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Chem. Res. Toxicol. 1999, 12, 700-706
Cytotoxicity and DNA Interaction of the Enantiomers of 6-Amino-3-(chloromethyl)-1-[(5,6,7-trimethoxyindol-2-yl)carbonyl]indoline (Amino-seco-CI-TMI) Moana Tercel,* Michael A. Gieseg, Jared B. Milbank, Maruta Boyd, Jun-Yao Fan, L. Karin Tan, William R. Wilson, and William A. Denny Auckland Cancer Society Research Centre, Faculty of Medicine and Health Science, The University of Auckland, Private Bag 92019, Auckland, New Zealand Received April 22, 1999
The enantiomers of the previously reported racemic 6-amino-3-(chloromethyl)-1-[(5,6,7trimethoxyindol-2-yl)carbonyl]indoline (amino-seco-CI-TMI) were prepared via resolution of a precursor by chiral HPLC. The only detectable product isolated from reaction of the racemic compound with calf thymus DNA, followed by thermal cleavage, was shown by mass spectrometry and two-dimensional NMR spectroscopy to be the adenine N3 adduct. Polyacrylamide gel electrophoresis assays with the racemate and with each enantiomer also showed adenine to be the only site of alkylation. While the racemic amino compound exhibited sequence selectivity identical to that of the previously characterized phenol analogue, the enantiomers exhibited distinctly different sequence selectivities, allowing the (+) enantiomer to be assigned the “natural” S configuration. The (+)-(S) enantiomer is 3-fold more cytotoxic than the (-)-(R) enantiomer (IC50 values of 240 and 700 nM, respectively, in AA8 cells, after exposure for 4 h).
Introduction CC-1065 and the duocarmycins constitute a group of extremely potent antitumor antibiotics with a common mechanism of action (1). These compounds bind to DNA in the minor groove and alkylate at N3 of adenine in a highly sequence selective manner. This alkylation is presumed to be responsible for their remarkable cytotoxicity (picomolar IC50 values are observed against some tumor cell lines) and in vivo antitumor activity (2, 3). Extensive studies with the natural products and with related synthetic derivatives have led to the identification of 2 (Scheme 1) as the minimum common pharmacophore for this class (4, 5), comprising a simplified left-hand alkylating subunit (CI),1 and a right-hand minor groove binding subunit (here illustrated with the trimethoxyindole group common to the duocarmycins). The cyclopropyldienone structure of the alkylating subunit can be formed by ring closure of a seco precursor 1, and, in fact, for many analogues the ring-closed and seco forms exhibit identical biological properties (1). These properties include a distinctive sequence selectivity for the alkylation step, with the compounds preferentially binding in the narrower and deeper AT rich regions of the minor groove, where the number of consecutive AT base pairs matches the effective length of the particular compound. For the natural enantiomers (such as those illustrated in Scheme 1), this translates to alkylation at the 3′ end of an AT sequence (about three 1 Abbreviations: CI, 1,2,7,7a-tetrahydrocyclopropa[1,2-c]indol-4-one; TMI, 5,6,7-trimethoxyindole-2-carboxylate; CBI, 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one; DSA, duocarmycin SA alkylation subunit; NOESY, nuclear Overhauser effect spectroscopy; COSY, correlation spectroscopy; HMQC, heteronuclear multiple-quantum coherence; HMBC, heteronuclear multiple-bond coherence; EDCI, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide; HRMS, high-resolution mass spectrometry; PNK, polynucleotide kinase; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; PCR, polymerase chain reaction.
Scheme 1. Mechanism of DNA Alkylation by seco-CI-TMI
base pairs in length for compounds such as 1 and 2), while the unnatural enantiomers exhibit a sequence specificity extending in the opposite direction around the alkylated base (1). Alkylation occurs by addition of N3 of adenine to the least substituted carbon of the cyclopropane ring, causing ring opening and aromatization of the alkylating subunit. The initially formed product is heat labile, and simple thermal cleavage leads to strand breakage and the isolation of adducts such as that shown in Scheme 1. We have previously reported (6-8) the synthesis of a series of compounds in which the crucial seco-CI phenol is replaced by nitrogen or sulfur substituents, as, for example, in the aniline 7 (Scheme 2). We have also recently extended this work to the synthesis of the more
10.1021/tx990069o CCC: $18.00 © 1999 American Chemical Society Published on Web 07/14/1999
Toxicity and DNA Interaction of Amino-seco-CI-TMI
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Scheme 2. Synthesis of the Enantiomers of Amino-seco-CI-TMI
chromatography of the residue (CH2Cl2) and trituration of the product with Et2O gave 1-(tert-butyloxycarbonyl)-3-[[(methylsulfonyl)oxy]methyl]-6-nitroindoline [(()-4] (537 mg, 85%): mp 124-126 °C; 1H NMR (CDCl3) δ 8.68, 8.34 (2 br s, 1 H, H-7), 7.86 (dd, J ) 8.2, 2.1 Hz, 1 H, H-5), 7.35 (d, J ) 8.2 Hz, 1 H, H-4), 4.38 (dd, J ) 10.1, 6.1 Hz, 1 H, H-2), 4.32 (dd, J ) 10.1, 7.2 Hz, 1 H, H-2), 4.21 (dd, J ) 11.7, 10.0 Hz, 1 H, CHHCl), 3.96 (dd, J ) 11.7, 5.1 Hz, 1 H, CHHCl), 3.83 (dddd, J ) 10.0, 7.2, 6.1, 5.1 Hz, 1 H, H-3), 3.03 (s, 3 H, SO2CH3), 1.60 [br s, 9 H, C(CH3)3]; 13C NMR δ 151.7, 149.1, 144.4 (br), 135.7 (br) (C6, C-8, C-9, NCO), 124.8 (br) (C-7), 117.8, 110.0 (C-4, C-5), 82.4 (br) [C(CH3)3], 69.6 (CH2OSO2), 60.0 (br) (C-2), 39.3 (br) (C-3), 37.7 (OSO2CH3), 28.3 [C(CH3)3]; MS (electron impact, relative intensity) m/z 372 (M+, 4%), 57 (100%); HRMS (high-resolution mass spectrometry) 372.09997 (C15H20N2O7S requires 372.09912). Resolution of (()-4 on a Daicel Chiralcel OD column (2 cm × 25 cm), eluting with iPrOH/hexane (35/65) at a rate of 6.75 mL/ min, gave (in order of elution, retention times of 62 and 70 min, R ) 1.13) (-)-4 [mp 38-40 °C, [R]20 D -59.8° (c 0.348, THF)] and (+)-4 [mp 38-40 °C, [R]20 D +59.8° (c 0.302, THF)]. A mixture of (-)-4 (100 mg, 0.269 mmol), LiCl (55 mg, 1.30 mmol), and DMF (0.5 mL) was stirred at 80 °C for 1 h. Ice-cold water (4 mL) was added, and the precipitate was removed by filtration, washed with water, and dissolved in Et2O (10 mL). This solution was washed with water (two times), dried (MgSO4), and evaporated. Crystallization from petroleum ether gave (-)-1-(tert-butyloxycarbonyl)-3-(chloromethyl)-6-nitroindoline [(-)5] (76 mg, 90%): mp 94-96 °C, [R]20 D -35.2° (c 0.401, THF). A solution of (-)-5 (93 mg, 0.249 mmol) in dioxane (4 mL) was saturated with HCl, allowed to stand at room temperature for 1.5 h, and evaporated. The resulting solid was dissolved in dimethylacetamide (3 mL), and 5,6,7-trimethoxyindole-2-carboxylic acid (63 mg, 0.249 mmol) and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI.HCl) (0.143 g, 0.75 mmol) were added. The mixture was stirred at room temperature for 6 h, and then diluted with ice-cold water (20 mL). The precipitate was removed by filtration, washed with water, dried (desiccator), and purified by flash chromatography (5 to 90% EtOAc/hexane). The product crystallized from CH2Cl2/iPr2O to give (-)-3-(chloromethyl)-6-nitro-1-[(5,6,7-trimethoxyindol-2-yl)carbonyl]indoline [(-)-6] (68 mg, 49%): mp 189-191 °C, [R]20 D -68.0° (c 0.315, THF). Hydrogenation of (-)-6 (13 mg, 29 µmol) in THF (5 mL) over platinum oxide (5 mg) at 50 psi H2 for 1 h gave (-)-6-amino-3(chloromethyl)-1-[(5,6,7-trimethoxyindol-2-yl)carbonyl]indoline [(-)-7] (12 mg, 100%): mp 99-101 °C, [R]20 D -34.9° (c 0.172, THF). Similar treatment of (+)-4 gave (+)-5 (90%) [mp 94-96 °C, [R]20 D +36.0° (c 0.414, THF)] and then [(+)-6] (53%) [mp 189191 °C, [R]20 D +66.6° (c 0.317, THF)]. Hydrogenation of (+)-6 gave (+)-7 (93%) [mp 99-101 °C, [R]20 D +35.1° (c 0.174, THF)]. Isolation of Adduct 8 from Reaction of (()-7 with Calf Thymus DNA. A solution of (()-7 (5.0 mg, 12.0 µmol) in DMF (27 mL) was added to a solution of calf thymus DNA (405 mg, drug/DNA base pair ratio of 1/50) in 10 mM Tris buffer (pH 7.4, containing 1 mM EDTA, 243 mL), and the mixture was stirred at 37 °C for 65 h, and then at reflux for 30 min. The cooled solution was extracted with CH2Cl2 (four times), and the extracts were washed with water, dried (Na2SO4), and evaporated to remove the remaining DMF. The residue was dissolved in CH2Cl2 and washed with water, and the organic layer was dried (Na2SO4) and evaporated to give crude 8 as a white solid (6.5 mg, 104%). 1H NMR and HPLC analysis (320 nm) showed only a single adduct. The sample was triturated with petroleum ether (5 × 1 mL) and dried to constant weight to give pure 8 (5.4 mg, 87%): 1H NMR [(CD3)2SO] δ 11.28 (s, 1 H, NH), 8.17 (s, 1 H, A H-2), 7.90 (br s, 2 H, A NH2), 7.77 (s, 1 H, A H-8), 7.46 (br s, 1 H, H-7), 6.90 (s, 1 H, H-4′), 6.89 (d, J ) 2.0 Hz, 1 H, H-3′), 6.59 (d, J ) 8.1 Hz, 1 H, H-4), 6.24 (dd, J ) 8.1, 2.1 Hz, 1 H, H-5), 5.18 (s, 2 H, NH2), 4.48-4.41 (m, 2 H, H-3b), 4.41-4.34 (m, 2 H, H-2), 4.10-4.02 (m, 1 H, H-3), 3.92 (s, 3 H, 7′-OCH3), 3.81 (s, 3 H, 5′-OCH3), 3.79 (s, 3 H, 6′-OCH3); 13C NMR
complex and more potent amino-seco-CBI (9, 10) and amino-seco-DSA (11) analogues. It was hoped that these amino compounds would retain DNA alkylating abilities similar to those of their phenol analogues, while offering new opportunities for the preparation of stable, less toxic prodrug forms, particularly those that might be activated by bioreduction (7). Compound 7 (as the racemate) was shown to be a moderately potent cytotoxin (IC50 values of 0.3-0.5 µM), although considerably less potent (50120-fold) than the racemate of phenol 1 (6). We now report detailed studies of the interaction of racemic 7 and its enantiomers with DNA, including sequence selectivity and isolation of the adduct of 7 and calf thymus DNA. The results of these studies support a common mechanism of DNA interaction for the phenol and amino forms of these seco-CI-TMI agents.
Experimental Procedures NMR Spectroscopy. 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively. The two-dimensional phasesensitive 1H-1H NOESY (nuclear Overhauser effect spectroscopy) spectrum of adduct 8 was collected using an 800 ms mixing time and a 209.1 ms acquisition time, a sweep width of 12.25 ppm, 512 complete t1 data points, and 48 scans per increment. The two-dimensional phase-sensitive 1H-1H COSY (correlation spectroscopy) spectrum was collected using a standard pulse sequence with a 209.1 ms acquisition time, a sweep width of 12.25 ppm, 512 complete t1 data points, and 32 scans per increment. The two-dimensional 1H-13C HMQC (heteronuclear multiple-quantum coherence) spectrum and 1H-13C HMBC (heteronuclear multiple-bond coherence) spectrum were collected using a standard pulse sequence (12), with a 209.1 ms acquisition time, 512 complete t1 data points, 80 (HMQC) or 128 (HMBC) scans per increment, and a spectral width in the f1 dimension of 172.54 ppm. Synthesis of Enantiomers of 7 (Scheme 2). Methanesulfonyl chloride (0.24 mL, 3.1 mmol) was added to a cooled (ice/ water) solution of racemic 1-(tert-butyloxycarbonyl)-3-(hydroxymethyl)-6-nitroindoline (8) [(()-3] (0.50 g, 1.70 mmol) and Et3N (0.47 mL, 3.4 mmol) in CH2Cl2 (10 mL). After 20 min, water (10 mL) was added and the mixture was stirred for 2 h. The aqueous phase was separated and extracted with CH2Cl2 (two times). The combined extracts were washed with water and a saturated NaCl solution, dried (MgSO4), and evaporated. Flash
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Scheme 3. Formation of the DNA Adduct of Amino-seco-CI-TMI
δ 160.0 (C-2a′), 154.9 (A C-6), 152.4 (A C-8), 149.8 (A C-4), 149.1 (C-6 or C-5′), 149.0 (C-6 or C-5′), 144.1 (C-7a), 143.5 (A C-2), 139.6 (C-6′), 139.0 (C-7′), 131.2 (C-2′), 125.1 (C-7a′), 124.5 (C4), 123.1 (C-3a′), 120.4 (A C-5), 118.6 (C-3a), 109.5 (C-5), 105.7 (C-3′), 103.0 (C-7), 97.9 (C-4′), 61.1 (7′-OCH3), 60.9 (6′-OCH3), 55.9 (5′-OCH3), 53.9 (C-2), 53.2 (C-3b), 38.8 (C-3); HRMS (fast atom bombardment) m/z 515.2146 (C26H27N8O4 requires 515.2155). Labeling of DNA and thermal cleavage assays were performed as previously described (13). Growth Inhibition Assay. Cell lines were maintained as monolayers in exponential phase growth using R-minimal essential medium containing fetal bovine serum (5% v/v) without antibiotics. Drug stock solutions were stored frozen in Me2SO and diluted into culture medium immediately before use to give final Me2SO concentrations of
