Supporting Information

© Wiley-VCH 2008

69451 Weinheim, Germany

1

Proximicins A, B, and C – Antitumor Furan Analogues of Netropsin from the Marine Actinomycete Verrucosispora – Induce Upregulation of p53 and the

Cyclin Kinase Inhibitor p21 in AGS Cells

Kathrin Schneider, Simone Keller, Falko E. Wolter, Lars Röglin, Winfried Beil, Oliver Seitz, Graeme Nicholson, Christina Bruntner, Julia Riedlinger, Hans-Peter Fiedler* and Roderich D. Süssmuth*

1. Structure elucidation

1.1 General Experimental Procedures.

LC-MS experiments were performed on a QTrap 2000 mass spectrometer (Applied Biosystems/MDS

Sciex, Darmstadt, Germany) or on a Bruker Esquire 3000plus mass spectrometer (Bruker-Daltonics,

Bremen, Germany) coupled to an Agilent 1100 HPLC system (Agilent, Waldbronn, Germany). ESI-FT-

ICR mass spectra were recorded on an APEX II FTICR mass spectrometer (4.7 T, Bruker-Daltonics,

Bremen, Germany). NMR experiments were recorded on an AMX600 NMR spectrometer (Bruker,

Karlsruhe, Germany) equipped with a 5-mm triple-resonance probehead with z gradients and some

experiments on two DRX600 NMR spectrometers (proximicin B (4)), on an AVANCE400 NMR

spectrometer (Bruker, Karlsruhe, Germany) (proximicin A (3), C (5)), on a DRX500 NMR spectrometer

(Bruker, Karlsruhe, Germany) equipped with a BBI probehead with z gradients (netropsin-proximicin-

hybrids 6, 7, 8).

1.2 Amino acid analysis.

Samples of proximicin A (3) and B (4) were hydrolysed under vacuum (200 µL 6 N HCl and 5 µL

phenol /110 °C /25 h), derivatized to the N-(O-)TFA ethyl esters ((i) + 200 µL 15 % acetyl chloride in

abs. ethanol /110 °C/30 min. ii) + 100 µL dichloromethane + 50 µL trifluoroacetic anhydride/ 110 °C/

10 min) and analysed by GC-MS (Agilent MSD) on a Chirasil capillary, (20 m x 0.25 mm, 0.13 µm

film).

In the case of proximicin C (5) no peaks were detected under the above conditions. For this reason,

further precautions were taken to minimize oxidative decompositions. Thus 250 µmol (i.e. as large

excess) of D-tryptophan were added to the sample of proximicin C (5) before the hydrolysis,

derivatization and GC-MS analysis were carried out as for proximicin A and B.

2

1.3 NMR data.

Proximicin A (3): colourless solid 1H NMR (DMSO-d6, 400 MHz) δH 10.69 (s, 1H, H-9), 9.80 (s, 1H, H-3), 8.12 (d, J = 0.53 Hz, 1H, H-11), 7,88 (bs, 1H, H-5), 7.84 (bs, 1H, H-15), 7.42 (bs, 1H, H-15), 7.17 (d, J = 0.53 Hz, 1H, H-12), 7.16 (bs, 1H, H-6), 3.67 (s, 3H, H-1).

13C NMR (DMSO-d6, 100 MHz) δc 158.9 (C-14), 155.0 (C-8), 153.7 (C-2), 145.7 (C-13), 145.0 (C-7), 133.0 (C-11), 131.7 (C-5), 127.2 (C-4), 125.8 (C-10), 108.4 (C-6), 107.7 (C-12), 51.9 (C-1). Proximicin B (4): colourless solid 1H NMR (DMSO-d6, 600 MHz) δH 10.64 (s, 1H, H-9), 9.75 (s, 1H, H-3), 9.12 (s, 1H, H-22), 8.41 (t, J = 5.71 Hz, 1H, H-15), 8.12 (s, 1H, H-11), 7.87 (bs, 1H, H-5), 7.17 (bs, 1H, H-6), 7.16 (s, 1H, H-12), 6.99 (d, J = 8.31 Hz, 1H, H-19), 6.67 (d, J = 8.31 Hz, 1H, H-20), 3.68 (s, 3H, H-1), 3.36 (m, 2H, H-16), 2.69 (t, J = 7.51 Hz, 2H, H-17).

13C NMR (DMSO-d6, 150 MHz) δc 157.4 (C-14), 155.6 (C-21), 155.2 (C-8), 153.9 (C-2), 146.0 (C-13), 145.2 (C-7), 133.0 (C-11), 132.0 (C-5), 129.4 (C-19), 129.3 (C-18), 127.3 (C-4), 126.0 (C-10), 115.1 (C-20), 108.5 (C-6), 107.7 (C-12), 52.0 (C-1), 40.4 (C-16), 34.2 (C-17). Proximicin C (5): colourless solid 1H NMR (DMSO-d6, 400 MHz) δH 10.81 (s, 1H, H-20), 10.71 (s, 1H, H-9), 9.82 (s, 1H, H-3), 8.57 (t, J = 5.66 Hz, 1H, H-15), 8.14 (s, 1H, H-11), 7.88 (s, 1H, H-5), 7.56 (d, J = 7.87 Hz, 1H, H-25), 7.32 (d, J = 7.87 Hz, 1H, H-22), 7.16 (bs, 3H, H-6, H-12, H-19), 7.05 (dd, J = 7.73, 7.20 Hz, 1H, H-23), 6.97 (dd, J = 7.11, 7.64 Hz, 1H, H-24), 3.67 (s, 3H, H-1), 3.48 (m, 2H, H-16), 2.91 (t, J = 7.48 Hz, 2H, H-17).

13C NMR (DMSO-d6, 100 MHz) δc 157.5 (C-14), 155.2 (C-8), 154.0 (C-2), 146.1 (C-13), 145.2 (C-7), 136.2 (C-21), 133.0 (C-11), 132.0 (C-5), 127.4 (C-4), 127.2 (C-26), 126.1 (C-10), 122.6 (C-19), 120.9 (C-23), 118.3 (C-25), 118.2 (C-24), 111.7 (C-18), 111.4 (C-22), 108.5 (C-6), 107.7 (C-12), 52.1 (C-1), 39.4 (C-16), 25.2 (C-17).

O

HNO

HN

OOH3C

O

HN O

OH

1

2

3 4

5

6 78

9 1011

12

1314

1516

17

1819

20

21

22

O

HNO

HN

OOH3C

O

H2N O

1

2

3 4

5

6 78

9 1011

12

1314

15

O

HNO

HN

OOH3C

O

HN O

NH23

24

1

2

3 4

5

6 78

9 1011

1213

14

1516

17

1819

202122

25 26

Proximicin A (3) Proximicin B (4) Proximicin C (5)

3

2. Synthesis of Netropsin-proximicin-hybrids (6-8)

NOH

O

BocHN

NOBn

O

RHN

N

OBn

O

HN

ON

RHN

10 R=Boc11 R=H HCl

12 R=Boc13 R=H HCl

N

OR

O

HN

ON

NHO

MeO

14 R=Bn15 R=H

N

NH2

O

HN

ON

NHO

MeO

N

HN

O

HN

ON

NHO

MeO

OH

N

HN

O

HN

ON

NHO

MeONH

9

6

7

8

1. CsCO3, EtOH/H2O2. BnBr, DMF

4 N HCl/dioxane

9, EDC,DMAP, DMF

4 N HCl/dioxane

TEA, MeOCOCl,THF

H2/Pd

1. DIC, HOBt2. 25 % NH3

Tyramine, EDC,DMAP, DMF

Tryptamine, EDC,DMAP, DMF

(83 %) (90 %) (93 %)

(13 %)

(64 %)

(93 %)

15

15

15

The synthesis of the netropsin-proximicin-hydrids (6-8) started from commercially available compound 9. This was transformed according to literature known procedures to compound 13.[ 1 , 2 ] Then we introduced at first the N-terminal methyl carbamate moiety followed by the modifications at the C-terminus found in the natural occuring proximicins A-C. General remarks: All reactions were carried out under an inert atmosphere with magnetic stirring. Anhydrous solvents (Acros) and all reagents were used as purchased (Acros, Sigma-Aldrich, Fluorochem Limited) for all reactions unless otherwise noted. Flash chromatography was performed with silica gel (for chromatography, 0.035-0.07 mm, 60A, Acros). Analytical thin-layer chromatography was performed with coated commercial silica gel plates (Merck, TLC aluminium sheets, silica gel 60 F254). Proton nuclear magnetic resonance (1H-NMR) data were acquired on a Bruker AV 400 (400 MHz) spectrometer and on a Bruker DRX500 (500 MHz) spectrometer. Chemical shifts are reported in delta (δ) units, in parts per million (ppm) with the residual proton signal as an internal standard.[3] Multiplets are designated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Carbon-13 nuclear magnetic resonance (13C-NMR) data were acquired on a Bruker AV 400 spectrometer at 100 MHz and on a Bruker DRX500 spectrometer at 125 MHz. Chemical shifts are reported in ppm relative to the solvent signal.[3] Infrared spectra were acquired in the ATR (attenuated total reflectance) mode on a Perkin-Elmer Infrared-Spectrometer 881. Absorbance frequencies are reported in reciprocal centimeters (cm-1). Mass spectra were collected using a Finnigan MAT 95 SQ with an EI ion source. [1] Grehn, L. and Ragnarsson, U. J. Org Chem. 1981, 46, 3492-3497. [2] Boger, D. L.; Fink, B. E.; Hedrick, M. P. J. Am. Chem. Soc. 2000, 122, 6382-6394. [3] Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem. 1997, 62, 7512-7515.

4

4-[(4-Methoxycarbonylamino-1-methyl-1H-pyrrole-2-carbonyl)-amino]-1-methyl-1H-pyrrole-2-carboxylic acid benzyl ester (14). Compound 13 (193 mg, 0.4963 mmol, 1.0 eq) was dissolved in 12 mL THF. TEA (153 µL, 1.0919mmol, 2.2 eq) was added and the reaction mixture was stirred for 10 min at RT. Methyl chloroformate (47 µL, 0.5956 mmol, 1.2 eq) was added dropwise and the mixture was stirred for additional 45 min at RT. The mixture was diluted with 25 mL EtOAc and washed with water (3 x 10 mL) and brine (5 x 10 mL). The organic extracts were dried over NaSO4 and the solvent was removed under reduced pressure yielding 190 mg (93 %) of the target compound. 1H NMR δH (400 MHz; DMSO-d6) 3.62 (s, 3H), 3.81 (s, 3H), 3.84 (s, 3H), 5.24 (s, 2H), 6.80 (bs, 1H), 6.92 (bs, 1H), 6.94 (d, 1H, J=1.9 Hz), 7.34-7.44 (m, 5H), 7.46 (d, 1H, J=1.9 Hz), 9.37 (s, 1H), 9.86 (s, 1H). 13C NMR δc (100 MHz; DMSO-d6) 36.1, 36.2, 51.5, 64.9, 103.8, 108.6, 117.3, 118.4, 121.0, 122.1, 122.7, 123.0, 127.8, 128.0, 128.5, 136.6, 154.0, 158.3, 160.1. EI-MS m/z 410 (M+•), 181, 149, 91. EI-HRMS m/z 410.1598 [M+•

, C21H22O5N4 requires 410.1590]. IR νmax (cm-1) 3312, 3133, 3032, 2951, 1702, 1647, 1586, 1555, 1433, 1405, 1243. 4-[(4-Methoxycarbonylamino-1-methyl-1H-pyrrole-2-carbonyl)-amino]-1-methyl-1H-pyrrole-2-carboxylic acid (15). Compound 14 (179 mg, 0.4361 mmol, 1.0 eq) was dissolved in 20 mL methanol. 40 mg of Pd/C (10 %) was added in one portion. The flask was immediately sealed with a rubber septum and it was flushed with H2 for 5 min. An H2-balloon was attached to the flask and the heterogenous mixture was vigorously stirred for 16 h at RT. Then the solution was filtered and evaporated to dryness yielding 126 mg (90 %) of the free acid. 1H NMR δH (400 MHz; DMSO-d6) 3.62 (s, 3H), 3.81 (s, 3H), 3.81 (s, 3H), 6.78 (d, 1H, J=1.9 Hz), 6.80 (s, 1H), 7.37 (d, 1H, J=1.9 Hz), 9.36 (s, 1H), 9.83 (s, 1H). 13C NMR δc (100 MHz; CDCl3) 36.1, 36.1 51.5, 103.8, 108.0, 117.1, 119.7, 121.2, 122.1, 122.5, 122.9, 154.1, 158.3, 162.3. EI-MS m/z 320 (M+•), 276, 181, 149. EI-HRMS m/z 320.1122 [M+•

, C14H16O5N4 requires 320.1121]. IR νmax (cm-1) 3432, 3374, 3260, 3114, 2952, 2922, 2851, 1737, 1693, 1582, 1568, 1434, 1235, 1201. Netropsin-proximicin-hybrid A (6). To acid 15 (30 mg, 0.0936 mmol, 1.0 eq) in DCM/DMF (4:1, 5 mL) was added HOBt (14 mg, 0.1030 mmol, 1.1 eq) and DIC (15 µL, 0.0983 mmol, 1.05 eq) and the mixture was stirred for 75 min at RT. 25 % Aq. NH3 (10 µL, 0.1546 mmol, 1.3 eq) was added and stirring was continued for 5 h. The pricipitate was removed by filtration, the filtrate was diluted with DCM (10 mL) and the solution was wahed with 5 % aq. NaHCO3 (3 x 5 mL), 5 % citric acid (2 x 5 mL), and H2O (5 mL), and dried over Na2SO4. The solvent was removed and the crude was purified by preparative HPLC (4 mg, 13 %). 1H NMR (DMSO-d6, 500 MHz) δΗ 9.83 (s, 1H), 9.35 (s, 1H), 7.19 (d, J = 1.4 Hz, 1H), 6.91 (bs,

1H), 6.81 (d, J = 1.5 Hz, 1H), 6.79 (bs, 1H), 3.80 (s, 3H), 3.78 (s, 3H), 3.61 (s, 3H). 13C NMR (DMSO-d6, 125 MHz) δC 163.0, 158.3, 154.0, 123.0, 122.6, 122.1, 121.6, 118.1, 116.8,

104.8, 103.6, 51.3, 35.9. EI-MS m/z 319 (M+•), 287, 181, 149. EI-HRMS m/z 319.1277 [M+•

, C14H17O5N4 requires 319.1281]. IR νmax (cm-1) 3328, 2924, 2853, 1704, 1646, 1600, 1436, 1404, 1248.

5

Netropsin-proximicin-hybrid B (7). Compound 15 (32 mg, 0.1 mmol, 1.0 eq) was dissolved in 2 mL DMF and EDCI (39 mg, 0.2 mmol, 2.0 eq), DMAP (25 mg, 0.2 mmol, 2.0 eq), and tyramine (14 mg, 0.1 mmol, 1.0 eq) were added sequentially. The flask was flushed with nitrogen and the mixture was stirred at RT for 16 h. The reaction mixture was diluted with EtOAc (20 mL) and washed with 10 % HCl (3 x 20 mL) and with saturated NaHCO3 (3 x 20 mL). The organic phase was dried over Na2SO4, filtered, and evaporated to yield a brown oil which gave after lyophilisation a white-fawn solid (28 mg, 64 %). 1H NMR (DMSO-d6; 500 MHz) δH 9.84 (s, 1H), 9.34 (s, 1H), 9.19 (s, 1H), 8.03 (t, J = 5.34 Hz,

1H), 7.16 (bs, 1H), 7.00 (d, J = 8.2 Hz, 2H), 6.91 (bs, 1H), 6.79 (bs, 2H), 6.66 (d, J = 8.2 Hz, 2H), 3.79 (s, 3H), 3.77 (s, 3H), 3.62 (s, 3H), 3.31 (not detectable), 2.66 (t, J = 7.4 Hz, 2H).

13C NMR (DMSO-d6, 125 MHz) δC 161.2, 158.3, 155.6, 154.1, 129.7, 129.2, 122.9, 121.9, 117.6, 116.9, 114.9, 103.8, 51.3, 40.4, 35.9, 35.8, 34.4.

EI-MS m/z 439 (M+•), 407, 319, 303, 271, 181, 149. EI-HRMS m/z 439.1850 [M+•

, C22H25O5N5 requires 439.1856]. IR νmax (cm-1) 3303, 2950, 1704, 1637, 1558, 1515, 1435, 1251. Netropsin-proximicin-hybrid C (8). Compound 15 (32 mg, 0.1 mmol, 1.0 eq) was dissolved in 2 mL DMF and EDCI (39 mg, 0.2 mmol, 2.0 eq), DMAP (25 mg, 0.2 mmol, 2.0 eq), and tryptamine (17 mg, 0.1 mmol, 1.0 eq) were added sequentially. The flask was flushed with nitrogen and the mixture was stirred at RT for 16 h. The reaction mixture was diluted with EtOAc (20 mL) and washed with 10 % HCl (3 x 20 mL) and with saturated NaHCO3 (3 x 20 mL). The organic phase was dried over Na2SO4, filtered, and evaporated to yield a brown oil which gave after lyophilisation a white-fawn solid (43 mg, 93 %). 1H NMR (DMSO-d6, 500 MHz) δH 10.79 (s, 1H), 9.84 (s, 1H), 9.35 (s, 1H), 8.13 (t, J = 5.7 Hz,

1H), 7.57 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.02 Hz, 1H), 7.17 (d, J=1.43 Hz, 1H), 7.15 (d, J=1,6 Hz, 1H), 7.06 (t, J = 7.4 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 6.91 (bs, 1H), 6.83 (d, J = 1.5 Hz, 1H), 6.79 (bs, 1H), 3.80 (s, 6H), 3.61 (s, 3H), 3.44 (m, 2H), 2.89 (t, J = 7.4 Hz, 2H).

13C NMR (DMSO-d6; 125 MHz) δc 161.3, 158.4, 154.0, 136.3, 127.3, 123.0, 122.4, 122.1, 120.7, 118.1, 117.6, 116.9, 112.0, 111.1, 103.9, 103.6, 51.3, 39.3, 35.8, 25.2.

EI-MS m/z 462 (M+•), 430, 319, 303, 287, 271, 181, 149, 143, 130. EI-HRMS m/z 462.2015 [M+•

, C24H26O4N6 requires 462.2016]. IR νmax (cm-1) 3299, 2969, 2929, 1703, 1637, 1587, 1524, 1459, 1434, 1249.

6

3. Biological profiling with tumor cell lines

The inhibitory activities of the proximicins on the growth of tumor cells was tested according to NCI

(USA) guidelines[4] with the human tumor cell lines AGS and HM02 (gastric adenocarcinoma), MCF7

(breast adenocarcinoma), HepG2 (hepatocellular carcinoma) and Huh7 (from hepatocellular carcinoma

with mutated p53).[5] The cells were cultivated in 96-well microtiter plates in RPMI 1640 with 10 %

fetal calf serum in a humidified atmosphere of 5 % CO2 in air. After 24 hours the test compounds

(0.1~50 µg/mL) were added to the cells and the cells cultivated for additional 48 hours. The cell count

was surveyed by protein determination with sulforhodamine B. From the resulting concentration-

activity curves, the GI50 (concentration at which half of the cells were inhibited in their growth) and TGI

values (concentration at which a total inhibition of cell growth was observed) were obtained.

Cell cycle distributions were determined by staining DNA with propidium iodide. AGS and Huh7 cells

were incubated for 24 or 40 hours with the test compounds in 12-well plates with RPMI 1640 with 10 %

fetal calf serum, harvested by trypsination, washed with RPMI 1640 containing 1 % fetal calf serum and

resuspended in 100 µL staining solution (propidium iodide 150 µg/mL, 4 mM Na-citrate pH 7.0, 1 %

Triton X-100, BSA 1 %). After 15 min incubation at RT in the dark, 100 µL RNAse solution (10

mg/mL Ribonuclease A in Tris/HCl buffer, pH 7.5) were added. Thirty min later cells were analyzed

using a Becton Dickinson FACSscan.

Expression of cell cycle regulatory proteins: AGS cells were incubated for 6 and 13 hours with 2, 5 and

8, harvested by scraping into ice-cold PBS and lysed for 10 min on ice in 20 mM Hepes (pH 7.4), 50

mM NaCl, 10 % glycerol, 2 mM EDTA, 10 mM NaF, 10 mM NaPPi, 100 µM Na3VO4, 10 µM

leupeptin, 10 nM okadaic acid, 1 mM DTT, and 0.1 % Triton. Cells were disrupted by sonication (three

5-s bursts, power 20 watt) and extracts were cleared by centrifugation (13.000 x g at 4 °C). 20 µg (p53,

Cyclin E) or 50 µg (p21) proteins of cell lysates were subjected to SDS-PAGE using 10 % (p53, cyclin

E) and 15 % (p21) polyacrylamide gels. Proteins were electrophoretically transferred to PVDF

membranes and membranes were blocked for 1 hour in RotiR-Block. Mouse monoclonal antibodies to

p53, cyclin E (Pharmingen, dilution 1:1000), p21 (Calbiochem, dilution 1:1000) and β-tubulin (Cell

signal, dilution 1 : 4000) were applied for 1 h in TTBS + 1 % BSA. After rinsing blots were incubated

with the appropriate AP-conjugated second antibody (1:10.000 in TTBS + 5 % nonfat dry milk) and

visualized using the Amersham ECL system.

[4] Grever, M. R, Shepartz, S. A., Chabner, B. A. Semin. Oncol. 1992, 19, 622-638. [5] Müller, M., Strand, S., Hug, H., Heinemann, E.M., Walczak, H., Hofmann, W.J., Stremmel, W., Krammer, P.H., Galle, P.R. J Clin Invest 1997, 99, 403-413.

7

3.1 Cytostatic Activities of (1-8) against Selected Human Tumor Cell Lines.

AGS HepG2 MCF7 HM02

Substance

GI50 [µM]

TGI [µM]

GI50 [µM]

TGI [µM]

GI50 [µM]

TGI [µM]

GI50 [µM]

TGI [µM]

Netropsin (1) >116.2 >116.15 60.4 116.2 74.3 106.9 60.4 106.9Distamycin (2) >103.8 >103.84 54.0 >103.8 14.3 66.5 32.2 >103.8Proximicin A (3) 2.0 >34.1 2.8 >34.1 24.6 >34.1 - -Proximicin B (4) 3.6 >24.2 23.0 >24.2 12.1 >24.2 - -Proximicin C (5) 0.6 >22.9 1.8 >22.9 20.6 >22.9 - -Hybrid A (6) 93.9 119.0 119.0 >156.6 131.5 >156.6 96.5 131.5Hybrid B (7) 28.4 41.0 34.1 81.9 41.0 77.4 30.7 72.4Hybrid C (8) 17.7 36.8 21.6 36.8 24.9 34.6 20.5 34.6

AGS: gastric adenocarcinoma; HepG2: hepatocellular carcinoma, MCF7: breast adenocarcinoma. GI50: 50 % growth inhibition. TGI: 100 % growth inhibition. 3.2.1 Cell cycle analysis of AGS cells exposed to (1), (2), (5) and (8) for 24h.

Substance Sub G1 (Apoptosis) G0/G1 S G2/M

control (1 % MeOH) 1.6 ± 0.15 58.3 ± 0.4 20.4 ± 0.4 19.6 ± 0.3

Netropsin (1) (116,2 µM) 1.7 ± 0,5 49.1 ± 2.7* 22.7 ± 0.7* 26.5 ± 2.7*

Distamycin (2) (103,8 µM) 1.4 ± 0,13 51.3 ± 1,4* 22.3 ± 0.8* 25.0 ± 2.0*

control (1 % DMSO) 1.9 ± 0.4 58.9 ± 0.8 18.6 ± 0.95 20.7 ± 1.1

Proximicin C (5) (1,1 µM) 2.1 ± 0.35 64.5 ± 0.3* 17.1 ± 0.47 16.5 ± 0.7*

control (1 % MeOH) 2.0 ± 0.13 56.6 ± 1.7 20.4 ± 2.1 21.1 ± 0.3

Hybrid C (8) (21,6 µM) 2.3 ± 0.17 78 ± 1.3* 7.2 ± 1.3* 12.4 ± 0.95*

Data represent percentage of cells in each stage of the cell cycle Values are means ± S.E. of three independent experiments. * p< 0.05 versus control (t-test)

8

3.2.2 Cell cycle analysis of AGS cells exposed to (1), (2), (5) and (8) for 40 h

Sub G1

G0/G1 S G2/M

Control 5.3 ± 1.1 58.1 ± 2 18.6 ± 1.1 17.9 ± 2

Netropsin (1) (116,2 µM) 4.9 ± 1.6 48.6 ± 0.9* 17.0 ± 0.7 29.2 ± 0.5*

Distamycin (2) (103,8 µM) 4.4 ± 1.6 37.6 ± 2.9* 11.0 ± 2* 46.8 ± 6*

Proximicin C (5) (1,1 µM) 8.2 ± 1.5* 56.0 ± 1.3 18.3 ± 1.8 18.4 ± 0.3

Hybrid C (8) (21,6 µM) 15.1 ± 4* 59.6 ± 6 8.6 ± 1.1* 16.9 ± 1.8

Data represent percentage of cells in each stage of the cell cycle Values are means ± S.E. of three independent experiments. *p< 0.05 versus control (t-test) 3.2.3 Cell cycle analysis of Huh7 cells exposed to (1), (2), (5) and (8) for 24 h

Sub G1

G0/G1 S G2/M

Control 1.2 ± 0.1 62 ± 1.8 22.5 ± 1.3 16.0 ± 0.8

Netropsin (1) (58.1µM) 1.0 ± 0.15 53.2 ± 1.8* 25.0 ± 3.3 20.4 ± 1.7*

Distamycin (2) (51.9 µM) 3.1 ± 0.2* 39.3 ± 2.4* 33.1 ± 1.4* 24.7 ± 1.2*

Proximicin C (5) (4,4 µM) 0.9 ± 0.1 66.2 ± 1.2 17.8 ± 4 15.1 ± 1.1

Hybrid C (8) (21,6 µM) 1.5 ± 0.6 58.7 ± 3.5 19.7 ± 2.4 19.6 ± 1.6

Data represent percentage of cells in each stage of the cell cycle Values are means ± S.E. of three independent experiments. *p< 0.05 versus control (t-test)

9

3.3.1 Effect of (8) on p21, p53 and cyclin E expression in AGS cells. Cells were collected after 6 and 13 h incubation with 21.6 µM (8). Cyclin E

p53

p21

Control Compound 8

6h 13h 6h 13h

10

3.3.2 Effect of (2) on p21, p53 and cyclin E expression in AGS cells. Cells were collected after 6 and 13 h incubation with 103.8 µM (2). Cyclin E

p53

p21

Control Distamycin

6h 13h 6h 13h

11

3.4 Thermal denaturation experiments: Denaturation experiments were performed on a Cary 100 Bio UV/Vis spectrophotometer (Varian) equipped with a peltier thermostatted cell changer. We used a DNA-Duplex (DNA-1: cgcaaatttgcg and DNA-2: catggccatg) concentration of 2 µM (diluted from 0.5 mM stock-solutions in 20 mM NaH2PO4, 100 mM NaCl, pH 7.4) in a freshly degassed 20 mM NaH2PO4, 100 mM NaCl buffer at pH 7.4 and the drug concentration was varied between 0 and 6 µM (diluted from 1.0 mM stock-solutions in DMSO). Denaturation was monitored at a detection wavelength of λ=260 nm with a heating rate of 0.5 °Cmin-1. The given TM-value is the mean of at least three denaturation curves.

12

Netropsin (1): DNA 1

DNA 2

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

13

Distamycin (2): DNA 1

DNA 2

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

14

Proximicin A (3): DNA 1

DNA 2

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

15

Proximicin B (4): DNA 1

DNA 2

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

16

Netropsin-proximicin-hybrid A (6): DNA 1

DNA 2

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

17

Netropsin-proximicin-hybrid B (7): DNA 1

DNA 2

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

18

Netropsin-proximicin-hybrid C (8): DNA 1

DNA 2

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM

0 µM 1 µM 2 µM

3 µM 4 µM 6 µM