetate (9, 2.13 mmol, 0.19 mL) were added. After stirring for 18 h at room temperature, the solution was concentrated

in

vacuum

and

the

residue

was

purified

by

column

chromatography (CH2Cl2 → 2% MeOH/CH2Cl2) to yield ester 11a (1.47 mmol, 0.64 g, 69% yield) as a beige solid. Rf (CH2Cl2/MeOH 10%) = 0.54. m.p = 103105 ºC. 1H NMR (600 MHz, CDCl3, presence of rotamers) δ 9.09-8.89 (2 br s, 1H), 7.35-7.30 (m, 5H), 7.13 (br s, 1H), 5.82 and 5.58 (2 br s, 1H), 5.13 (s, 2H), 4.72 (d, J = 9.5 Hz, 1H), 4.06-3.84 (m, 4H), 3.72 (s, 3H), 2.24-2.12 (m, 1H), 2.07 (s, 3H), 1.02-0.91 (m, 6H).13C NMR (150 MHz, CDCl3, presence of rotamers): δ 174.2; 171.2; 170.3; 168.3; 156.6; 136.0; 128.5; 128.0; 67.3; 64.3; 52.4; 43.3; 40.9; 27.2; 20.9; 19.8; 19.3. HRMS (ESI) m/z: calcd. for [M+Na]+ C20H28N4O7Na: 459.1856; found: 459.1848.

S5

Compound 11b To a stirred solution of Boc-glycine hydrazide (3b, 0.50 mmol, 0.094 g) in methanol (1.0 mL) was added isobutyraldehyde (8a, 0.50 mmol, 0.45 mL) and stirring was continued for 2 h at room temperature. After the solvent was evaporated and the imine was dissolved in trifluoroethanol (2.0 mL), acetic acid (10b, 0.25 mmol, 0.014 mL) and methyl isocyanoacetate (9, 0.25 mmol, 0.023 mL) were added. After stirring for 43 h at room temperature, the solution was concentrated

in

vacuum

and

the

residue

was

purified

by

column

chromatography (CH2Cl2 → 2% MeOH/CH2Cl2) to yield ester 11b (0.293 mmol, 0.118 g, 59%) as a white solid. Rf (CH2Cl2/MeOH 10%) = 0.51. m.p. = 160-162 ºC. 1H NMR (300 MHz, CDCl3, presence of rotamers) δ 8.99 (br s, 1H), 6.83 (br s, 1H), 5.16 (br s, 1H), 4.64 (br s, 1H), 3.97 (s, 2H), 3.82 (s, 2H), 3.73 (s, 3H), 2.27-2.14 (m, 1H), 2.07 (s, 3H), 1.42 (s, 9H), 1.02 (s, 3H), 0.95 (d, J = 6.6 Hz, 3H).

13

C NMR (150 MHz, DMSO-d6, presence of rotamers): δ 172.3, 170.8,

169.9, 169.1, 155.8, 78.3, 63.4, 51.6, 42.4, 40.5, 28.1, 26.9, 20.6, 19.1, 18.7. HRMS (ESI) m/z: calcd. for [M+Na]+ C19H27N3O6Na: 425.2012; found: 425.2012.

Compound 11c To a stirred solution of hydrazine 3b (1.59 mmol, 0.30 g) in trifluoroethanol (2.0 mL) was added ketone 8b (3.18 mmol, 0.23 mL) and stirring was continued for 2 h at room temperature. Then were added sodium sulfate (0.30 g), propanoic acid (10c, 0.79 mmol, 0.059 mL) and methyl isocyanoacetate (9, 0.79 mmol, 0.072 mL). After stirring for 40 h at room temperature, the solution was concentrated

in

vacuum

and

the

residue

was

purified

by

column S6

chromatography (CH2Cl2 → 4% MeOH/CH2Cl2) to yield ester 11c (0.714 mmol, 0.287 g, 90%) as a viscous light yellow oil. Rf (CH2Cl2/MeOH 10%) = 0.47. 1H NMR (600 MHz, CDCl3, presence of rotamers) δ 9.65 (br s, 1H), 8.73 (br s, 1H), 5.70 (br s, 1H), 4.11 (dd, J = 5.9 and 18.0 Hz, 1H), 3.97-3.96 (m, 2H), 3.92 (dd, J = 5.1 and 18.0 Hz, 1H), 3.71 (s, 3H), 2.42-2.36 and 2.25-2.19 (2m, 2H), 1.45 (s, 12H), 1.36 (s, 3H), 0.99 (t, J = 7.3 Hz, 3H).

13

C NMR (150 MHz, CDCl3,

presence of rotamers): δ 175.6, 175.1, 172.6, 170.3, 156.2, 80.4, 64.5, 52.1, 42.9, 41.6, 28.2, 26.2, 24.5, 22.2, 8.3. HRMS (ESI) m/z: calcd. for [M+Na]+ C17H30N4O7Na: 425.2012; found: 425.2013.

Compound 11d To a stirred solution of hydrazine 3b (0.50 mmol, 0,094 g) in trifluoroethanol (1.0 mL) was added ketone 8b (1.0 mmol, 0.073 mL) and stirring was continued for 1 h at room temperature. Then were added sodium sulfate (0.50 g), formic acid (10a, 0.25 mmol, 0.009 mL) and methyl isocyanoacetate (9, 0.25 mmol, 0.023 mL). After stirring for 24 h at room temperature, the solution was filtrated, concentrated

in

vacuum

and

the

residue

was

purified

by

column

chromatography (CH2Cl2 → 4% MeOH/CH2Cl2) to yield ester 11d (0.25 mmol, 0.084 g, 90%) as a viscous light yellow oil. Rf (CH2Cl2/MeOH 10%) = 0.48. 1H NMR (600 MHz, CDCl3, presence of rotamers) δ 9.50 (br s, 1H), 8.69 and 8.59 (2 br s, 1H), 8.29 and 8.00 (2s, 1H), 5.52 (br s, 1H), 4.03-3.96 (m, 4H), 3.73 (s, 3H), 1.58 (s, 3H), 1.46-1.44 (m, 12H).

13

C NMR (150 MHz, CDCl3, presence of

rotamers): δ 174.4, 170.4, 164.2, 161.4, 156.1, 80.4, 63.9, 52.1, 42.6, 41.5, 28.2, 25.5, 23.6. HRMS (ESI) m/z: calcd. for [M+Na]+ C15H26N4O7Na: 397.1699; found: 397.1703. S7

Compound 11e To a stirred solution of hydrazine 3c (2.13 mmol, 0,524 g) in methanol (4.0 mL) was added isobutyraldehyde (8a, 2.13 mmol, 0.19 mL) and stirring was continued for 1 h at room temperature. After the solvent was evaporated and the imine was dissolved in TFE (4.0 mL), were added acetic acid (10b, 2.13 mmol, 0.12 mL) and methyl isocyanoacetate (9, 2.13 mmol, 0.19 mL). After stirring for 48 h at room temperature, the solution was concentrated in vacuum and the residue was purified by column chromatography (CH2Cl2 → 2% MeOH/CH2Cl2) to yield the ester 11e (1.55 mmol, 0.710 g, 73%) as a yellow solid. Rf (CH2Cl2/MeOH 10%) = 0.38. m.p = 57-59 ºC. 1H NMR (600 MHz, CDCl3, presence of rotamers) δ 9.32 and 9.25 (2s, 1H), 7.53 and 7.49 (2s, 1H), 5.63 and 5.53 (2s, 1H), 4.61 (d, J = 9.2 Hz, 1H), 4.00 and 3.87 (2 br s, 6H), 3.75 (s, 3H), 2.25-2.15 (m, 1H), 2.08 (s, 3H), 1.44 (s, 9H), 1.10-0.97 (m, 6H).

13

C

NMR (150 MHz, CDCl3, presence of rotamers): δ 174.1, 171.0, 170.4, 169.7, 168.3, 156.5, 80.5, 65.2, 52.4, 44.2, 41.9, 41.0, 27.2, 20.9, 19.8, 19.4, 19.2. HRMS (ESI) m/z: calcd. for [M+Na]+ C19H33N5O8Na: 482.2227; found: 482.2228.

Compound 11f To a stirred solution of hydrazine 3c (1.62 mmol, 0,398 g) in methanol (3.0 mL) was added isobutyraldehyde (8a, 1.62 mmol, 0.15 mL) and stirring was continued for 1 h at room temperature. After the solvent was evaporated and the imine was dissolved in TFE (3.0 mL), were added propanoic acid (10c, 1.62 mmol, 0.12 mL) and methyl isocyanoacetate (9, 1.62 mmol, 0.15 mL). After stirring for 51 h at room temperature, the solution was concentrated in vacuum S8

and the residue was purified by column chromatography (CH2Cl2 → 4% MeOH/CH2Cl2) to yield ester 11f (1.15 mmol, 0.543 g, 71%) as a light yellow solid. Rf (CH2Cl2/MeOH 10%) = 0.46. m.p = 53-55 ºC. 1H NMR (600 MHz, CDCl3, presence of rotamers) δ 9.27 and 9.20 (2 br s, 1H), 7.58 (br s, 1H), 7.33 (br s, 1H), 5.68 and 5.57 (2 br, 1H), 4.63 (s, 1H), 4.20-3.78 (m, 6H), 3.74 (s, 3H), 2.47-2.31 (m, 2H), 2.27-2.17 and 2.14-2.05 (2m, 1H), 1.44 (s, 9H), 1.140.91 (m, 9H).

13

C NMR (150 MHz, CDCl3, presence of rotamers): δ 177.1,

171.3, 170.4, 169.6, 168.4, 156.4, 80.5, 65.6, 52.4, 44.2, 41.9, 41.0, 28.3, 27.3, 25.7, 20.0, 19.5, 8.6. HRMS (ESI) m/z: calcd. for [M+Na]+ C20H35N5O8Na: 496.2383; found: 496.2379.

Hydrazide 12a Compound 12a was prepared following the general procedure for the preparation of hydrazides (refluxing 5 h) using ester 11b (0.71 mmol, 0.286 g), hydrazine hydrate (2.84 mmol, 0,14 mL) and 3.3 mL of ethanol. Purification by column chromatography (CH2Cl2 → 15% MeOH/CH2Cl2) furnished hydrazide 12a in 72% yield (0.51 mmol, 0.205 g) as a white solid. Rf (CH2Cl2/MeOH 15%) = 0.29. m.p = 59-61 ºC. 1H NMR (600 MHz, CD3OD, presence of rotamers) δ 4.63 (d, J = 9.5 Hz, 1H), 3.96-3.68 (m, 4H), 2.24-2.12 (m, 1H), 1.45 (s, 9H), 1.01-0.89 (m, 6H).

13

C NMR (150 MHz, CD3OD, presence of rotamers) δ 175.9,

173.1, 171.7, 170.6, 158.6, 80.9, 64.9, 43.4, 42.9, 28.8, 27.9, 20.9, 20.5, 19.5. HRMS (ESI) m/z: calcd. for [M+Na]+ C16H30N6O6Na: 425.2125; found: 425.2124.

S9

Hydrazide 12b Compound 12b was prepared following the general procedure for the preparation of hydrazides (refluxing 5 h) using ester 11f (0.890 mmol, 0.421 g), hydrazine hydrate (3.56 mmol, 0.17 mL) and 4.0 mL of ethanol. Purification by column chromatography (CH2Cl2 → 15% MeOH/CH2Cl2) furnished hydrazide 12b in 72% yield (0.64 mmol, 0.304 g) as a white solid. Rf (CH2Cl2/MeOH 15%) = 0.31. m.p = 101-103 ºC. 1H NMR (600 MHz, CD3OD) δ 4.67 (d, J = 9.5 Hz, 1H), 4.04-3.66 (m, 6H), 2.46-2.40 (m, 1H), 2.32-2.13 (m, 2H), 1.45 (s, 9H), 1.07 (t, J = 7.5 Hz, 3H), 1.02-0.91 (m, 6H).

13

C NMR (150 MHz, CD3OD) δ 178.6,

173.4, 172.1, 171.7, 170.5, 158.6, 80.7, 64.8, 44.7, 42.8, 42.2, 28.7, 27.8, 26.7, 20.3, 19.4, 9.4. HRMS (ESI) m/z: calcd. for [M+H]+ C19H36N7O7: 474.2676; found: 474.2670.

Compound 14a To a stirred solution of hydrazide 12a (0.472 mmol, 0.190 g) in trifluoroethanol (2.0 mL) was added ketone 8b (1.89 mmol, 0.14 mL) and stirring was continued for 1 h at room temperature. Then were added propanoic acid (10c, 0.472 mmol, 0.035 g) and methyl isocyanoacetate (9, 0.472 mmol, 0.043 mL). After stirring for 45 h at room temperature, the solution was concentrated in vacuum and the residue was purified by column chromatography (CH2Cl2 → 8% MeOH/CH2Cl2) to yield compound 14a (0.281 mmol, 0.173 g, 60% yield) as a yellow solid. Rf (CH2Cl2/MeOH 10%) = 0.48. m.p = 118-120 ºC. 1H NMR (600 MHz, CD3OD) δ 4.64 (d, J = 9.5 Hz, 1H), 4.31-3.74 (m, 6H), 3.72 and 3.69 (2s, 3H), 2.43-2.32 (m, 1H), 2.26-2.13 (m, 2H), 2.07-2.02 (m, 3H), 1.46-1.31 (m, 15H), 1.03-0.88 (m, 9H).

13

C NMR (150 MHz, DMSO-d6) δ 175.3, 173.8, 173.6, S10

172.1, 170.1, 169.2, 168.7, 155.8, 78.2, 63.5, 61.5, 51.6, 51.4, 48.7, 40.8, 28.0, 25.7, 24.0, 22.4, 20.6, 19.3, 18.8, 8.4. HRMS (ESI) m/z: calcd. for [M+Na]+ C26H45N7O10Na: 638.3126; found: 638.3115.

Compound 14b To a stirred solution of hydrazide 12b (0.361 mmol, 0.171 g) in trifluoroethanol (2.0 mL) was added ketone 8b (1.44 mmol, 0.11 mL) and stirring was continued for 3 h at room temperature. Then were added propanoic acid (10c, 0.180 mmol, 0.013 mL) and methyl isocyanoacetate (9, 0.180 mmol, 0.016 mL). After stirring for 40 h at room temperature, the solution was concentrated in vacuum and the residue was purified by column chromatography (CH2Cl2 → 8% MeOH/CH2Cl2) to yield compound 14b (0.161 mmol, 0.076 g, 90% yield) as a yellow solid. Rf (CH2Cl2/MeOH 10%) = 0.39. m.p = 107-109 ºC. 1H NMR (600 MHz, CDCl3) δ 9.81 and 9.74 (2br s, 1H), 9.67 and 9.63 (2br s, 1H), 8.88 and 8.79 (2 br s, 1H), 8.58 and 8.26 (2 br s, 1H), 7.83 and 7.76 (2 br s, 1H), 5.88 and 5.81 (2 br s, 1H), 4.60 and 4.50 (s, 1H), 4.10-3.75 (m, 8H), 3.73 and 3.71 (2s, 3H), 2.49-2.39 (m, 1H), 2.35-2.18 (m, 4H), 1.46-1.34 (m, 15H), 1.06-0.92 (m, 12H).

13

C NMR (150 MHz, CDCl3) δ 175.4; 175.2; 174.8; 171.4; 171.2;

170.8; 170.5; 170.1; 169.3; 80.9; 71.8; 64.5; 52.1; 49.4; 44.6; 42.2; 41.7; 41.2; 29.7; 28.3; 24.9; 22.0; 19.2; 14.1; 8.7. HRMS (ESI) m/z: calcd. for [M+Na]+ C29H50N8O11Na: 709.3497; found: 709.3495.

Compound 15a A sealed 10 mL glass tube containing a solution of ester 11a (0.47 mmol, 0.205 g) in THF/H2O (2:1, 7.5 mL) and LiOH (1.18 mmol, 0.028 g) at room S11

temperature was introduced in the cavity of a microwave reactor (CEM Co., Discover) and irradiated at 60 ºC for 5 min under magnetic stirring. The reaction mixture was then acidified with a 2 M solution of NaHSO 4 to pH 2 and extracted twice with ethyl acetate (2 × 30 mL). The organic phase was dried with sodium sulfate, filtered and concentrated to yield acid 13a (0.44 mmol, 0.185 g, 94%), which was used without further purification. A mixture of acid 13a (0.44 mmol, 0.185

g),

methanol

(1.5

mL),

anhydrous

sodium

sulfate

(0.20

g),

paraformaldehyde (0.88 mmol, 0.0264 g), benzylamine (0.88 mmol, 0.094 g) and methyl isocyanoacetate (0.44 mmol, 0.040 mL) was introduced in the cavity of a microwave reactor (CEM Co., Discover) and irradiated at 80 ºC for 3 min under magnetic stirring. The product was filtered, concentrated in vacuum and purified by column chromatography (CH2Cl2 → 4% MeOH/CH2Cl2) to yield compound 15a (0.207 mmol, 0.133 g, 47% yield) as a yellow solid. Rf (CH2Cl2/MeOH 10%) = 0.53. m.p = 87-89 ºC. 1H NMR (600 MHz, CDCl3) δ 9.90 and 9.57 (2 br s, 1H), 7.42 and 7.11 (m, 12H), 5.82 and 5.66 (2 br s, 2H), 5.10 (s, 2H), 4.82 (br s, 1H), 4.66-4.59 (m, 2H), 4.10-3.84 (m, 8H), 3.71 (s, 3H), 2.082.00 (m, 4H), 1.06-0.94 (m, 6H).

13

C NMR (150 MHz, CDCl3) δ 171.0, 170.4,

170.3, 170.1, 168.5, 167.9, 156.7, 136.1, 134.7, 129.2, 128.9, 128.6, 128.3, 128.2, 126.8, 67.3, 64.4, 52.4, 51.8, 49.7, 43.5, 41.2, 41.0, 29.7, 27.1, 20.9, 19.6. HRMS (ESI) m/z: calcd. for [M+Na]+ C31H40N6O9Na: 663.2754; found: 663.2755.

Compound 15b To a solution of ester 11b (1.96 mmol, 0.788 g) in THF/H2O (2:1, 69 mL) was added LiOH (9.8 mmol, 0.235 g) at 0 ºC. The reaction was stirred for 2.5 h at 0 S12

ºC, acidified with a 2 M solution of NaHSO4 to pH 2 and extracted three times with ethyl acetate. The combined organic phases were dried with sodium sulfate and concentrated to yield the respective acid 13b (1.96 mmol, 0.761 g, quantitative yield), which was used without further purification. A sealed 10 mL glass tube containing a mixture of acid 13b (1.63 mmol, 0.633 g), methanol (2.0 mL), benzylamine (3.25 mmol, 0.348 g), anhydrous sodium sulfate (0.975 g), paraformaldehyde (3.25 mmol, 0.0975 g) and methyl isocyanoacetate (9, 1.63 mmol, 0.15 mL) was introduced in the cavity of a microwave reactor (CEM Co., Discover) and irradiated at 80 ºC for 3 min (ramp time: 100 s) under magnetic stirring. The residue was filtered, concentrated in vacuum and purified by column chromatography (CH2Cl2 → 4% MeOH/CH2Cl2) to yield compound 15b (1.23 mmol, 0.748 g, 76% yield) as a yellow solid. Rf (CH2Cl2/MeOH 15%) = 0.53. m.p = 79-81 ºC. 1H NMR (600 MHz, CDCl3) δ 9.96 and 9.59 (2s, 1H), 7.37-7.22 (m, 5H), 4.84-4.76 (m, 1H), 4.71-4.60 (m, 2H), 4.25-3.78 (m, 8H), 3.74 (s, 3H), 2.16-1.95 (m, 4H), 1.43 (s, 9H), 1.11-1.02 (m, 2H), 0.95 (d, J = 6.2 Hz, 4H). 13C NMR (150 MHz, CDCl3) δ 174.0, 170.9, 170.3, 168.9, 168.5, 168.0, 156.2, 134.8, 129.1, 128.8, 128.4, 128.0, 126.8, 80.4, 64.7, 52.4, 51.7, 49.5, 43.1, 41.2, 41.1, 28.2, 27.2, 20.9, 19.6. HRMS (ESI) m/z: calcd. for [M+Na]+ C28H42N6O9Na: 629.2911; found: 629.2905.

Compound 15c To a solution of ester 11c (0.803 mmol, 0.323 g) in THF/H2O (2:1, 39 mL) was added LiOH (0.401 mmol, 0.096 g) at 0 ºC. The reaction was stirred for 2 h at 0 ºC, acidified with a 2 M solution of NaHSO4 to pH 2 and extracted three times with ethyl acetate. The combined organic phases were dried with sodium sulfate S13

and concentrated to yield the respective acid 13c (0.775 mmol, 0.301 g, 96% yield), which was used without further purification. To a solution of benzylamine (1.55 mmol, 0.166 g) in methanol (10 mL) were added sodium sulfate (0.20 g) and paraformaldehyde (1.55 mmol, 0.0465 g) and stirring was continued for 1 h at rt. Acid 13c (0.775 mmol, 0.301 g) was added and after 10 min methyl isocyanoacetate (0.775 mmol, 0.070 mL) was added. The reaction was stirred for 24 h at rt. After filtration, the solution was concentrated in vacuum and the residue was purified by column chromatography (CH2Cl2 → 4% MeOH/CH2Cl2) to yield compound 15c (0.425 mmol, 0.258 g, 55% yield) as a yellow solid. Rf (CH2Cl2/MeOH 10%) = 0.44. m.p = 84-86 ºC. 1H NMR (600 MHz, CDCl3, presence of rotamers) δ 9.83 (br s, 1H), 8.58 (br s, 1H), 7.48 and 7.34 (2 br s, 1H), 7.28 and 7.10 (m, 6H), 5.87 and 5.78 (br s, 1H), 4.71-4.54 (m, 2H), 4.173.82 (m, 8H), 3.63 and 3.61 (2s, 3H), 2.33-2.26 and 2.16-2.09 (2m, 2H), 1.371.20 (m, 15H), 0.89-0.86 (m, 3H).

13

C NMR (150 MHz, CDCl3, presence of

rotamers) δ 175.2, 172.0, 170.4, 170.1, 169.2, 168.6, 156.5, 135.3, 129.1, 128.7, 128.4, 128.0, 126.8, 80.6, 64.6, 52.3, 50.0, 49.4, 42.9, 41.8, 41.1, 28.3, 26.5, 24.4, 22.6, 8.4. HRMS (ESI) m/z: calcd. for [M+Na]+ C28H42N6O9Na: 629.2911; found: 629.2903.

References 1. Khan, K. M.; Rasheed, M.; Ullah, Z.; Hayat, S.; Kaukab, F.; Choudhary, M. I.; Rahman, A.-U.; Perveen, S. Bioorg. Med. Chem. 2003, 11, 1381-1387. 2. Caplar, V.; Zinir, M.; Pozzo, J.-L.; Fages, F.; Mieden-Gundert, G.; Vogtle, F. Eur. J. Org. Chem. 2004, 4048-4059. 3. Ottersbach, P. A.; Schnakenburg, G.; Gϋtschow, M. Chem. Commun. 2012, 48, 5772-5774. 4. Gaccioli, F.; Franchi-Gazzola, R.; Lanfranchi, M.; Marchio, L.; Metta, G.; Pellinghelli, M. A.; Tardito, S.; Tegoni, M. J. Inorg. Biochem. 2005, 99, 15731584. S14

Spectra of compounds

1.0 Cbz-Glicina-Me-Hidrazina-1H

O H2N

0.9

N H

0.8

NHCbz 3a

Normalized Intensity

0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 5.00 8.0

7.5

2.25 7.0

6.5

6.0

5.5

5.0

2.04 4.5 4.0 3.5 Chemical Shift (ppm)

3.0

2.5

2.0

1.5

1.0

0.5

0

Figure S1: 1H NMR (300 MHz, CD3OD) spectrum of compound 3a.

S15

O H2N

N H

NHCbz 3a

Figure S2: 13C NMR (75.46 MHz, CD3OD) spectrum of compound 3a.

S16

V-09.001.esp 0.55

O

0.50

H2N

Methanol 0.45


0.40

N H 3b

NHBoc

0.35 0.30 0.25 0.20 0.15 0.10

Methanol

0.05 0 1.85 7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5 3.0 Chemical Shift (ppm)

9.00 2.5

2.0

1.5

1.0

0.5

0

-0.5

Figure S3: 1H NMR (300 MHz, CD3OD) spectrum of compound 3b.

S17

28.82

V-09.013.esp

O H2N

0.07

N H 3b

NHBoc

0.05

43.49


0.06

0.04

80.88

0.02

158.55

171.91

0.03

0.01

200

192

184

176

168

160

152

144

136

128

120

112 104 96 88 Chemical Shift (ppm)

80

72

64

56

48

40

32

24

16

8

0

Figure S4: 13C NMR (75.46 MHz, CD3OD) spectrum of compound 3b.

S18

1.0 R-40-1H

H N

BocHN

0.9

O

0.8

O N H

NH2

3c


0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 1.99 2.01 7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5 Chemical Shift (ppm)

9.35 3.0

2.5

2.0

1.5

1.0

0.5

0

Figure S5: 1H NMR (300 MHz, CD3OD) spectrum of compound 3c.

S19

R-40=13C 0.8

0.7

BocHN

0.6

O

N H

28.79

NH2

3c

0.5

0.4

81.04

173.26 170.92

0.2

0.1

44.92 42.36

0.3

158.75


O

H N

0 176

168

160

152

144

136

128

120

112

104

96 88 80 Chemical Shift (ppm)

72

64

56

48

40

32

24

16

8

0

Figure S6: 13C NMR (75.46 MHz, CD3OD) spectrum of compound 3c.

S20

1.0 Angelica_R-69.001.esp 0.9

O

0.8

MeO


0.7

H N

N

11a O

0.6

H N O

NHCbz O

0.5 0.4 0.3 0.2 0.1 0 0.95 9.5

9.0

5.14 0.56 8.5

8.0

7.5

7.0

0.89 6.5

6.0

2.00 5.5

0.66


4.11 2.90 4.0

3.91 3.5

3.0

2.5

2.0

6.00 1.5

1.0

0.5

0

Figure S7: 1H NMR (300 MHz, CDCl3, presence of rotamers) spectrum of compound 11a.

S21

128.49 128.00

Angelica_R69-13C.013.esp

MeO

H N

N

11a O

H N O

0.20

NHCbz O

220

210

200

190

180

170

160

140

27.21

43.28 40.91

52.37

136.01 150

64.34

0.05

156.59

174.16 171.23 170.30 168.33

0.10

19.29

0.15

67.29


0.25

130

120


80

70

60

50

40

30

20.87 19.82

O

20

10

0

-10

-20

Figure S8: 13C NMR (75.46 MHz, CDCl3, presence of rotamers) spectrum of compound 11a.

S22

O MeO

H N 11a O

N

H N O

NHCbz O

Calc. [M+Na]+ : 459.1856 Calc. [M+H]+ : 437.2036

Figure S9: ESI-HRMS of compound 11a.

S23

0.7

Angelica_R-81.001.esp

O

H N

0.6

MeO


N

11b O

0.5

H N O

NHBoc O

0.4

0.3

0.2

0.1

0 0.69 9.0

0.66 8.5

8.0

7.5

7.0

0.68 6.5

6.0

5.5

1.03

3.89 3.31

5.0 4.5 4.0 Chemical Shift (ppm)

0.93 3.29 3.5

3.0

2.5

2.0

9.00

5.59

1.5

1.0

0.5

0

Figure S10: 1H NMR (300 MHz, CDCl3, presence of rotamers) spectrum of compound 11b.

S24

28.06

11b O

H N O

40.48

51.58

O

NHBoc

168

160

152

144

136

128

120

112

104

96

88 80 Chemical Shift (ppm)

42.43

63.35

78.34

155.84

169.09

172.29

0.05

72

64

56

48

40

32

19.10

N

18.66

H N

MeO

20.60

0.10

26.90

O

169.94


Angelica_R81.013.esp

24

16

8

0

Figure S11: 13C NMR (75.46 MHz, DMSO-d6, presence of rotamers) spectrum of compound 11b.

S25

O MeO

H N 11b O

N

H N O

NHBoc O

Calc. [M+Na]+ : 425.2012

Figure S12: ESI-HRMS of compound 11b.

S26

V-18-1H.001.esp 0.45

O

0.40

MeO


0.35

H N

11c

N O

H N

O

NHBoc O

0.30 0.25 0.20 0.15 0.10 0.05 0 0.86 9.5

1.00 9.0

0.91 8.5

8.0

7.5

7.0

6.5

6.0

4.42 2.91 5.5


4.0

2.59 3.5

3.0

2.5

12.58 2.82 3.00 2.0

1.5

1.0

0.5

0

Figure S13: 1H NMR (600 MHz, CDCl3, presence of rotamers) spectrum of compound 11c.

S27

28.18 64.53

V-18-13C

O

O 52.07

O

NHBoc

24.53

0.20

42.87

80.35

156.16

175.06

0.15

0.10

8.30

11c

0.25

N

22.15

170.28

MeO

H N

172.58


0.30

H N

26.22

O

41.58

0.35

175.56

0.40

0.05

0 176

168

160

152

144

136

128

120

112

104


72

64

56

48

40

32

24

16

8

0

Figure S14: 13C NMR (150 MHz, CDCl3, presence of rotamers) spectrum of compound 11c.

S28

O MeO

H N

11c

N O

H N O

NHBoc O

Calc. [M+Na]+ : 425.2012

Figure S15: ESI-HRMS of compound 11c.

S29

1.0 V-19-1H.esp

O

0.9

MeO

0.8

N

11d O H

0.7


H N

H N O

NHBoc O

0.6 0.5 0.4 0.3 0.2 0.1 0 0.48 9.5

1.13 9.0

8.5

1.32 8.0

1.17 7.5

7.0

6.5

6.0

5.5


4.0

3.47 11.87 3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

Figure S16: 1H NMR (600 MHz, CDCl3, presence of rotamers) spectrum of compound 11d.

S30

28.15

V-19-13C

O MeO

0.55

H N

N

11d O H

0.50

H N O

NHBoc O

0.40 0.35

25.54 23.63

42.61

0.10

41.50

63.94 164.16 161.38

0.15

80.41

0.20

156.14

174.36

0.25

52.09

0.30

170.35


0.45

0.05 0 200

192

184

176

168

160

152

144

136

128

120

112


80

72

64

56

48

40

32

24

16

8

0

Figure S17: 13C NMR (150 MHz, CDCl3, presence of rotamers) spectrum of compound 11d.

S31

O MeO

H N

N

11d O H

H N O

NHBoc O

+

Calc. [M+Na] : 397.1699

Figure S18: ESI-HRMS of compound 11d.

S32

Angelica_R-100-1.001.esp

O

0.45

MeO

H N

0.40

N O

11e

O

H N O

O

N H

NHBoc


0.35 0.30 0.25 0.20 0.15 0.10 0.05 0 0.80 10.0

9.5

1.23 9.0

8.5

8.0

7.5

0.82 7.0

6.5

6.0

5.5


5.92 2.92 4.0

0.76 2.72 3.5

3.0

2.5

2.0

9.02 1.5

6.16 1.0

0.5

0

-0.5

Figure S19: 1H NMR (600 MHz, CDCl3, presence of rotamers) spectrum of compound 11e.

S33

O

H N

Angelica_R-100-13C.013.esp

MeO

O

O

NHBoc

N H

0.15

220

210

200

190

180

170

160

150

140

130

120


80

52.42

65.25

80.51

156.49

174.07 171.05 170.43 169.66 168.26

0.05

70

60

50

40

27.25 20.94 19.85 19.42 19.20

0.10

44.19 41.90 41.02


O

11e

0.20

N

O

H N

30

20

10

0

-10

-20

Figure S20: 13C NMR (150 MHz, CDCl3, presence of rotamers) spectrum of compound 11e.

S34

O

H N

MeO 11e

N O

O

H N O

O

N H

NHBoc

Calc. [M+Na]+ : 482.2227

Figure S21: ESI-HRMS of compound 11e.

S35

0.7


O MeO

0.6

11f

0.5


H N

N O

O

H N O

O

N H

NHBoc

0.4

0.3

0.2

0.1

0 0.83 9.5

1.69 9.0

8.5

8.0

7.5

0.77 7.0

6.5

6.0

5.5


5.89 3.10 4.0

1.59 1.25 3.5

3.0

2.5

2.0

9.00 1.5

9.29 1.0

0.5

0

Figure S22: 1H NMR (600 MHz, CDCl3, presence of rotamers) spectrum of compound 11f.

S36

28.26

Angelica_R-107-13C.013.esp

O

0.45

MeO

0.40


H N 11f

0.35

N O

O

H N O

O

N H

NHBoc

0.30 0.25 0.20

176

168

160

152

144

136

128

120

112

104


72

64

56

48

40

32

24

8.56

27.28 25.73 19.95 19.48

44.21 41.94 41.05

52.44

65.55

80.49

156.36

0.05

171.27 170.41 169.55 168.37

0.10

177.14

0.15

16

8

0

Figure S23: 13C NMR (150 MHz, CDCl3, presence of rotamers) spectrum of compound 11f.

S37

O

H N

MeO 11f

N O

O

H N O

O

N H

NHBoc

Calc. [M+Na]+ : 496.2383

Figure S24: ESI-HRMS of compound 11f.

S38

Angelica-R-139.001.esp

0.25

O


H2N 0.20

N H

Methanol

H N 12a

N O

H N O

NHBoc Methanol

O

0.15

0.10

0.05

0 0.50 7.5

7.0

6.5

6.0

5.5

5.0

4.5


1.00 2.71 3.0

2.5

2.0

8.82 1.5

6.00 1.0

0.5

0


S39

28.82


0.055 0.050

O


0.045

H2N

0.040

N H

0.035

H N 12a

N O

H N O

NHBoc O

0.030 0.025

0.005

220

210

200

190

180

170

160

150

140

130

120


80

70

27.92 20.95 20.51 19.54

0.010

43.36 42.92

80.90

175.86 173.13 171.69 170.59

0.015

64.94

158.60

0.020

60

50

40

30

20

10

0

-10

-20

Figure S26: 13C NMR (150 MHz, CD3OD, presence of rotamers) spectrum of compound 12a.

S40

R-112.001.esp

O H2N

0.30

N H

H N 12b


0.25

N O

O

H N O

O

N H

NHBoc

0.20

0.15

0.10

0.05

0 0.50 7.5

7.0

6.5

6.0

5.5

5.0

5.80 4.5

4.0


0.87 1.82 2.5

9.00 2.0

1.5

2.97 5.74 1.0

0.5

0

-0.5

Figure S27: 1H NMR (600 MHz, CD3OD) spectrum of compound 12b.

S41

28.73

R-112.013.esp

O H2N 0.06

N H

H N 12b

N O

O

H N O

O

N H

NHBoc

0.04

184

176

168

160

152

144

136

128

120

112

104


72

64

20.29

9.35

19.45

27.85 26.69

42.84 42.20

64.80

158.61

173.42 172.14 171.67 170.49

178.63

0.02

0.01

44.74

0.03

80.89


0.05

56

48

40

32

24

16

8

0

Figure S28: 13C NMR (150 MHz, CD3OD) spectrum of compound 12b.

S42


O

0.30

MeO


N H

O

0.25

O

O N

N H 14a

H N

N O

H N O

NHBoc O

0.20

0.15

0.10

0.05

0 1.07 7.5

7.0

6.5

6.0

5.5

5.0

4.5

6.15

3.05


0.60 1.65 3.25 3.0

2.5

2.0

15.40 1.5

9.01 1.0

0.5

0

-0.5


S43

27.99

Angelica-R145-13C.013.esp

0.055

N H 14a

0.030 0.025

0.005

O

O

NHBoc O

160

78.22 152

144

136

128

120

112

104


72

64

51.56 48.66

168

63.50 61.51

176

155.76 169.21 168.69

0.010

175.26 173.82 173.55 172.11

0.020 0.015

N

H N

56

48

40

32

24

16

8.43

O

N H

H N

24.03 22.35 20.57 19.32 18.77

0.035

O N

25.72

MeO

0.040

O

40.75

O

170.12


0.045

51.44

0.050

8

0

Figure S30: 13C NMR (150 MHz, DMSO-d6) spectrum of compound 14a.

S44

O MeO O

O

N H

O N

H N

N H 14a

Calc.

[M+Na]+ :

N O

H N O

NHBoc O

638.3126


S45

1.0 Angelica_R-163.001.esp

O

0.9

MeO 0.8

O

O

O N

N H

N H

14b

0.7


H N

N O

O

H N O

O

N H

NHBoc

0.6 0.5 0.4 0.3 0.2 0.1 0 1.49 10.5

10.0

0.95 9.5

9.0

8.5

1.67 8.0

1.07 7.5

7.0

6.5

6.0

1.09


8.45 3.01 4.0

1.41 3.72 3.5

3.0

2.5

14.61 2.0

1.5

11.81 1.0

0.5

0

Figure S32: 1H NMR (600 MHz, CDCl3) spectrum of compound 14b.

S46

Angelica-R142.013.esp 0.13

0.12

0.11

0.10


0.09

O MeO

0.08

O N

N H

O

O

H N

N H

14b

0.07

N O

O

H N O

O

N H

NHBoc

0.06

0.05

0.04

0.03

0.02

0.01

0 176

168

160

152

144

136

128

120

112

104


72

64

56

48

40

32

24

16

8

0

-8

Figure S33: 13C NMR (150 MHz, CDCl3) spectrum of compound 14b. S47

O MeO O

N H

O

O N

N H

H N 14b

N O

O

H N O

O

N H

NHBoc

Calc. [M+Na]+ : 709.3497


S48


O

0.9

MeO

0.8

O

H N

N O

H N

N O

0.7

H N O

NHCbz O


15a 0.6 0.5 0.4 0.3 0.2 0.1 0 0.82 10.0

12.12 9.5

9.0

8.5

8.0

7.5

0.83 7.0

6.5

6.0

2.20 0.88 2.21 5.5 5.0 4.5 Chemical Shift (ppm)

7.73 3.00 4.0

4.33 3.5

3.0

2.5

2.0

6.10 1.5

1.0

0.5

0

-0.5

Figure S35: 1H NMR (600 MHz, CDCl3) spectrum of compound 15a.

S49

O Angelica_R-71.013.esp

MeO

O

H N

N O

H N

N O

H N O

0.20

NHCbz O

128.58 126.83

0.15

184

176

168

152

144

136

128

120

112

104


72

64

56

48

20.92 19.64

41.15 41.04

29.71 27.07

136.10 134.72

156.71 160

67.28 64.36

0.05

43.52

52.43 51.83 49.67

129.23

0.10

171.02 170.43 170.26 170.12 168.48 167.89


15a

40

32

24

16

8

0

Figure S36: 13C NMR (150 MHz, CDCl3) spectrum of compound 15a.

S50

O MeO

O

H N

N O

H N

N O

H N O

NHCbz O

15a Calc. [M+Na]+ : 663.2754


S51


O

0.9

MeO

O

H N

N O

0.8

N O

H N O

NHBoc O

15b

0.7


H N

0.6 0.5 0.4 0.3 0.2 0.1 0 0.39 10.0

7.07 9.5

9.0

8.5

8.0

7.5

0.82 1.68 7.0

6.5

6.0


7.53 3.00 4.0

3.60 3.5

3.0

2.5

2.0

9.48 1.5

6.01 1.0

0.5

0

Figure S38: 1H NMR (600 MHz, CDCl3) spectrum of compound 15b.

S52

O MeO

O

H N

N

126.80

O

N O H3C

H N O

NHBoc O

15b

28.21

R-85.013.esp

H N

0.10

176

168

160

152

144

136

128

120

112

104


72

64

56

48

20.89 19.65

43.11

51.73

64.66

80.37

134.79

156.19

173.95 170.96 170.26 168.90 168.49 168.03

0.05

27.22

49.54

41.09

52.35

129.13


0.15

40

32

24

16

8

0

Figure S39: 13C NMR (150 MHz, DMSO-d6) spectrum of compound 15b.

S53

O MeO

O

H N

N O

H N

N O

H N O

NHBoc O

15b Calc. [M+Na]+ : 629.2911


S54

R-117.001.esp 0.55 0.50

O 0.45

MeO


0.40

O

H N

N O

0.35

H N

N O

H N O

NHBoc O

15c

0.30 0.25 0.20 0.15 0.10 0.05 0 0.59 10.0

0.67 9.5

9.0

8.5

1.09 6.40 8.0

7.5

7.0

0.60 6.5

6.0


8.42 4.0

3.00 3.5

1.91 3.0

2.5

15.41 2.0

1.5

2.86 1.0

0.5

0

Figure S41: 1H NMR (600 MHz, CDCl3, presence of rotamers) spectrum of compound 15c.

S55

MeO

O

H N

H N

N O

0.40

N O

H N O

28.28

O Angelica_R-117-2-Ugi--.013.esp

NHBoc O

15c 129.09 126.83

0.30 0.25

168

152

144

136

128

120

112

104


72

64

56

40

8.40

48

24.41 22.58

80.57

64.65

135.28 160

42.87 41.78 41.06

176

156.52

0.05

172.00 170.40 170.14 169.17 168.60

0.10

175.23

0.15

26.46

52.28

0.20

50.05 49.37


0.35

32

24

16

8

0

Figure S42: 13C NMR (150 MHz, CDCl3, presence of rotamers) spectrum of compound 15c.

S56

O MeO

O

H N

N O

H N

N O

H N O

NHBoc O

15c Calc. [M+Na]+ : 629.2911

Figure S43: ESI-HRMS of compound 15c.

S57