Supplementary MaterialS1 Supplementary Material for Bipiperidine conjugates as soluble sugar...

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Supplementary Material

for

Bipiperidine conjugates as soluble sugar surrogates in DNA-

intercalating antiproliferative polyketides

Nico Ueberschaar,a,b Florian Meyer,a Hans-Martin Dahse,a and Christian Hertwecka,b*

a Leibniz Institute for Natural Product Research and Infection Biology, Hans‐Knöll‐Institute (HKI), Beutenbergstr. 11a, D-07745, Jena, Germany.

b Friedrich Schiller University, Jena, Germany

*Correspondence and requests for materials should be addressed to

Christian Hertweck (e‐mail: christian.hertweck@hki‐jena.de).

Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2016

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Content Instrumentation ............................................................................................................................................ 3

General methods ........................................................................................................................................... 3

UV-Titration ................................................................................................................................................... 3

Determination of growth inhibition and cytotoxicity of HUVEC, K-562 and HeLa ........................................ 4

Metabolomics analysis on K-562 cells ........................................................................................................... 4

Preparation and analytical data .................................................................................................................... 5

Daunorubicin-10-1,4'-bipiperidine-1'-carboxylate (3) .............................................................................. 5

Daunorubicin aglycone (4) ........................................................................................................................ 6

Resistomycin-10-1,4'-bipiperidine-1'-carboxylate (8) ............................................................................... 6

Benastatin A-11-1,4'-bipiperidine-1'-carboxylate (9) ................................................................................ 7

Benastatin B-11-1,4'-bipiperidine-1'-carboxylate (10) .............................................................................. 8

Chartarin-10-1,4'-bipiperidine-1'-carboxylate (11) and chartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (13) .......................................................................................................................................................... 10

Bromochartarin-10-1,4'-bipiperidine-1'-carboxylate (18) and bromochartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (20)................................................................................................................................... 11

Norchartarin-10-1,4'-bipiperidine-1'-carboxylate (19) and norchartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (21) ....................................................................................................................................... 12

Assigning the regiochemistry of chartreusin derivatives ........................................................................ 13

Spectral data:............................................................................................................................................... 16

Daunorubicin-10-1,4'-bipiperidine-1'-carboxylate (3) ............................................................................ 16

Resistomycin-10-1,4'-bipiperidine-1'-carboxylate (8) ............................................................................. 18

Benastatin A-11-1,4'-bipiperidine-1'-carboxylate (9) .............................................................................. 20

Benastatin B-11-1,4'-bipiperidine-1'-carboxylate (10) ............................................................................ 23

Chartarin-10-1,4'-bipiperidine-1'-carboxylate (11) ................................................................................. 26

Chartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (13) .......................................................................... 29

Bromochartarin-10-1,4'-bipiperidine-1'-carboxylate (18) ....................................................................... 31

Norchartarin-10-1,4'-bipiperidine-1'-carboxylate (19) ........................................................................... 33

Bromochartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (20) ................................................................ 35

Norchartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (21) .................................................................... 37

LogD and Log S values ................................................................................................................................. 39

References: .................................................................................................................................................. 51

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Instrumentation All 1D (1H, 13C, DEPT) and 2D NMR (1H-1H COSY, 1H-13C HSQC, 1H-13C HMBC) were recorded in deuterated

solvents on a Bruker AVANCE II 300, a AVANCE III 500 or 600 MHz instrument equipped with a Bruker Cryo

Platform. The chemical shifts are reported in ppm relative to the solvent residual peak (δ 1H (DMSO-

D6) = 2.50 ppm, δ 13C (DMSO-D6) = 39.52 ppm, δ 1H (CD3OD) = 3.31 ppm, δ 13C (CD3OD) = 49.15 ppm, δ 1H

(CD2Cl2) = 5.32 ppm, δ 13C (CD2Cl2) = 54.00 ppm, δ 1H (D3CCN) = 1.94 ppm, δ 13C (D3CCN) = 118.69 ppm).

Following abbreviations are used for multiplicities of resonance signals: s = singlet, d = doublet, t = triplet,

dd = doublet of doublet, q = quartet, qt = quintet, m = multiplet. br broad. HR-ESI-MS measurements were

conducted on either a Thermo Exactive or a Q-Exactive apparatus. Semi-preparative HPLC purification was

achieved by using a Agilent 1260 device equipped with a quaternary Pump, a UV/Vis detector and a

fraction collector (Column: Zorbax Eclipse XDB-C8, 5 µm, 250 x 9.4 mm, eluent: H2O/0,1% HCOOH, MeOH).

Preparative HPLC for the chartreusin derivatives was performed on a Gilson 321 Pump with a UV/VIS 156

detector system using a Phenomenx Kinetix C18 column 5 μm, Ø 21.2 mm × 250 mm at 21 mL/min with a

gradientent from 10 to 83% acetonitrile in water containing 0.1% formic acid.

General methods

All reactions were carried out in standard glassware with magnetic stirrer. Syntheses requiring an inert

reaction atmosphere were carried out under a positive stream of Argon applying the Schlenk technique.

All solvents were dried and distilled under an inert atmosphere before being used. Anhydrous pyridine

was purchased from Sigma-Aldrich. All other reagents were purchased from commercial available

suppliers and used without further purification. Reaction progresses were monitored by thin layer

chromatography (TLC) GC-MS or HPLC-MS. Analytical thin-layer chromatography for reaction monitoring

was performed on pre-coated aluminum-backed silica gel plates (silica gel 60 F254, Merck KGaA,

Darmstadt), visualized with an UV lamp (254 nm) or with a 4-anisaldehyde-solution (1 mL 4-anisaldehyde

in a mixture of 100 mL of methanol, acetic acid, sulfuric acid in a ratio of 85:10:5).

UV-Titration

A 1 mg/mL solution of chartreusin or its analogues in DMSO were diluted to 1 mL with PBS‐buffer (pH 7.5)

to a final concentration of 50 μmol/L in a fused quartz cuvette. Then a 10 mg/mL herring sperm DNA

solution (GC content ≈43%, Mmid. = 649.66 g/mol) in PBS buffer were added to give the ratios substrate :

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DNA as 1:0, 1:0.5, 1:1, 1:2, 1:5, 1:10, 1:20, 1:30, 1:50 and 1:100. After each dilution step a UV‐VIS spectrum

between 300 and 500 nm was recorded.1

Determination of growth inhibition and cytotoxicity of HUVEC, K-562 and

HeLa

Compounds were assayed using human umbilical vein endothelial cells HUVEC (ATCC CRL-1730) and

human chronic myeloid leukemia cells K-562 (DSM ACC 10) for their antiproliferative effects (GI50) and

using human cervix carcinoma cells HeLa (DSM ACC 57) for their cytotoxic effects (CC50) as previously

described.2

Metabolomics analysis on K-562 cells

A culture of K-562 (5 mL) was treated with 21 (10 mg / L in 10% aq. NH4Cl solution) to a final concentration

of 3.125 µg / mL. After 48 h of cultivation (conditions see above) 5 mL ethanol were added and the

suspension was shaken for 1 h followed by evaporation to dryness. Then 200 µL of a 1:1 mixture of 10%

aq. NH4Cl solution and methanol were added followed by filtration through a 0.2 µm filter. The samples

were analyzed using HPLC/HRMS.

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Preparation and analytical data

Daunorubicin-10-1,4'-bipiperidine-1'-carboxylate (3)

A solution of daunorubicin aglycone (12 mg, 30 µmol, 1 eq. 4), 4-piperidinopiperidine-1-carbonyl chloride

(15.6 mg, 68 µmol, 2.25 eq.) and DMAP (12.4 mg, 102 µmol, 3.38 eq.) in pyridine (2 mL) was stirred for

72 h at 45 °C under argon. Subsequently the pyridine was removed under reduced pressure and the

remaining solid was dissolved in a mixture of MeOH/HCl (1 M in water, 200/1) and purified by preparative

HPLC yielding compound 3 (9.5 mg, 16 µmol, 53%) as a red solid.

1H NMR (500 MHz, CD3OD): δ = 7.94 (d, 1H, J = 7.8 Hz, CH-aryl), 7.83 (t, 1H, J = 8.1 Hz, CH-aryl), 7.57

(d, 1H, J = 8.3 Hz, CH-aryl), 6.17 (m, 1H, CHOCON), 4.36 and 4.16 (m, 2H, CONCH2), 4.02 (s, 3H,

OCH3), 3.47 and 3.00 (m, 4H, NCH2), 3.37 (m, 1H, NCH), 3.09 and 2.71 (m, 2H, CCH2C), 2.85

(m, 2H, CONCH2), 2.35 (s, 3H, COCH3), 2.31 and 2.22 (m, 2H, CCH2CH), 2.05, 1.76 and 1.50 (m,

4H, NCHCH2), 2.00 and 1.76 (m, 4H, NCH2CH2), 1.85 and 1.51 (m, 2H, NCH2CH2CH2) ppm.

Spectrum: see Figure S1 on page S16.

13C NMR (150 MHz, CD3OD): δ = 214.3 (CH3CO), 188.5 (CO), 188.1 (CO), 162.6 (C-OH-aryl), 157.1 (NCO),

156.6 (C-OH-aryl), 156.2 (C-OH-aryl), 137.3 (CH-aryl), 136.5 (C-aryl), 136.5 (C-aryl), 134.0 (C-

aryl), 121.6 (C-aryl), 120.6 (CH-aryl), 120.4 (CH-aryl), 112.6 (C-aryl), 112.6 (C-aryl), 76.6

(CCOCH3), 65.9 (CHOCON), 65.1 (NCH), 57.1 (OCH3), 51.3 and 51.1 (NCH2), 43.9 and 43.7

(CONCH2), 36.0 (CCH2CH), 32.8 (CCH2C), 27.4 and 27.3 (NCHCH2), 24.6 (CH3CO), 24.5

(NCH2CH2), 22.9 (NCH2CH2CH2) ppm. Spectrum: see Figure S2 on page S17.

HRMS (ESI+) calc. for C32H37N2O9 [M+H]+: 593.2476, found 593.2480.

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Daunorubicin aglycone (4)

A solution of daunorubicin (75 mg, 142 μmol, 1) in 1 M aqueous HCl was stirred at 80 °C for 2 h. The

precipitated solid was filtered off, washed with water and dried under reduced pressure. 4 was obtained

as a red solid quantitatively.

1H NMR (500 MHz, CDCl3): δ = 13.90 (s, 1H, OH), 13.21 (s, 1H, OH), 7.98 (dd, 1H, J1 = 7.7 Hz, J2 = 0.9 Hz,

CH-aryl), 7.76 (dd, 1H, J1 = 7.8 Hz, J2 = 7.8 Hz, CH-aryl), 7.37 (m, 1H, CH-aryl), 5.29 (m, 1H, CH),

4.07 (s, 3H, OCH3), 3.14 (dd, 1H, J1 = 18.5 Hz, J2 = 2.1 Hz, CH2), 2.89 (d, 1H, J = 18.5 Hz, CH2),

2.41 (s, 3H, CH3), 2.32 (m, 1H, CH2), 2.13 (dd, 1H, J1 = 14.5 Hz, J2 = 4.9 Hz, CH2) ppm.

13C NMR (125 MHz, CDCl3): δ = 211.9 (CH3CO), 187.0 (CO), 186.6 (CO), 161.0 (C-aryl), 156.0 (C-aryl),

155.8 (C-aryl), 136.0 (C-aryl), 135.8 (CH-aryl), 135.5 (C-aryl), 133.6 (C-aryl), 120.8 (C-aryl),

119.8 (CH-aryl), 118.4 (CH-aryl), 111.5 (C-aryl), 111.1 (C-aryl), 76.8 (CCOCH3), 61.9 (CHOH),

56.7 (OCH3), 35.3 (CH2), 33.2 (CH2), 24.6 (CH3).

Resistomycin-10-1,4'-bipiperidine-1'-carboxylate (8)

A solution of resistomycin (3.0 mg, 8.0 µmol, 1 eq. 5), 4-piperidinopiperidine-1-carbonyl chloride (2.1 mg,

9.2 µmol, 1.15 eq.) and DMAP (1.7 mg, 13.8 µmol, 1.75 eq.) in pyridine (1 mL) was stirred for 24 h at room

temperature under argon. Subsequently the pyridine was removed under reduced pressure and the

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remaining solid was dissolved in a mixture of THF/MeOH/HCl (1 M in water, 100/100/1) and purified by

preparative HPLC yielding compound 8 (3.4 mg, 6.0 µmol, 75%) as a yellow solid.

1H NMR (600 MHz, CD2Cl2): δ = 14.67 (s, 1H, OH), 14.27 (s, 1H, OH), 13.90 (s, 1H, OH), 7.41 (s, 1H, CH-

aryl), 7.16 (s, 1H, CH-aryl), 6.52 (s, 1H, CH-aryl), 4.64 and 4.50 (m, 2H, CONCH2), 3.60 (m, 2H,

NCH2), 3.45 (m, 1H, NCH), 3.22 and 3.01 (m, 2H, CONCH2), 2.85 (s, 3H, CH3), 2.83 (m, 2H,

NCH2), 2.30 and 2.21 (m, 2H, NCHCH2), 1.98 (m, 4H, NCH2CH2), 1.94 and 1.88 (m, 2H, NCHCH2),

1.92 and 1.44 (m, 2H, NCH2CH2CH2), 1.68 (s, 6H, CH3) ppm. Spectrum: see Figure S3 on page

S18.

13C NMR (150 MHz, CD2Cl2): δ = 205.9 (CO), 187.7 (CO), 171.8 (C-OH-aryl), 171.1 (C-OH-aryl), 169.4 (C-

OH-aryl), 153.2 (C-aryl), 153.0 (NCOO), 151.3 (C-aryl), 148.7 (C-aryl), 141.0 (C-aryl), 130.0 (C-

aryl), 123.6 (CH-aryl), 119.5 (C-aryl), 119.1 (CH-aryl), 115.5 (C-aryl), 108.2 (C-aryl), 108.2 (C-

aryl), 105.3 (C-aryl), 103.5 (CH-aryl), 64.0 (NCH), 51.3 and 50.3 (NCH2), 47.3 [C(CH3)2], 44.0 and

43.6 (CONCH2), 29.1 [C(CH3)2], 27.3 and 26.3 (NCHCH2), 25.3 (CH3), 23.6 and 23.5 (NCH2CH2),

22.8 (NCH2CH2CH2) ppm. Spectrum: see Figure S4 on page S19.


Benastatin A-11-1,4'-bipiperidine-1'-carboxylate (9)

A solution of benastatin A (8.0 mg, 16 µmol, 1 eq. 6), 4-piperidinopiperidine-1-carbonyl chloride (4.2 mg,

18.4 µmol, 1.2 eq.) and DMAP (3.4 mg, 27.6 µmol, 1.7 eq.) in pyridine (3.0 mL) was stirred for 24 h at room



HPLC yielding compound 9 (5.2 mg, 7.5 µmol, 47%) as a yellow solid.

1H NMR (600 MHz, DMSO-D6): δ = 13.59 (s, 1H, OH), 12.83 (s, 1H, OH), 10.20 (s, 1H, CH-aryl), 8.12 (d,

1H, J = 9.1 Hz, CH-aryl), 7.57 (d, 1H, J = 9.1 Hz, CH-aryl), 7.19 (d, 1H, J = 2.2 Hz, CH-aryl), 6.86

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(s, 1H, CH-aryl), 6.77 (d, 1H, J = 2.2 Hz, CH-aryl), 4.29 and 4.14 (m, 2H, CONCH2), 3.20 (t, 2H,

J = 2.2 Hz, CH2), 3.06 and 2.92 (m, 2H, CONCH2), 2.92 (m, 4H, NCH2), 1.76 (s, 6H, CH3), 2.51 (m,

1H, NCH), 2.00-1.20 (m, 10H, CH2), 1.58 (m, 2H, CH2), 1.31 (m, 4H, CH2), 0.86 (t, J = 6.9 Hz,

CH3) ppm. Spectrum: see Figure S5 on page S20.

1H NMR (600 MHz, CD3OD): δ = 9.64 (s, 1H, CH-aryl), 8.35 (d, 1H, J = 9.1 Hz, CH-aryl), 7.57 (d, 1H,

J = 9.1 Hz, CH-aryl), 7.21 (s, 1H, CH-aryl), 7.11 (d, 1H, J = 2.0 Hz, CH-aryl), 6.72 (d, 1H,

J = 2.0 Hz, CH-aryl), 4.50 and 4.39 (m, 2H, CONCH2), 3.56 and 3.09 (m, 4H, NCH2), 3.48 (m, 1H,

NCH), 3.16 and 3.01 (m, 2H, CONCH2), 3.10 (t, 2H, J = 2.2 Hz, CH2), 2.20 and 1.82 (m, 4H,

NCHCH2), 2.02 and 1.84 (m, 4H, NCH2CH2), 1.87 and 1.55 (m, 2H, NCH2CH2CH2), 1.80 (s, 6H,

CH3), 1.67 (m, 2H, CH2), 1.40 (m, 4H, CH2), 0.93 (t, J = 7.0 Hz, CH3) ppm. Spectrum: see Figure

S6 on page S21.

13C NMR (150 MHz, CD3OD): δ = 193.1 (CO), 176.3 (COOH), 166.7 (C-OH-aryl), 165.7 (C-OH-aryl), 162.0

(C-OH-aryl), 159.1 (C-aryl), 156.0 (C-aryl), 153.8 (NCO), 148.2 (C-aryl), 146.3 (C-aryl), 140.4 (C-

aryl), 138.0 (C-aryl), 127.0 (CH-aryl), 125.2 (CH-aryl), 122.9 (CH-aryl), 121.9 (C-aryl), 118.3 (CH-

aryl), 118.2 (C-aryl), 113.0 (C-aryl), 112.7 (CH-aryl), 110.1 (C-aryl), 109.8 (C-aryl), 109.3 (CH-

aryl), 64.9 (NCH), 51.4 (NCH2), 44.3 and 43.9 (CONCH2), 40.8 [C(CH3)2], 38.0 (CH2), 34.9

[C(CH3)2], 33.3 (CH2), 32.9 (CH2), 27.6 and 27.2 (NCHCH2), 24.6 (NCH2CH2), 23.6 (CH2), 22.9

(NCH2CH2CH2), 14.4 (CH3) ppm. Spectrum: see Figure S7 on page S22.


Benastatin B-11-1,4'-bipiperidine-1'-carboxylate (10)

A solution of benastatin B (5 mg, 9.9 µmol, 1 eq.7;), 4-piperidinopiperidine-1-carbonyl chloride (2.8 mg,

11.9 µmol, 1.2 eq.) and DMAP (2.2 mg, 17.9 µmol, 1.8 eq.) in pyridine (2.5 mL) was stirred for 24 h at room


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HPLC yielding compound 10 (5.3 mg, 7.6 µmol, 76%) as a yellow solid.

1H NMR (600 MHz, CD2Cl2): δ = 16.30 (s, b, 1H, OH), 12.99 (s, 1H, OH), 12.81 (s, 1H, OH), 8.58 (s, 1H,

CH-aryl), 6.95 (d, 1H, J = 2.2 Hz, CH-aryl), 6.80 (m, 1H, CH-aryl), 6.57 (s, 1H, CH-aryl), 4.48 (m,

2H, CONCH2), 3.54 and 2.85 (m, 4H, NCH2), 3.47 (m, 1H, NCH), 3.13 (m, 2H, CH2), 3.13 and

2.99 (m, 2H, CONCH2), 2.60-1.20 (various m, 10H, CH2), 1.76 (s, 6H, CH3), 1.64 (m, 2H, CH2),

1.37 (m, 4H, CH2), 0.92 (t, 3H, J = 7.0 Hz, CH3) ppm. Spectrum: see Figure S8 on page S23.

1H NMR (600 MHz, CD3OD): δ = 8.40 (s, 1H, CH-aryl), 7.07 (d, 1H, J = 2.2 Hz, CH-aryl), 6.74 (s, 1H, CH-

aryl), 6.69 (d, 1H, J = 2.2 Hz, CH-aryl), 4.49 and 4.36 (m, 2H, CONCH2), 3.56 and 3.06 (m, 4H,

NCH2), 3.50 (m, 1H, NCH), 3.16 and 3.02 (m, 2H, CONCH2), 2.97 (m, 2H, CH2), 2.81 (m, 2H,

CH2), 2.74 (m, 2H, CH2), 2.24 and 1.85 (m, 4H, NCHCH2), 1.99 and 1.88 (m, 4H, NCH2CH2), 1.86

and 1.54 (m, 2H, NCH2CH2CH2), 1.70 (s, 6H, CH3), 1.61 (m, 2H, CH2), 1.37 (m, 4H, CH2), 0.91 (t,

3H, J = 7.1 Hz, CH3) ppm. Spectrum: see Figure S9on page S24.

13C NMR (150 MHz, CD3OD): δ = 192.9 (CO), 175.5 (COOH), 165.5 (C-OH-aryl), 162.8 (C-OH-aryl), 159.9

(C-OH-aryl), 158.3 (C-aryl), 156.0 (C-aryl), 153.9 (NCO), 150.1 (C-aryl), 149.3 (C-aryl), 148.0 (C-

aryl), 142.2 (C-aryl), 124.3 (C-aryl), 123.5 (CH-aryl), 120.7 (C-aryl), 119.3 (CH-aryl), 112.9 (C-

aryl), 112.8 (C-aryl), 112.8 (C-aryl), 112.2 (C-aryl), 109.3 (CH-aryl), 64.7 (NCH), 51.3 (NCH2),

44.3 and 43.9 (CONCH2), 40.2 [C(CH3)2], 37.7 (CH2), 34.3 [C(CH3)2], 33.3 (CH2), 32.9 (CH2), 30.7

(CH2), 27.5 and 27.2 (NCHCH2), 24.5 (NCH2CH2), 23.4 (CH2), 22.9 (NCH2CH2CH2), 20.7 (CH2),

14.4 (CH3) ppm. Spectrum: see Figure S10 on page S25.


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Chartarin-10-1,4'-bipiperidine-1'-carboxylate (11) and chartarin-6,10 di-1,4'-bipiperidine-

1'-carboxylate (13)

Chartarin (10 mg; 30 µM, 1.0 eq. 12), 1,4'-bipiperidin-1'-carbonylchloride (27.6 mg; 120 µM; 4.0 eq.) and

N,N-dimethyl4-aminopyridin (22 mg; 180 µM; 6 eq.) were placed in a Schlenk tube and flushed with argon.

Pyridin (2.4 mL) was added and the solution was stirred for 24 h at 60 °C. After cooling the solution was

evaporated under reduced pressure to yield the crude product which was taken up in a mixture of

methanol, THF and 10% aq. NH4Cl solution (1:1:1; v/v/v). The mixture was suspended to preparative

column chromatography monitoring the wavelength of 254 and 400 nm.

data for chartarin-10-1,4'-bipiperidine-1'-carboxylate (11)

1H NMR (600 MHz; D3CCN): see Table S1 on page S14; spectrum: see Figure S11 on page S26.

13C NMR (150 MHz; D3CCN): see Table S2 on page S15; spectrum: see Figure S12 on page S27.

UV-Vis (from LC/MS run using acetonitrile/water with 0.1% formic acid) λmax = 234, 264, 327, 371,

394, 414 nm.

HRMS (ESI+) calc. for C31H31O7N4 [M+H]+: 543.2126, found. 543.2138.

data for chartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (13)

1H NMR (600 MHz; D3CCN): see Table S1 on page S14; spectrum: see Figure S14 on page s29.



383, 401 nm.

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HRMS (ESI+) calc. for C41H48O8N4 [M+H]+: 723.3388, found. 723.3395.

(ESI+) calc. for C41H48O8N4 [M+2H]2+: 362.1731, found. 362.1738.

Bromochartarin-10-1,4'-bipiperidine-1'-carboxylate (18) and bromochartarin-6,10 di-1,4'-

bipiperidine-1'-carboxylate (20)

The preparation of (18 and 20) follows the procedure of Chartarin-10-1,4'-bipiperidine-1'-carboxylate (11)

and chartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (13) (page S10) using 15 mg starting material (using

the same molar equivalents).

data for bromochartarin-10-1,4'-bipiperidine-1'-carboxylate (18)

1H NMR (500 MHz; D3CCN): see Table S1 on page s14; spectrum: see Figure S16 on page S31.



376, 394, 416 nm.

HRMS (ESI+) calc. for C29H2679BrO7N2 [M+H]+: 593.0918, found 593.0918.

data for bromochartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (20)



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383, 403 nm.

HRMS (ESI+) calc. for C40H4479BrO8N4 [M+H]+: 787.2337, found 787.2325.

(ESI+) calc. for C40H4479BrO8N4 [M+2H]2+: 394.1205, found 394.1202.

Norchartarin-10-1,4'-bipiperidine-1'-carboxylate (19) and norchartarin-6,10 di-1,4'-

bipiperidine-1'-carboxylate (21)

The preparation of (19 and 21) follows the procedure of Chartarin-10-1,4'-bipiperidine-1'-carboxylate (11)

and chartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (13) (page S10) using 10 mg starting material (using

the same molar equivalents).

data for norchartarin-10-1,4'-bipiperidine-1'-carboxylate (19)




369, 388, 409 nm.

HRMS (ESI+) calc. for C29H27O7N2 [M+H]+: 515.1813; found: 515.1807.

data for norchartarin-6,10 di-1,4'-bipiperidine-1'-carboxylate (21)

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303, 317, 337, 351, 380, 397 nm.

HRMS (ESI+) calc. for C40H46O8N4 [M+H]+: 709.3232; found: 709.3221.

(ESI+) calc for C40H46O8N4 [M+2H]2+: 355.1652; found: 355.1649.

Assigning the regiochemistry of chartreusin derivatives

Because of the possibility that two phenol groups may be converted into the carbamate we tried to use

HBMC NMR experiments to visualize couplings from the core aglycon to the carbamate residue and vice

versa to elucidate the position of the modified phenol group. Unfortunately, this approach enables us not

to elucidate the structure. Next, we tried to incorporate a methyl group into the remaining phenol but

during synthesis, decomposition of the starting material hampers this approach. So we used 13C-NMR shift

prediction for the structure elucidation. Comparing the 13C shift of the phenol carbon atom of both possible

isomers (R2 = pip in position 6 and R3 = pip in position 10) with the measured values it becomes clear, that

compound 11 is modified in position 10 which is equal position to the glycoside residue of chartreusin (5)

[ref: ACD CNMR predictor version 8.15]. Due to overlapping NMR signals we are not able to assign the 13C

carbon atom shifts unambiguously for the other derivatives. Due to the almost same structure and similar

behavior during analysis we assume the same substitution for the compounds 18 and 19.

For detailed table of the calculated and measured values including the corresponding HMBC-NMR

spectrum and a structure with the basic couplings see Figure S13 on page S28.

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Table S1: 13C NMR shifts of chartreusin (2) and the derivatives 11, 13, 18-21 a: pyridine-D5, b: D3CCN, c: CD3OD. (δ [ppm], J [Hz], *: signals are overlapping)

2a 13b 11b 20c 18b 21b 18b

pos. δ ∫ m J1 J2 δ ∫ m δ ∫ m J1 J2 δ ∫ m J1 J2 δ ∫ m J1 J2 δ ∫ m δ ∫ m J1 J2

1 - - - - - 8.1-7.9 m 8.13 1 dd 7.7 0.9

1a 2.73 3 s 2.65 3 s 2.66 3 s - - - -

2 7.32 1 d 8.8 7.53 1 br m 7.48 1 d 8.3 8.08 1 d 9.5 7.97 1 d 8.8 7.75 m 7.75 1 m

3 7.47 1 d 8.3 7.53 1 br m 7.52 1 d 8.2 7.65 1 d 9.5 7.57 1 d 8.8 7.65 m 7.56 1 dd 7.6 0.9

7 8.36 1 dd 8.3 0.6 8.1-7.9 1 br m 8.29 1 d 8.3 8.2-8.1 1 m 8.38 1 dd 8.2 1.2 8.1-7.9 m 8.42 1 dd 8.6 0.8

8 7.66 1 t 8.1 7.68 1 br m 7.66 1 dd 8.3 7.5 7.8-7.9 1 m 7.74 1 dd 8.3 7.7 7.75 m 7.81 1 dd 8.0 7.8

9 7.77 1 d 7.8 7.45 1 br m 7.44 1 d 7.5 7.6-7.7 1 m 7.51 1 dd 7.6 1.2 7.44 m 7.77 1 dd 8.2 0.9

1' 5.83 1 d 4.1 - - - - - -

2' 5.08 1 dd 9.5 7.7

4.62; 4.23; 3.28; 2.97

8 m

4.58; 4.23; 3.27; 2.94

4 m

4.66 4.56 4.29 4.23

m

4.61; 4.26; 3.24; 3.00

4 m

4.63; 4.28; 3.36; 3.01

8 m

4.63; 4.28; 3.36; 3.01

4 m

3' 4.34 1 dd 9.6 3.5 2.21; 1.86

8 m 2.07; 1.86

4 m 2.40; 2.26

m 2.18; 2.09

4 m 2.20; 1.90

8 m 2.18; 1.92

4 m

4' 4.22 1 d 3.4 3.48 2 m 3.46 1 m 3.64* m 3.46 1 m 3.46 2 m 3.46 1 m

5' 4.12 1 q 6.5 - - - - - -

6' 1.58 3 d 6.5 - - - - - -

1'' 6.56 1 d 4.1 - - - - - -

2'' 4.57 1 dd 10.0 4.1 3.53; 2.90

8 m 3.58; 3.51; 3.02

4 m 3.65*;

3.17 m

3.55; 3.51, 3.02

4 m 3.54; 3.00

8 m 3.54; 2.99

4 m

3'' 3.87 1 dd 10.1 3.1 1.94 8 m 1.91 4 m 2.09; 1.92

m 1.94 4 m 1.92 8 m 1.94 4 m

3''OMe 3.34 3 s - - - - - -

4'' 4.16 1 d 1.7 1.84; 1.51

4 m 1.86; 1.56

2 m 1.59; 1.61

m 1.82; 1.49

1 m 1.84; 1.50

4 m 1.83; 1.50

1 m

5'' 5.03 1 dq 6.3 0.9 - - - - - -

6'' 1.59 3 d 6.5 - - - - - -

S15

Table S2: 13C NMR shifts of chartreusin (2) and the derivatives 11, 13, 18-21 a: pyridine-D5, b: D3CCN, c: CD3OD. (δ [ppm], J [Hz], *: signal under solvent residual signal)

2a 11b 13b 18b 19b 820c 21b

1 139.8 141.3 140.8 119.8 126.8 120.5 126.9

1a 22.5 22.6 22.6 - - - -

2 133.2 135.0 135.3 138.3 132.6 138.9 132.8

3 121.1 122.7 122.8 123.9 122.9 124.2 122.9

3a 147.2 148.1 148.4 149.4 148.3 149.0 148.4

3a1 120.5 120.6 119.5 120.7 120.3 120.7 120.6

5 165.1 165.9 161.7 165.3 166.0 157.6 161.6

5a 97.8 98.4 109.8 98.0 98.8 109.0 109.8

5a1 109.5 111.0 112.4 110.3 111.0 111.4 112.4

6 159.5 158.46 153.3 158.7 158.6 153.7 152.3

6a 127.7 127.52 131.7 127.8 127.8 131.7 126.9

7 119.9 123.33 122.8 123.4 123.4 123.4 122.9

8 128.8 129.45 130.8 130.0 129.4 131.6 130.6

9 115.5 127.43 126.8 127.7 127.6 127.6 126.8

10 155.5 148.3 148.1 148.3 148.3 148.9 148.0

10a 119.7 122.9 121.8 122.8 123.5 121.3 121.9

10b 139.9 138.4 143.4 138.6 138.5 142.9 143.2

12 157.4 159.9 159.2 161.1 160.5 156.8 159.7

12a 118.5 118.8* 118.0 119.2 121.5 117.6 119.2

carbamate - 154.5 153.3; 154.4 154.4 154.7 155.0; 155.4 154.7; 154.4

1' 101.6 - - - - - -

2' 80.7 44.5; 44.0 45.1; 44.6; 44.5; 44.0 44.5; 44.0 44.6; 44.0 44.9; 44.5; 44.1; 43.9 45.1; 44.5; 44.0

3' 74.6 26.7; 26.5 27.1; 27.0; 26.6; 26.1 26.7; 26.5 26.8; 26.7 27.0; 26.6; 26.4; 26.2 26.4; 27.0; 27.7

4' 73.1 65.1 65.1; 64.7 65.1 65.0; 64.9 64.8; 64.4 65.0; 64.8; 64.7; 64.4

5' 72.3 - - - - - -

6' 17.5 - - - - - -

1'' 102.3 - - - - - -

2'' 67.8 51.4; 50.8 51.8; 51.7; 50.8; 50.7; 50.5 51.5; 51.0 51.4; 51.3; 51.1; 51.0 51.6; 51.4; 51.2 50.7; 51.7

3'' 82.0 24.4 22.4; 22.3 24.4 24.4 24.5; 24.4 24.3

3''OMe 57.2 - - - - - -

4'' 69.4 23.2 23.1 23.2 23.2 22.9; 22.8 23.1

5'' 67.2 - - - - - -

6'' 17.5 - - - - - -

2.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm

1.48

1.49

1.50

1.51

1.52

1.54

1.72

1.72

1.73

1.75

1.76

1.77

1.78

1.80

1.83

1.86

1.98

2.01

2.24

2.25

2.29

2.30

2.32

2.34

2.35

2.99

3.00

3.02

3.07

3.11

3.16

3.43

3.44

3.44

3.44

3.47

4.02

7.56

7.58

7.81

7.83

7.85

7.93

7.95

2.00

3.29

1.74

3.80

1.06

0.99

3.03

3.06

3.24

1.13

2.31

3.17

2.22

0.99

1.04

1.05

1.00

PC 1.00GB 0.3LB −0.30 HzSSB 0WDW GMSF 500.3000157 MHzSI 131072SFO1 500.3045027 MHzPL1W 7.71660900 WPL1 5.50 dBP1 14.00 usecNUC1 1H======== CHANNEL f1 ========

TD0 1D1 1.00000000 secTE 295.9 KDE 6.50 usecDW 50.000 usecRG 645AQ 3.2768500 secFIDRES 0.152588 HzSWH 10000.000 HzDS 2NS 128SOLVENT MeODTD 65536PULPROG zg30PROBHD 5 mm PABBO BB−INSTRUM spectTime 10.41Date_ 20150302PROCNO 1EXPNO 5NAME 5NFM377_p

1H am 2.3./MeOD5NFM377_p

O

OOMe

OH

OH O

OH

O

O

N

N

Figure S1 1H NMR (CD3OD) of daunorubicin-10-1,4‘-bipiperidine-1‘-carboxylate (3)

S16

220 200 180 160 140 120 100 80 60 40 20 ppm

22.9

24.5

27.3

27.4

32.8

36.0

43.9

51.1

51.3

57.1

65.1

65.9

76.6

112.6

120.4

120.6

121.6

134.0

136.5

136.5

137.3

156.2

156.6

157.1

162.6

188.1

188.5

214.3

PC 1.40GB 0LB 5.00 HzSSB 0WDW EMSF 150.9378005 MHzSI 262144SFO2 600.2724011 MHzPL12W 0.03154787 WPL2W 5.00000000 WPL12 26.00 dBPL2 4.00 dBPCPD2 100.00 usecNUC2 1HCPDPRG2 waltz16======== CHANNEL f2 ========

SPOFFS8 0.00 HzSPOFFS2 0.00 HzSPOAL8 0.500SPOAL2 0.500SPNAM8 Crp60,0.5,20.1SPNAM2 Crp60comp.4SP8 5.24 dBSP2 5.24 dBSFO1 150.9553699 MHzPL1W 75.60778046 WPL1 0.00 dBP26 500.00 usecP12 2000.00 usecP1 14.00 usecNUC1 13C======== CHANNEL f1 ========

TD0 1D20 10.00000000 secD12 0.00002000 secD11 0.03000000 secD1 3.00000000 secTE 298.8 KDE 20.00 usecDW 13.800 usecRG 2050AQ 0.3293180 secFIDRES 1.518520 HzSWH 36231.883 HzDS 4NS 4711SOLVENT MeODTD 23860PULPROG udeftPROBHD 5 mm CPTCI 1H−INSTRUM spectTime 6.07Date_ 20150304PROCNO 1EXPNO 50NAME 6NFM377pCA

NFM377p/CD3OD/Meyer/1H

O

OOMe

OH

OH O

OH

O

O

N

N

Figure S2 13C NMR (CD3OD) of daunorubicin-10-1,4‘-bipiperidine-1‘-carboxylate (3)

S17

23456789101112131415 ppm

1.26

1.35

1.42

1.43

1.44

1.45

1.68

1.86

1.88

1.88

1.91

1.94

1.98

2.01

2.04

2.06

2.19

2.22

2.29

2.31

2.74

2.85

2.99

3.01

3.04

3.20

3.22

3.25

3.42

3.45

3.47

3.59

3.61

4.48

4.51

4.63

4.65

6.52

7.16

7.16

7.41

13.90

14.27

14.67

1.15

6.26

7.38

1.06

1.08

8.28

4.23

1.36

1.06

1.07

2.03

1.04

1.02

0.90

1.01

1.01

1.00

1.00

1.00

PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 500.3000204 MHzSI 131072SFO1 500.3045027 MHzPL1W 7.71660900 WPL1 5.50 dBP1 14.00 usecNUC1 1H======== CHANNEL f1 ========

TD0 1D1 1.00000000 secTE 298.0 KDE 6.50 usecDW 50.000 usecRG 912AQ 3.2768500 secFIDRES 0.152588 HzSWH 10000.000 HzDS 2NS 36SOLVENT CD2Cl2TD 65536PULPROG zg30PROBHD 5 mm PABBO BB−INSTRUM spectTime 12.26Date_ 20140606PROCNO 1EXPNO 1NAME 5NFM278−P7PR

NFM278−P7/Cd2Cl2/Meyer/1H

OH O OH

OH

O O

ON

N

Figure S3 1H NMR (CD2Cl2) of resistomycin-10-1,4‘-bipiperidine-1‘-carboxylate (8)

S18

200 180 160 140 120 100 80 60 40 20 ppm

22.8

23.5

23.6

25.3

26.3

27.3

29.1

43.6

44.0

47.3

50.3

51.4

64.0

103.5

105.3

108.2

115.5

119.1

119.5

123.6

130.0

141.0

148.7

151.3

153.0

153.2

169.4

171.1

171.8

187.7

205.9



TD0 1D20 10.00000000 secD12 0.00002000 secD11 0.03000000 secD1 3.00000000 secTE 298.0 KDE 6.50 usecDW 16.800 usecRG 2050AQ 0.3359828 secFIDRES 1.488393 HzSWH 29761.904 HzDS 4NS 12288SOLVENT CD2Cl2TD 19996PULPROG udeftPROBHD 5 mm PABBO BB−INSTRUM spectTime 14.32Date_ 20140606PROCNO 1EXPNO 1NAME 5NFM278−P7MU

NFM278−P7/Cd2Cl2/Meyer/13C

OH O OH

OH

O O

ON

N

Figure S4 13C NMR (CD2Cl2) of resistomycin-10-1,4‘-bipiperidine-1‘-carboxylate (8)

S19

234567891011121314 ppm

0.85

0.86

0.88

1.22

1.29

1.30

1.31

1.32

1.33

1.48

1.56

1.57

1.58

1.59

1.60

1.61

1.65

1.65

1.76

1.90

1.95

1.95

1.96

2.89

2.92

2.93

2.93

3.06

3.19

3.20

3.22

6.77

6.77

6.86

7.19

7.19

7.56

7.58

8.11

8.12

8.13

10.20

12.83

13.59

3.71

6.61

10.6

66.

422.

52

6.67

3.89

2.06

1.98

1.08

0.98

1.33

1.05

1.00

1.00


TD0 1D1 1.00000000 secTE 298.0 KDE 6.50 usecDW 41.600 usecRG 8AQ 2.7263477 secFIDRES 0.183399 HzSWH 12019.230 HzDS 2NS 16SOLVENT DMSOTD 65536PULPROG zg30PROBHD 5 mm CPTCI 1H−INSTRUM spectTime 13.14Date_ 20141015PROCNO 1EXPNO 10NAME 6NFM325_P3PR

NFM325_P3/DMSO−d6/Meyer/1H

O

OH O OH

HOCOOH

n-C5H11N O

N

Figure S5 1H NMR (DMSO-D6) of benastatin A-11-1,4‘-bipiperidine-1‘-carboxylate (9)

S20

12345678910 ppm

0.92

0.93

0.94

1.27

1.39

1.40

1.40

1.41

1.66

1.67

1.68

1.80

1.82

1.85

1.85

1.86

1.88

2.00

2.03

2.19

2.21

3.07

3.08

3.10

3.11

3.24

3.29

3.30

3.30

3.30

3.34

3.55

3.57

4.85

6.72

6.72

7.11

7.11

7.21

7.57

7.58

8.35

8.36

9.64

3.40

1.17

4.25

1.25

2.18

11.2

82.

232.

060.

964.

111.

170.

201.

092.

04

2.01

0.99

1.02

1.04

1.01

0.99

1.00

PC 1.00GB 0LB 0.30 HzSSB 0WDW EMSF 600.2700197 MHzSI 262144SFO1 600.2754024 MHzPL1W 5.00000000 WPL1 4.00 dBP1 8.00 usecNUC1 1H======== CHANNEL f1 ========

TD0 1D1 1.00000000 secTE 298.8 KDE 6.50 usecDW 41.600 usecRG 11.3AQ 2.7263477 secFIDRES 0.183399 HzSWH 12019.230 HzDS 2NS 128SOLVENT MeODTD 65536PULPROG zg30PROBHD 5 mm CPTCI 1H−INSTRUM spectTime 11.38Date_ 20150123PROCNO 1EXPNO 1NAME 6NFM368_pPR

FM368p/CD3OD/Meyer/1H

O

OH O OH

HOCOOH

n-C5H11N O

N

Figure S6 1H NMR (CD3OD) of benastatin A-11-1,4‘-bipiperidine-1‘-carboxylate (9)

S21

200 180 160 140 120 100 80 60 40 20 ppm

22.9

23.6

24.6

27.2

27.6

32.9

33.3

34.8

38.0

40.8

43.9

44.3

51.4

64.9

109.3

109.8

110.1

112.7

113.0

118.2

118.3

121.9

122.9

125.2

127.0

138.0

140.4

146.3

148.2

153.8

156.0

159.1

162.0

165.7

166.7

176.3

193.1



TD0 1D20 10.00000000 secD12 0.00002000 secD11 0.03000000 secD1 3.00000000 secTE 298.8 KDE 20.00 usecDW 13.800 usecRG 2050AQ 0.3293180 secFIDRES 1.518520 HzSWH 36231.883 HzDS 4NS 1316SOLVENT MeODTD 23860PULPROG udeftPROBHD 5 mm CPTCI 1H−INSTRUM spectTime 12.53Date_ 20150123PROCNO 1EXPNO 50NAME 6NFM368_pCA

FM368p/CD3OD/Meyer/13C

O

OH O OH

HOCOOH

n-C5H11N O

N

Figure S7 13C NMR (CD3OD) of benastatin A-11-1,4‘-bipiperidine-1‘-carboxylate (9)

S22

234567891011121314151617 ppm

0.88

0.91

0.92

0.93

1.26

1.27

1.35

1.36

1.36

1.37

1.38

1.38

1.39

1.39

1.40

1.41

1.42

1.61

1.63

1.64

1.65

1.76

1.86

1.87

1.89

2.14

2.16

2.73

2.74

2.75

2.85

3.13

3.47

5.32

5.32

5.32

5.33

6.57

6.80

6.94

6.95

8.58

12.81

12.99

4.18

6.95

5.98

3.20

0.96

0.76

2.20

2.86

1.15

2.93

1.81

0.96

1.93

0.20

0.98

0.89

1.01

1.00

0.95

0.96

0.70


TD0 1D1 1.00000000 secTE 298.0 KDE 6.50 usecDW 41.600 usecRG 8AQ 2.7263477 secFIDRES 0.183399 HzSWH 12019.230 HzDS 2NS 128SOLVENT CD2Cl2TD 65536PULPROG zg30PROBHD 5 mm CPTCI 1H−INSTRUM spectTime 11.12Date_ 20140804PROCNO 1EXPNO 1NAME 6NPW002_P2PR

PW002_P2/Cd2Cl2/Meyer/1H

O

OH O OH

HOCOOH

n-C5H11N O

N

Figure S8 1H NMR (CD2Cl2) of benastatin B-11-1,4‘-bipiperidine-1‘-carboxylate (10)

S23

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm

0.90

0.91

0.92

0.93

1.36

1.37

1.37

1.38

1.60

1.61

1.70

1.84

1.86

1.87

1.89

1.89

1.91

1.97

2.00

2.01

2.23

2.25

2.68

2.72

2.73

2.74

2.75

2.81

2.81

2.82

2.83

2.96

2.97

2.97

2.98

3.06

3.55

3.57

6.69

6.69

6.74

7.07

7.08

8.40

3.10

4.10

1.09

2.05

6.01

5.17

2.09

2.02

1.97

1.99

2.09

2.95

1.10

1.02

1.97

1.01

0.99

0.97

1.00

0.99

1.00


TD0 1D1 1.00000000 secTE 298.8 KDE 6.50 usecDW 41.600 usecRG 25.4AQ 2.7263477 secFIDRES 0.183399 HzSWH 12019.230 HzDS 2NS 64SOLVENT MeODTD 65536PULPROG zg30PROBHD 5 mm CPTCI 1H−INSTRUM spectTime 6.08Date_ 20150205PROCNO 1EXPNO 10NAME 6NFM374_p2PR

FM374_p2/CD3OD+DCl/Meyer/1H

O

OH O OH

HOCOOH

n-C5H11N O

N

Figure S9 1H NMR (CD3OD) of benastatin B-11-1,4‘-bipiperidine-1‘-carboxylate (10)

S24

180 160 140 120 100 80 60 40 20 ppm

22.9

23.5

24.5

27.2

27.5

30.7

32.9

33.3

34.3

37.7

40.2

43.9

44.3

51.3

64.7

109.3

112.2

112.8

112.9

119.3

120.7

123.5

124.3

142.2

148.0

149.3

150.1

153.9

156.0

158.9

159.9

162.8

165.5

175.5

192.9



TD0 1D20 10.00000000 secD12 0.00002000 secD11 0.03000000 secD1 3.00000000 secTE 298.8 KDE 20.00 usecDW 13.800 usecRG 2050AQ 0.3293180 secFIDRES 1.518520 HzSWH 36231.883 HzDS 4NS 8192SOLVENT MeODTD 23860PULPROG udeftPROBHD 5 mm CPTCI 1H−INSTRUM spectTime 4.04Date_ 20150205PROCNO 1EXPNO 50NAME 6NFM374_p2CA

FM374_p2/CD3OD+DCl/Meyer/13C

O

OH O OH

HOCOOH

n-C5H11N O

N

Figure S10 13C NMR (CD3OD) of benastatin B-11-1,4‘-bipiperidine-1‘-carboxylate (10)

S25

23456789101112 ppm

2.35

2.66

2.92

3.01

3.02

3.44

3.46

3.48

3.51

3.52

3.53

3.58

3.60

3.60

4.22

4.24

4.57

4.59

7.29

7.29

7.44

7.45

7.47

7.48

7.51

7.53

7.66

7.67

7.68

8.28

8.30

10.70

11.56

11.56

1.55

2.33

1.99

13.9

54.

064.

121.

413.

75

1.14

1.09

3.12

1.05

1.00

0.96

0.88

Current Data ParametersNAME 6NNU350PREXPNO 1PROCNO 1

F2 - Acquisition ParametersDate_ 20131129Time 11.13INSTRUM spectPROBHD 5 mm CPTCI 1H-PULPROG zg30TD 65536SOLVENT CD3CNNS 64DS 2SWH 12019.230 HzFIDRES 0.183399 HzAQ 2.7262976 secRG 16DW 41.600 usecDE 6.50 usecTE 300.0 KD1 1.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 1HP1 8.00 usecPL1 4.00 dBPL1W 5.00000000 WSFO1 600.2754024 MHz

F2 - Processing parametersSI 262144SF 600.2700202 MHzWDW EMSSB 0LB 0.30 HzGB 0PC 1.00

NNU350/ACN-d8/Ueberschaar/1HNU814f

O

O

O

O

O

OH

O

N

N

Figure S11 1H NMR (CD3CN) of chartarin-10-1,4‘-bipiperidine-1‘-carboxylate (11)

S26

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm22.6

23.2

24.4

26.5

26.7

44.0

44.5

50.9

51.5

65.1

98.4

111.0

120.6

122.7

122.9

123.3

127.4

129.4

135.0

138.4

141.3

148.1

148.3

154.5

158.5

159.9

165.9 Current Data Parameters

NAME 6NNU350CAEXPNO 50PROCNO 1

F2 - Acquisition ParametersDate_ 20131130Time 15.01INSTRUM spectPROBHD 5 mm CPTCI 1H-PULPROG udeftTD 23860SOLVENT CD3CNNS 8192DS 4SWH 36231.883 HzFIDRES 1.518520 HzAQ 0.3292680 secRG 2050DW 13.800 usecDE 20.00 usecTE 300.1 KD1 3.00000000 secD11 0.03000000 secD12 0.00002000 secD20 10.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 13CP1 14.00 usecP12 2000.00 usecP26 500.00 usecPL1 0 dBPL1W 75.60778046 WSFO1 150.9553699 MHzSP2 5.24 dBSP8 5.24 dBSPNAM[2] Crp60comp.4SPNAM[8] Crp60,0.5,20.1SPOAL2 0.500SPOAL8 0.500SPOFFS2 0 HzSPOFFS8 0 Hz

======== CHANNEL f2 ========CPDPRG[2 waltz16NUC2 1HPCPD2 100.00 usecPL2 4.00 dBPL12 26.00 dBPL2W 5.00000000 WPL12W 0.03154787 WSFO2 600.2724011 MHz


NNU350/ACN-d6/Ueberschaar/13CNU814f

O

O

O

O

O

OH

O

N

N

Figure S12 13C NMR (CD3CN) of chartarin-10-1,4‘-bipiperidine-1‘-carboxylate (11)

S27

ppm

7.57.67.77.87.98.08.18.28.3 ppm

115

120

125

130

135

140

145

150

155

160

165

NAME 6NNU350HMEXPNO 10PROCNO 1Date_ 20131130Time 16.26INSTRUM spectPROBHD 5 mm CPTCI 1H-PULPROG hmbcgplpndqfTD 4096SOLVENT CD3CNNS 64DS 0SWH 8417.509 HzFIDRES 2.055056 HzAQ 0.2433524 secRG 2050DW 59.400 usecDE 6.50 usecTE 300.1 KCNST2 145.0000000CNST13 10.0000000D0 0.00000300 secD1 1.50000000 secD2 0.00344828 secD6 0.05000000 secD16 0.00010000 secIN0 0.00001505 sec

======== CHANNEL f1 ========NUC1 1HP1 8.00 usecP2 16.00 usecPL1 4.00 dBPL1W 5.00000000 WSFO1 600.2742019 MHz

======== CHANNEL f2 ========NUC2 13CP3 14.00 usecPL2 0.00 dBPL2W 75.60778046 WSFO2 150.9546152 MHz

====== GRADIENT CHANNEL =====GPNAM1 SINE.100GPNAM2 SINE.100GPNAM3 SINE.100GPZ1 50.00 %GPZ2 30.00 %GPZ3 40.10 %P16 1000.00 usecND0 2TD 192SFO1 150.9546 MHzFIDRES 172.968826 HzSW 220.000 ppmFnMODE QFSI 2048SF 600.2700426 MHzWDW SINESSB 0LB 0.00 HzGB 0PC 1.40SI 1024MC2 QFSF 150.9377842 MHzWDW SINESSB 0LB 0.00 HzGB 0

O

O

O

O

O

OH

O

N

N

2

3

78

910

6a

6

3a10a

H-8H-7H-9

C-6

C-10C-3a

C-9

H-3H-2

C-10a

C-8

C-7

C-6a

C-5

C-12

C-1

C-3

C-2

10b

C-12aunder solventresidual peak

carbabamate

C-3a1

C-5a1

position of C-atom subtitution in position 6 subtitution in position 10 measured shift6 146.94 157.16 158.46

6a 126.34 126.46 127.527 120.47 118.26 123.338 125.79 124.67 129.459 107.06 112.26 127.43

10 154.33 151.23 148.2510a 119.73 119.70 122.85

Figure S13 1H-13C HMBC NMR (CD3CN) of chartarin-10-1,4‘-bipiperidine-1‘-carboxylate (11)

S28

11 10 9 8 7 6 5 4 3 2 1 ppm

1.50

1.51

1.83

1.83

1.83

1.85

2.05

2.05

2.06

2.19

2.21

2.65

3.00

3.28

3.54

4.21

4.24

4.61

7.42

7.43

7.47

7.49

7.53

7.65

7.70

7.96

8.05

10.58

10.83

1.97

2.75

4.69

16.8

9

1.49

1.67

0.82

1.16

0.86

1.06

1.42

0.63

Current Data ParametersNAME 6NNU349PREXPNO 1PROCNO 1




NNU349/ACN-d8/Ueberschaar/1HNU814e

O

O

O

O

O

O

O

N

N

O

N

N

Figure S14 1H NMR (CD3CN) of chartarin-6,10 di-1,4‘-bipiperidine-1‘-carboxylate (13)

S29

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

22.6

23.1

24.3

24.4

26.1

26.6

27.0

27.1

44.0

44.5

44.6

45.1

50.5

50.7

50.8

51.7

51.8

64.7

65.1

109.8

112.4

117.3

118.0

119.5

121.8

122.8

122.9

126.8

130.6

131.7

135.3

140.8

143.4

148.1

148.4

153.3

154.3

154.6

157.3


NAME 6NNU349CAEXPNO 50PROCNO 1





NNU349/ACN-d6/Ueberschaar/13CNU814e

O

O

O

O

O

O

O

N

N

O

N

N

Figure S15 13C NMR (CD3CN) of chartarin-6,10 di-1,4‘-bipiperidine-1‘-carboxylate (13)

S30

234567891011 ppm

1.80

1.85

2.08

2.21

2.37

2.96

3.02

3.29

3.47

3.51

3.53

3.59

3.62

4.25

4.28

4.59

4.62

7.50

7.50

7.52

7.52

7.56

7.58

7.72

7.74

7.74

7.75

7.96

7.98

8.37

8.37

8.38

8.39

10.41

2.55

3.64

5.80

2.30

5.09

2.06

1.95

1.01

1.13

1.16

1.03

1.00

0.88

Current Data ParametersNAME 5NNU352EXPNO 1PROCNO 1

F2 - Acquisition ParametersDate_ 20131207Time 14.34INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT CD3CNNS 32DS 2SWH 10000.000 HzFIDRES 0.152588 HzAQ 3.2767999 secRG 1030DW 50.000 usecDE 6.50 usecTE 299.9 KD1 1.00000000 secTD0 1


F2 - Processing parametersSI 131072SF 500.3000151 MHzWDW noSSB 0LB 0 HzGB 0PC 1.00

5NNU3521H/MeCN

O

O

O

O

O

OH

BrO

N

N

Figure S16 1H NMR (CD3CN) of bromochartarin-10-1,4‘-bipiperidine-1‘-carboxylate (18)

S31

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm23.2

24.4

26.5

26.7

44.0

44.5

51.0

51.5

65.1

98.0

110.3

119.3

120.8

123.4

123.9

127.8

127.8

130.0

138.3

138.6

148.4

154.4

158.7


NAME 5NNU352EXPNO 2PROCNO 1

F2 - Acquisition ParametersDate_ 20131208Time 0.50INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG udeftTD 19996SOLVENT CD3CNNS 24000DS 4SWH 29761.904 HzFIDRES 1.488393 HzAQ 0.3359328 secRG 2050DW 16.800 usecDE 6.50 usecTE 300.0 KD1 3.00000000 secD11 0.03000000 secD12 0.00002000 secD20 10.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 13CP1 13.00 usecP12 2000.00 usecP26 500.00 usecPL1 3.30 dBPL1W 44.17614746 WSFO1 125.8150021 MHzSP2 9.18 dBSP8 9.18 dBSPNAM[2] Crp60comp.4SPNAM[8] Crp60,0.5,20.1SPOAL2 0.500SPOAL8 0.500SPOFFS2 0 HzSPOFFS8 0 Hz



5NNU35213C/MeCN

O

O

O

O

O

OH

BrO

N

N

Figure S17 13C NMR (CD3CN) of bromochartarin-10-1,4‘-bipiperidine-1‘-carboxylate (18)

S32

12 11 10 9 8 7 6 5 4 3 2 1 ppm

0.94

1.42

1.44

1.83

2.05

2.51

2.99

3.01

3.54

4.27

4.29

4.62

4.64

4.96

4.98

7.55

7.56

7.73

7.75

7.76

7.77

7.79

7.81

7.82

8.12

8.13

8.42

8.43

10.56

11.54

1.17

3.94

6.44

6.37

1.47

1.39

1.00

1.96

1.23

1.07

1.00

0.92

0.88

Current Data ParametersNAME 6NNU354MUEXPNO 1PROCNO 1

F2 - Acquisition ParametersDate_ 20131205Time 8.07INSTRUM spectPROBHD 5 mm CPTCI 1H-PULPROG zg30TD 65536SOLVENT CD3CNNS 32DS 2SWH 12019.230 HzFIDRES 0.183399 HzAQ 2.7262976 secRG 11.3DW 41.600 usecDE 6.50 usecTE 300.1 KD1 1.00000000 secTD0 1



6NNU3541H/MeCN

O

O

O

O

O

OH

O

N

N

Figure S18 1H NMR (CD3CN) of norchartarin-10-1,4‘-bipiperidine-1‘-carboxylate (19)

S33

170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm23.1

24.4

26.8

44.0

44.5

51.0

51.2

51.4

65.0

98.8

111.0

120.3

121.5

122.9

123.4

123.5

126.6

127.6

127.8

129.3

132.6

138.5

148.3

150.1

154.7

158.6

160.5


NAME 6NNU354MUEXPNO 50PROCNO 1





6NNU35413C/MeCN

O

O

O

O

O

OH

O

N

N

Figure S19 13C NMR (CD3CN) of norchartarin-10-1,4‘-bipiperidine-1‘-carboxylate (19)

S34

1.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5 ppm

1.59

1.61

1.92

2.09

2.26

2.40

3.17

3.62

3.65

4.23

4.26

4.29

4.56

4.64

4.66

7.62

7.64

7.66

7.68

7.92

7.94

8.07

8.09

8.13

8.14

8.20

18.0

110

.92

9.98

13.3

7

12.3

9

4.09

4.00

3.34

2.22

1.53

2.09

Current Data ParametersNAME 5NNU351EXPNO 5PROCNO 1

F2 - Acquisition ParametersDate_ 20150312Time 8.00INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG zg30TD 65536SOLVENT MeODNS 64DS 2SWH 10000.000 HzFIDRES 0.152588 HzAQ 3.2767999 secRG 228DW 50.000 usecDE 6.50 usecTE 295.9 KD1 1.00000000 secTD0 1


F2 - Processing parametersSI 131072SF 500.2999990 MHzWDW noSSB 0LB 0 HzGB 0PC 1.00

5NNU3511H/MeOD + DCl

O

O

O

O

O

O

BrO

N

N

O

N

N

Figure S20 1H NMR (CD3OD+DCl) of bromochartarin-6,10 di-1,4‘-bipiperidine-1‘-carboxylate (20)

S35

200 180 160 140 120 100 80 60 40 20 ppm

22.8

22.9

24.4

24.5

26.2

26.4

26.6

27.1

43.9

44.1

44.5

44.9

64.4

64.8

108.9

109.0

111.2

117.6

120.5

120.8

121.3

123.4

124.3

127.6

131.6

138.9

142.9

147.3

147.9

148.9

153.8

155.0

155.4

156.9


NAME 5NNU351EXPNO 6PROCNO 1

F2 - Acquisition ParametersDate_ 20150312Time 14.24INSTRUM spectPROBHD 5 mm PABBO BB-PULPROG udeftTD 19996SOLVENT MeODNS 10000DS 4SWH 29761.904 HzFIDRES 1.488393 HzAQ 0.3359328 secRG 2050DW 16.800 usecDE 6.50 usecTE 297.8 KD1 3.00000000 secD11 0.03000000 secD12 0.00002000 secD20 10.00000000 secTD0 1

======== CHANNEL f1 ========NUC1 13CP1 13.00 usecP12 2000.00 usecP26 500.00 usecPL1 3.30 dBPL1W 44.17614746 WSFO1 125.8150021 MHzSP2 9.18 dBSP8 9.18 dBSPNAM[2] Crp60comp.4SPNAM[8] Crp60,0.5,20.1SPOAL2 0.500SPOAL8 0.500SPOFFS2 0 HzSPOFFS8 0 Hz



5NNU35113C udeft/MeOD + DCl

O

O

O

O

O

O

BrO

N

N

O

N

N

Figure S21 13C NMR (CD3OD+DCl) of bromochartarin-6,10 di-1,4‘-bipiperidine-1‘-carboxylate (20)

S36

11 10 9 8 7 6 5 4 3 2 1 ppm

1.49

1.50

1.51

1.83

1.83

1.85

2.16

2.18

2.21

2.28

2.98

3.00

3.26

3.28

3.30

3.40

3.45

3.54

4.23

4.61

7.42

7.46

7.65

7.75

7.95

7.99

10.56

10.80

2.07

3.90

5.07

5.30

2.02

2.37

1.88

1.81

1.00

2.62

1.89

1.52

0.61

Current Data ParametersNAME 6NNU353MUEXPNO 1PROCNO 1




6NNU3531H/MeCN

O

O

O

O

O

O

O

N

N

O

N

N

Figure S22 1H NMR (CD3CN) of norchartarin-6,10 di-1,4‘-bipiperidine-1‘-carboxylate (21)

S37

180 160 140 120 100 80 60 40 20 ppm23.1

24.3

26.4

27.0

27.7

44.0

44.5

45.1

50.7

51.7

64.4

64.7

64.8

65.0

109.8

112.2

119.2

120.6

121.8

122.0

122.9

126.3

126.9

126.9

130.6

131.8

132.8

143.2

148.0

148.4

150.2

153.2

153.4

154.4

154.7

157.4


NAME 6NNU353MUEXPNO 50PROCNO 1





6NNU35313C/MeCN

O

O

O

O

O

O

O

N

N

O

N

N

Figure S23 13C NMR (CD3CN) of norchartarin-6,10 di-1,4‘-bipiperidine-1‘-carboxylate (21)

S38

S39

LogD and Log S values

All logD and logS values were calculated using the ChemAxon calculation PlugIn.3 LogD options: lopP method: Consensus; Electrolyte concentration:

0.1 mol/L Cl−; 0.1 mol/L Na+, K+.

compound logD logS

daunorubicin (1)

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH

S40

chartreusin (2)

Daunorubicin-10-1,4'-bipiperidine-1'-

carboxylate (3)

daunorubicin aglycon (4)

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S41

resistomycin (5)

benastatin A (6)

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S42

benastatin B (7)

resistomycin-10-1,4'-bipiperidine-1'-

carboxylate (8)

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S43

Benastatin A-11-1,4'-bipiperidine-1'-

carboxylate (9)

Benastatin B-11-1,4'-bipiperidine-1'-

carboxylate (10)

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S44

chartarin-10-1,4'-bipiperidine-1'-

carboxylate (11)

chartarin (12)

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S45

chartarin-6,10 di-1,4'-bipiperidine-1'-

carboxylate (13)

bromochartreusin (14)

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S46

norchartreusin (15)

bromochartarin (16)

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S47

norchartarin (17)

bromchartarin-10-1,4'-bipiperidine-1'-

carboxylate (18)

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S48

norchartarin-10-1,4'-bipiperidine-1'-

carboxylate (19)

bromochartarin-6,10 di-1,4'-bipiperidine-

1'-carboxylate (20)

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S49

norchartarin-6,10 di-1,4'-bipiperidine-1'-

carboxylate (21)

irinotecan

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S50

ciprofloxacin

-3

0

3

6

4 5 6 7 8 9

pH-9

-6

-3

0

4 5 6 7 8 9

pH

S51

References:

1. N. Ueberschaar, Z. Xu, K. Scherlach, M. Metsä-Ketelä, T. Bretschneider, H.-M. Dahse, H. Goerls and C. Hertweck, J. Am. Chem. Soc., 2013, 135, 17408-17416.

2. K. Scherlach, L. P. Partida-Martinez, H. M. Dahse and C. Hertweck, J. Am. Chem. Soc., 2006, 128, 11529-11536.

3. ChemAxon, MarvinSketch, (2015) ChemAxon Ltd.

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