Structural Necessity of Indole C5-O-Substitution of seco-Duocarmycin Analogs for Their Cytotoxic Activity

A series of racemic indole C5-O-substituted seco-cyclopropylindole (seco-CI) compounds 1-5 were prepared by coupling in the presence of EDCI of 1-(tert-butyloxycarbonyl)-3-(chloromethyl)indoline (seg-A) with 5-hydroxy-, 5-O-methylsulfonyl, 5-O-aminosulfonyl, 5-O-(N,N-dimethylaminosulfonyl)- and 5-O-benzyl-1H-indole-2-carboxylic acid as seg-B. Compounds 1-5 were tested for cytotoxic activity against four human cancer cell lines (COLO 205, SK-MEL-2, A549, and JEG-3) using a MTT assay. Compounds 2 and 3 with small sized sulfonyl substituents like 5-O-methylsulfonyl and 5-O-aminosulfonyl exhibit a similar level of activity as doxorubicin against all cell lines tested.

The cytotoxicity of DUMs and CC-1065 results from the reaction of the CPI moiety with adenine-N3 groups in the minor groove of AT-rich sequences of DNA [4]. Even though (+)-CC-1065 has OPEN ACCESS potential cytotoxic properties, its use as an anticancer drug is hampered by delayed lethal toxicity to animals at therapeutic doses [5]. DUM SA seems to be superior to CC-1065 due to the lack of a delayed fetal hepatotoxicity like that with (+)-CC-1065 [6]. Moreover, DUM SA is the most hydrolytically stable member of this class of compounds [6][7][8], but it is toxic to the bone marrow [9]. Therefore, there is a strong interest in the design and development of novel analogs of duocarmycin that effectively kill cancer cells and have reduced toxicity to the host. One attempt [10,11] to design novel analogs involves the synthesis of a spirocyclic 1,2,7,7a-tetrahydrocycloprop [1,2-c]indol-4-one [(CI-numbering), CI] as the minimum size pharmacophore [12], which can be formed by ring closure of a seco-CI parent (Scheme 1). Seco compounds possess a similar alkylating selectivity and efficiency as the corresponding natural product (+)-enantiomers [13] since they can form the cyclopropane moiety in situ by an intramolecular Winstein cyclization [14]. Scheme 1. Proposed mechanism of activation and DNA alkylation of the seco-CI moiety [14]. Most duocarmycins exhibit more pronounced distinctions between enantiomers, but the spirocyclic 1,2,7,7a-tetrahydro-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]cycloprop [1,2-c]indol-4-one (CI-TMI) has been observed to show similar biological potency and relative DNA alkylation efficiency [13,15]. There is no difference of cytotoxic activity between (+)-CI and (-)-CI enantiomers, therefore no resolution is required to obtain a more active enantiomer.
Some previous results indicated that the seg-A ring structure influences the electrophilicity of cyclopropane [16]. On the other hand, the seg-B one has been considered to play some important roles in noncovalent binding to DNA [17][18][19]. Especially, the C5 position of seg-B was known as the most important site potentiating the cytotoxic activity. An adequate indole C5 substituent may not only extend the rigid length of the compound enhancing the DNA binding and thus accelerating the rate of DNA alkylation, but also benefit close contacts within a minor groove hydrophobic pocket [20]. Boger reported that a single 5-methoxy group on the indole as a minor groove binding unit was sufficient to maintain potency and a series of dimethylaminoethoxy group substituted analogs retained the cytotoxicity of the TMI analog, while providing increased aqueous solubility [16,21].
Indole C5 sulfone derivatives were synthesized and evaluated against the L1210 cell line and as a result, the C5 methanesulfonyl derivatives proved exceptionally potent, being 15-20 fold more active than the corresponding thiomethyl or methoxy derivatives [20]. Herein we describe the synthesis and cytotoxic activity of indole-C5-O-substituted and O-sulfonyl seco-CI analogs as different minor groove binding side chains.

Cytotoxic activity
Cytotoxic activity was determined as IC 50 values in four cell lines (COLO 205, SK MEL-2, A549 and JEG-3) for a 48 h drug exposure using a growth inhibition microassay. The results of the evaluation of the in vitro cytotoxic activity of compounds 1-5 are presented in Table 1 and Figure 2.    [20]. This trend has been explained by the fact that inclusion of an extended aromatic unit in the CI unit, as in the case of CBI, significantly enhances chemical stability and cytotoxicity [21]. In conclusion, both the alkylation part (CBI unit) and a (+)-enantiomer are seem to be important factors for enhanced activity compared with racemic and a seco-CI unit.

General
Moisture-or air-sensitive reactions were conducted under nitrogen in distilled solvents. The commercial reagents were purchased from Aldrich, Fluka, or Sigma. Melting points were measured on a Gallenkamp melting point apparatus and are not corrected. 1 H-(400 MHz), 13 C-NMR (100 MHz), HSQC, and HMQC spectra were taken on a Varian AS 400 MHz spectrometer. Chemical shifts (δ) are in parts per million (ppm) relative to tetramethylsilane, and coupling constants (J) are in Hertz. GC/MS spectra were obtained on a Shimadzu MS-QP2010 mass spectrometer. The MPLC apparatus used was a Yamazen YFLC-AI-580. Analytical TLC was performed on pre-coated silica gel 60 F 254 plates (Merck). Column chromatography was carried out on Merck silica gel 9385 (230-400 mesh) and the eluting solvent is indicated in each entry. (9). According to the method of Fukuda et al. [3], to a solution of metal Na (2.2 g, 94.35 mmol) in methanol (100 mL) was added a solution of 3-hydroxybenzaldehyde (6, 5.0 g, 23.58 mmol) and methyl azidoacetate (7, 10.9 g, 94.35 mmol) in methanol (100 mL) at −20 °C for 30 min. The reaction mixture was stirred at −20 °C for 3 h and at 0 °C for 12 h and then added with water to form a precipitate. This precipitate was filtered off and washed with ice water to obtain compound 8, which was refluxed in xylene (50 mL) for 1 h. The remaining xylene was evaporated to yield the crude compound which was recrystallized from ethyl acetate/n-hexane to afford 9 as a pale pink powder. Yield  (10). A suspension of methyl 5-hydroxy-1H-indole-2carboxylate (9, 1 g, 5.23 mmol) in methanol (10 mL) was treated with a solution of 10 % NaOH (10 mL) and heated at 60 °C for 1 h. After cooling to room temperature, the solution was acidified with 10% HCl and the precipitate formed was isolated by filtration and washed with water to give the crude compound which was recrystallized with methanol/ethyl acetate to afford 10 as a white powder. Yield: 908 mg (98%); mp: 245-247 (dec.) °C, (249 °C (dec.), commercial available compound). (11). According to the method of Schmidhammer et al. [26], a suspension of K 2 CO 3 (14.8 g, 107.13 mmol) in chloroform/methanol (2:1, 300 mL) was refluxed for 15 min and added a mixture of 6 (3.0 g, 24.57 mmol) and benzyl bromide (5.0 g, 29.48 mmol) in chloroform/ methanol (2:1, 90 mL) over 30 min. The reaction mixture was refluxed for 8 h to yield a precipitate which was filtered and the residue was concentrated to give a crude oily compound. This was diluted with dichloromethane and the organic solvent was washed with brine, dried with anhydrous MgSO 4 , filtered and concentrated to give the crude compound which was recrystallized from ethyl acetate/nhexane to give 11 as a white powder. Yield: 5.2 g (99%); mp: 54-56 °C (56-58 °C) [27]. (14). To a solution of Na (2.2 g, 94.35 mmol) in absolute methanol (100 mL) was added a solution of 3-benzyloxybenzaldehyde (7, 5.0 g, 23.58 mmol) and methyl azidoacetate (7, 10.9 g, 94.35 mmol) in methanol (100 mL) at −20 °C, and the mixture was stirred at 0 °C for 12 h. After addition of cold water, the resulting precipitate was collected by filtration. The solid was washed with water and dried to give methyl 2-azido-3-(3-benzyloxyphenyl)acrylate (12, 6.4 g, 88 %) as pale yellow crystals. To refluxing xylene (50 mL) was added a xylene solution (50 mL) of acrylate (6.4 g) and the mixture was refluxed for 1 h. After cooling, the solvent was removed in vacuo to give the crude oily compound which was purified by column chromatography (ethyl acetate/n-hexane = 1:9) to afford methyl 7benzyloxy-1H-indole-2-carboxylate (13) (15). A suspension of methyl 5-benzyloxy-1H-indole-2carboxylate (14, 1 g, 3.56 mmol) in methanol (10 mL) was reacted with a solution of 10 % NaOH (10 mL) and heated at 60 °C for 1 h. The resulting mixture was worked up conditions like compound 10 to afford 15 as a white powder. Yield: 930 mg (98%); mp: 193-195 °C (192 °C, commercially available compound) (17). 5-Benzyloxy-1-chloromethyl-N-(tert-butyloxycarbonyl)indoline (16, 50 mg, 0.13 mmol) [22] was dissolved in 4M HCl in ethyl acetate (5 mL) and stirred at room temperature for 1 h. The reaction mixture was treated with 20% NaOH and extracted with ethyl acetate and the organic layer was dried with anhydrous MgSO 4 and filtered, concentrated to give the residue which was dried under vacuum for 1 h. The resulting amine hydrochloride (5-benzyloxy-1-chloromethyl-3H-indoline·HCl) was dissolved in dimethylformamide (2 mL) and 5-substituted-1H-indole-2-carboxylic acid (50.4 mg, 0.20 mmol) and EDCI (74.0 mg, 0.40 mmol) were added. The reaction mixture was stirred for 14 h at room temperature and added with water (3 mL) and obtained precipitates were purified by column chromatography (ethyl acetate:n-hexane=1:5) to afford 17 as a white solid.

Cytotoxicity
Synthetic compounds were dissolved in DMSO to obtain 10 mM stock solutions, which were diluted with RPMI 1640 to obtain 100 µM solutions. These solutions were then further diluted with 1% DMSO in RPMI 1640 to prepare solutions from concentrations of 50 µM to 10 nM. These compound solutions (10 µL) were added to wells containing 90 µL media/cell suspension.
Cell concentrations were adjusted to 5 × 10 4 cells/mL (COLO 205, SK-MEL-2), 1 × 10 5 cells/mL (A549) and 2 × 10 4 cells/mL (JEG-3) after counting the cells with a hemocytometer. Cell suspentions (90 µL) were seeded onto 96-well flat-bottom cell culture plates. After 24 h in a 5% CO 2 atmosphere at 37 °C, the drug solutions diluted 1% DMSO in RPMI 1640 were added to each well (10 µL/well). The plates were incubated for 48 h at 37 °C in a 5% CO 2 atmosphere. MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) was dissolved in PBS (2 mg/mL). After the indicated cell incubation period, 20 μL of this stock MTT solution was added to each well and the plates were further incubated for 4 h at 37 °C in a 5% CO 2 . The contents of the well were shaken and the plates were read on an ELISA reader, utilizing Sunrise xfluor4 V.4.51, with a test wavelength of 570 nm. The dose inhibiting the growth by 50% (IC 50 ) was extrapolated from curves generated based of the average of the absorbance data (seven points/concentration).