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The synthesis and in vitro anti-tumor 60 cell lines screen of a novel series of anthracenyl isoxazole amides (AIMs)¥ (22–33) is described. The molecules consist of an isoxazole that pre-organizes a planar aromatic moiety and a simple amide and/or lexitropsin-oligopeptide. The new conjugate molecules were prepared via doubly activated amidation modification of Weinreb’s amide formation technique, using SmCl3 as an activating agent which produces improved yields for sterically hindered 3-aryl-4-isoxazolecarboxylic esters. The results of the National Cancer Institute’s (NCI) 60 cell line screening assay show a distinct structure activity relationship (SAR), wherein a trend of the highest activity for molecules with one N-methylpyrrole peptide. Evidence consistent with a mechanism of action via the interaction of these compounds with G-quadruplex (G4) DNA, and a structural based rational for the observed selectivity of the AIMs for G4 over B-DNA is presented.
Small molecules that bind DNA have found application in medicine, notably as cancer chemotherapeutic agents.1, 2 That these agents are also highly cytotoxic is attributed to their tendency to bind rather indiscriminately to DNA, and any beneficial effect arises from the more rapid death rates for faster replicating cancer cells.3 Thus, it seems a reasonable proposition that agents which are able to selectively recognize specific sequences of DNA could potentially have higher therapeutic indices.4 In fact, considerable effort has been devoted towards the conjugation of DNA binding pharmacophores in an attempt to recognize and selectively bind specific DNA sequences.5, 6 The bulk of the focus of this work has, until recently, been on B-DNA.7–10 While this has produced a greatly increased understanding of the principles relating to the binding of small molecules to DNA, only limited progress in the development of new chemotherapeutic agents has been achieved.
The initial rationale for our work involved the use of an isoxazole to pre-organize B-DNA binding groups and thereby increase anti-cancer efficacy.11, 12 While our initial foray into this arena met with encouraging anti-tumor activity in screens provided by the National Cancer Institute’s Developmental Therapeutics Program (NCI-DTP)13, 14 (vide infra), we will present evidence herein that our lead molecules did not appear to exert their bioactivity by binding B-DNA. Concurrent with our studies at this time, numerous workers recognized that non-B-DNA conformations are potentially feasible targets for anti-tumor drug development. Two prominent theories recognize the G-quadruplex (G-4) conformation of DNA as a potential molecular target. The first line of reasoning involves the inhibition of telomere maintenance via stabilization of the G-4 conformer.7–10, 15–18 The second theory postulates that G-4 may represent an ancient off-switch for gene expression in specific oncogenes, such as c-myc.19 It has been argued that molecules that selectively target G-4 could plausibly have unprecedented selectivity. Proof-of-concept has emerged in the form of a G-4 binder which advanced to clinical trials as an anti-cancer agent. 49 Based on this information, we have examined systematic structural changes in our initial lead compound to test this revised hypothesis, and describe in this work the synthesis and anti-tumor activity of these compounds in the NCI-DTP 60 cell line screening protocol. The structure activity relationship (SAR) that emerges is consistent with the G-4 binding working hypothesis, and is supported by evidence of G-4 interaction from spectroscopy, telomerase inhibition assays, and electrospray mass spectrometry.
The central strategy for the preparation of the target molecules necessitated improved synthetic methodology which could overcome formidable steric encumbrance, hindrance which was required by the premise of our working hypothesis. The route to these compounds (Scheme 1) involves the initial acetylation and nitration of N-methylpyrrole (1). Amide bond formation between 3 and an appropriate primary amine gave rise to the expected amidopyrroles (6 and 12). Reduction of the nitro group allows step-wise extension of the pyrrole chain to the desired chain length.
Linkage via an amide bond of the relatively unreactive heteroaromatic amines (7, 9, 11, 13, and 15) with rigid isoxazole carboxylates connected to dirigible-like aromatic moieties was accomplished using a modification of our previously reported double activation (Scheme 2). Based on this previously developed methodology,20,21 we prepared a set of pyrrole-type lexitropsin oligopeptides possessing at least one moiety which would be protonated at physiological pH.
The series which contained two tertiary amine groups on the end of the chain were suggested by molecular modeling studies docking target molecules, (23–26), with the solid state coordinates of the G-4 DNA reported by Neidle. Such studies indicated that the two “tails” could plausibly increase the interaction of a lexitropsin-type molecule with the DNA phosphate backbones.
The amide formation between lexitropsin and 3-(9′-anthracenyl) 5-methyl 4-isoxazolecarboxylate 17 (prepared via the 1,3-cycloaddition of 9-anthracenyl nitrile oxide and the enamine of ethyl acetoacetate)22 was achieved through doubly activated amidation12 This amidation is actually a modification of Weinreb’s amidation, which has been an effective method for direct conversion from ester to amide for many years. In classical Weinreb’s amidation,23 trialkyl aluminum is mixed with free primary or secondary amine to generate dialkylaluminum amide in situ, which not only increases the nucleophilicity of the amine but also makes carboxylic ester group liable to be attacked.
Double activated amidation was applied after we tried typical Weinreb’s amidation on the amide formation between an amino-lexitropsin and 3-(9′-antracenyl) 5-methyl 4-isoxazocarboxylic ester and obtained products in only modest yield. We had previously observed by NMR and that the ester group of 3-(9′-anthracenyl) 5-methyl 4-isoxazolcarboxylate is located proximal to the tricyclic aromatic system as evidenced by significant magnetic anisotropy.24 This has also been supported by subsequent x-ray studies. It is a reasonable expectation that the low reactivity arises from considerable steric hindrance preventing ester group interaction with the aluminum center of Weinreb’s amide. In our modified methodology, the carboxylic ester was pre-mixed with a mild Lewis acid (SmCl3) to avoid a coordinative interaction with the aluminum activated amine. In order for the Lewis acid to be compatible to the existence of basic tertiary amino group of lexitropsin as well as the utilization of alkyl aluminum, mild Lewis acid lanthanide chloride was applied. No or minimum additional coordination interaction between the lanthanide center and aluminum center is required. Two decades ago, in the study of the characteristics of lanthanide coordination catalysts for polymerization, it was suggested that SmCl3 and EuCl3 gave the lowest coordination interaction with alkylaluminum.25 This finding indicates that SmCl3 or EuCl3 may serve as ideal lanthanide Lewis acid for the doubly activated amidation.
Our work was initiated to find a compound that meets the needs of both DNA-intercalation and B-DNA’s minor-groove binding and thus targets HIV.22 It was reported that DNA minor groove binders (such as netropsin or distamycin analogues) linked with acridine showed greater binding affinity for DNA than either acridine or minor groove binders, and optimum linker length should consist of a chain of 5 atoms.29 An isoxazole ring was then designed to tether two biologically active portions, acridine (22) or anthracene (29) and lexitropsin peptide containing polyamidopyrroles, which exhibit preference for binding to poly-AT DNA and target HIV’s tat gene. The results from National Cancer Institute (NCI)’s anti-HIV test showed both 22 and 29 were inactive to HIV. But surprisingly, compound 29 showed slight activity against certain human tumor cells in NCI’s 60 cell line screen.13,14 This intrigued us to think about its mechanism, and COMPARE30, 31 analysis with the NCI Standard Agent Database was performed. However, it did not give a significant correlation with agents of known mechanism of action (all Pairwise Correlation Coefficients were <0.5); in contrast to the good correlations usually observed for intercalating agents.
A plethora of G-quadruplex ligands have been developed as potential molecular targets for cancer chemotherapy, such as PIPER, TMPyP, 2,6-diamidoanthraquinones, and bisamido acridines, etc. 15–19,32–34 Almost all of these G-quadruplex ligands have extended planar chromophores, and π-π stacking on the G-quartet end(s) is an important factor in their binding.19,32–35 The anthracene moiety in 29 may serve as an analogous π–electron rich planar chromophore, and provide binding to the top stack of G-quadruplex.35 The lexitropsin peptide moiety was presumed to bind the TTA loop of G-quadruplex.4,5,36,37 This explanation inspired us to design a series of analogues (23–28, 30–32) based on 29.
The isoxazole moiety plays a pivotal role in our working hypothesis.38 It is a rigid linker, pre-organizing the anthracene and peptide in three dimensions, and potentially can also function as a prodrug to deliver the bioconjugate molecule to the cellular DNA. Lexitropsin analogues were in different lengths, to test the hypothesis that given the binding site for ‘n’ pyrrole rings is ‘n+1’ base pairs long in terms of contacted base pairs,36,37 the relationship for the n= 0–3 series should be linear and increasing for B-DNA, but in contrast for G-4-DNA would be expected to be maximum at n=1 based on our working model (vide infra). Furthermore, introduction of an electron-pair bearing element (Cl) or group (phenyl) may provide interaction to cations in the G-quadruplex cavity, thus preferentially enhancing binding of these compounds to G-4 DNA.
Structure-activity relationship (SAR) data for the anthracenyl isoxazole amides synthesized (23–32) were acquired by evaluating their in vitro antitumor activity against NCI’s 60 human tumor cell lines.13,14,39,40 These cell lines have been derived from nine cancer types that adequately meet minimal quality assurance criteria representing leukemia, melanoma and cancers of the lung, colon, brain, ovary, breast, prostate, and kidney. The results are shown in Table 1. Several compounds were selected for re-screening by the Biological Evaluation Committee of NCI, and for those ranges are given (AIMs 23, 29, 30 and 32). Since single digit micromolar is a practical measure of encouraging anti-tumor activity, we use the total number of cell lines inhibited at single digit micromolar (N in Table 1) as an additional bench mark for overall activity. We also note those cell lines for which the anti-umor efficacy was within the nanomolar regime. We started the SAR by conjugating different lengths of lexitropsin peptide derivatives (23–32), and anticipating higher antitumor activity as peptide length increased. Even though varying the length of peptide had a dramatic effect on antitumor potency, the anti-tumor activity of this series of molecules had no linear relationship with the length of lexitropsin peptide. Inside each series, either double tail (23–26) or single tail (27–32), compounds which had one pyrrole (n=1) moiety gave the strongest anti-tumor activity and the broadest spectrum of inhibition at single digit micro-molar (μM) GI50. Compound 24, which had GI50 of 3.89 μM and inhibited 44 tumor cell lines, was 3.98 and 6.30 fold more potent than 25 and 26, which inhibited 7 and 0 cell lines, respectively. Compound 28, which had GI50 as 7.94 μM and inhibited 23 cell lines, was 4.06 and 1.32 fold more potent than 27 and 29, which inhibited 1 and 12 cell lines, respectively. This suggests the interaction of the conjugate molecule occurs with folded DNA structures possessing n+1 (two) ATT-rich bases instead of with duplex DNA’s minor groove, which requires long-chained base pair recognition structure for binding. On the other hand, compound 23, which has no pyrrole ring in the structure, has similar GI50 (4.57 μM) and inhibition spectrum (42 cell lines) to that of compound 24. Both 30 and 32 had less than 2 μM GI50 values, and inhibited almost all the tumor cell lines tested, indicating that introduction of electron-rich substituents at C10′ of anthracene (30 and 32) increased antitumor potency.
An interesting phenomenon is that compounds 23, 27, 30 and 32 showed higher activity against some colon cancer cell lines and non-small cell lung cancer cell lines, in which the c-myc oncogene is implicated as a potential contributing factor (Table 1).59 C-myc expression has been reported to be downregulated by G-quadruplex stabilization.19 This implies that compounds 23, 27, 30 and 32 inhibit more the cells, in which c-myc oncogene is over expressed, by stabilizing G-quadruplex.
In light of the observation that the in vitro activity lies outside the category of adequately studied antitumor agents, the lead compound 29 was selected for the hollow fiber in vivo screen.58 The total Score of 10 reflected some in vivo effect, however, the score usually required for further development is 20. The mouse toxicity assay indicated no acute toxicity for 29, and the animals showed weight gain at day 14 for all three dose regimens (100, 200 and 400 mg/kg). Therefore, further studies into SAR development to increase the efficacy (op cit) and understand the mechanism of action (vide infra) appeared warranted.
An oligonucleotide microarray experiment was conducted wherein the effect of 29 on Calf thymus DNA and an oligonucleotide designed by Hurley, (5′-CATGGTGGTTT(GGGTTA)4CCAC-3′) known to form a quadruplex in solutions of KCl and NaCl,41 was examined. The microarray fluorescence experiment for the mixture of Calf Thymus (CT) DNA with 29 indicates a quenching of the fluorescence but shows no bathochromic shift which would be consistent with an anthracene intercalation mechanism (Figure 2).54,55 The Hurley-Oligonucleotide experiment with 29 shows considerable quenching of the fluorescence which is consistent with a π-stacking interaction with the G-tetrad of the quadruplex structure that forms in situ under the experimental conditions.56,57
The oligonucleotide and CT-DNA solutions were prepared in a 10mM KCl solution containing a TE buffer at pH=7.0 with a final concentration of 400μg/μL. The oligonucleotide solution was hybridized using a cyanine dye (Cy3-AP3-dCTP) utilizing a 96-well plate. AIM-2 was in an aqueous 25% DMSO solution (10μM). All experiments were carried out at 37°C with oligonucleotide solutions being incubated for 5 minutes prior to printing on the slide for analysis. All spectra were taken at four excitation wavelengths (310nm, 380nm, 410nm, and 485nm) with 530nm emission. All microarray experiments were run on a GenePix 4000 microarray fluorescence scanner with 10μm resolution and a dynamic detection range of four orders of magnitude which is linear over three orders of magnitude.
An additional fluorescence experiment involving 29 with human DNA (1:1 M ratio), sans the histones, was performed(data not shown). The results of the second fluorescence experiment were an 11nm hypsochromic shift in the λmax which is inconsistent with an intercalative mechanism55 and is likely due to hydrogen bonding interactions and or salvation of 29 in the presence of the DNA. Further fluorescence-DNA-titration experiments are warranted in light of these results.
We used 29 as a probe or seed compound in the NCI’s COMPARE Algorithm to rank the similarity of responses of the 60 cell lines to the standard agent database.13,14, 39, 40 Similarity of pattern to that of the seed is expressed quantitatively as a Pearson correlation coefficient (PCC). The results obtained with the COMPARE algorithm indicate that compounds high in this ranking may possess a mechanism of action (MoA) similar to that of the seed compound. 30,31 COMPARE works quite well for intercalating/topoisomerase II inhibitors. The top ten matches for adriamycin, with a PCC range of 0.758 to 0.950, were all topoisomerase II inhibitors. In contrast, 29 – originally thought by our group to be a potential intercalating/minor groove binder – indicted no strong correlation to any consistent MoA, with the highest PCC being a relatively weak 0.517 (Table 2). Similar COMPARE analysis of 23–26 indicated no significant correlation with any MoA in the Standard Agent Data Base.
The dihedral angle between the tricyclic planar aromatic moiety and the isoxazole from our crystallographic studies of intermediates and analogs, is in the range of 74–80°.22, 26–8,44,45 The idealized helical pitch angle for B-DNA was estimated to be ca. 47.1° by Goodsell and Dickerson in their isohelical analysis of groove-binding drugs.46,47 Therefore, we calculated the energy associated with conformational changes to this dihedral angle in order to determine whether B-DNA binding seemed plausible. The rotational barrier calculations were performed using the torsion force constraint in the Discover module.48 The bond in question was rotated through 360 intervals (1° increments) using a force constant of 10. After each rotation, the structure was subjected to 1000 steps of minimization, or until an rms value of 0.01 was reached, using the VA09A algorithm. The results of the conformational searches were examined with the Analysis module by constructing a table of total energy verses dihedral angle. Calculations from the INSIGHT II program suggests that this energetic cost is high, precisely in the vicinity of the Helical pitch angle requisite for binding of a C-3 aromatic isoxazole to B-DNA (Table 3).
In combination with the COMPARE analysis, we felt it was prudent to consider alternatives to B-DNA as a molecular target.
In light of the increasingly numerous reports postulating G-4 binding as a mechanism for anti-tumor activity, and given an overall similarity of some of the salient structural features of our compounds, we conducted a number of computational studies with the G-4 coordinates available from Neidle’s elegant crystallographic studies.50–51
In a typical example, the minimum energy structure of NSCD 694332 and quadrulex DNA50 was calculated in a SGI INSIGHT 2000 docking study, using the cvff forcefield, and all the solution molecules (H2O) were removed. The whole DNA molecule was constrained, the distance between first potassium and C-10 hydrogen was fixed to <4.00 angstrom. After 3000 iterations using steepest descent minimization, in most cases the drug-receptor interaction had converged to a final energy, which the program reported as consisting of separate electrostatic and VdW components. If the minimization did not reach convergence at a set number of iterations, reasonable and slight adjustments were made to the ligand structure, and minimization was repeated until a convergent structure solution was obtained.26
Two of the possible docking modes we considered are illustrated in Figure 2. In the first we initiated the docking with the anthracenyl moiety intercalated between the G-tetrads. During minimization, the anthracene of 29 displaced two of the guanines (in red). In the minimized structure (Figure 2a), the Isoxazole ring N is within 3 Å of the NH2 of the guanine of the lower, intact G-tetrad. Alternatively, we considered an external stacking (Figure 2b), and in the case of analog 30 which contained a group bearing lone pairs at C(10) of the anthracenyl group, C(10) tended to orient – again after minimization – within 2.62Å over the potassium in the cavity of the G-tetrad.
Similar features which emerged from both of these minimized structures were (1) the isoxazole nitrogen was within hydrogen bonding distance of the guanine 2-amino group of the G-tetrad, and (2) the amine of the dimethylamino tail would move slightly to associate proximal to a group peripheral to the intact G-tetrad, as seen in the case of 30, with the phosphate in the sugar-phosphate TTA loop.
Thus, there is a specific structure-based reason for the expectation that the AIMs should be selective for G4 DNA at the expense of B. The energy cost of B-DNA binding is increased by the mismatch with the helical pitch angle.
Stabilizing G-quadruplex inhibits telomerase activity, and this has been correlated with inhibition of cancer growth. Thus, until recently the TRAP assay has been widely used to assess G-quadruplex interaction. We reported in 2004 that the fluorescence analysis TRAPese assay was marred by the inhibition of taq polymerase by 32,52 and this recently has been verified by Mergny’s group.53 In the more reliable gel electrophoresis TRAP analysis, AIM 30 did indeed appear to inhibit telomere elongation rather than taq polymerase (data for both TRAPese and TRAP is shown in the Supplementary Material), however, we sought a more direct method of determining G4 interaction with our compounds, and therefore also examined electrospray mass spectrometry (vide infra).
Total ions of G-quadruplex DNA, i-motif DNA, and duplex DNA were measured under the same conditions (Fig. 6.7). In the absence of G-4 stabilizing ligands (negative control), ion intensities of single-stranded DNA dropped dramatically, while duplex formation is rather fast. Rehvbridization in the absence of G4 stabilizing ligands proceeded to 50% completion in 10 minutes and was essentially complete in 45 minutes (negative control, shown in Supplementary Material). When incubated with either TMPyP4 (positive control), 30 or 32, the decrease of single-stranded DNA signals and increase of duplex signals were dramatically slowed. Although no DNA-ligand complex ion was detected, the inhibition of duplex formation indicated interference from both TMPyP, 30, and 32 in DNA annealing, which implies their ability for G-quadruplex stabilization. The slopes of ion intensity trend lines give approximate inhibition abilities of TMPyP4 and 32. For example, slopes in total duplex ion intensity trends showed that TMPyP4 inhibited duplex formation at a comparable rate to 32, where both took about 80 minutes to proceed to 50% hybridization, and significant ions corresponding to single stranded oligonuclotides were still present even at 2 hours.
The new compounds reported in this study showed in vitro antitumor activity, and the mean GI50 against the NCI60 cell line panel for the length of the oligopyrrole moiety (n= the number of pyrroles) was observed as 1 > 0 2 > 3 for the bis-dimethylaminopropyl series 23–26) and 1>20 for the mono-dimethylaminopropyl series (27–29); and for the anthracenyl C(10) position was Ph≈Cl H > Br (28, 30–32), with the most efficacious examples having average activity comparable to agents currently in general medical practice.13 The activity correlates inversely with the length of lexitropsin oligopeptides within the conjugate molecules, that is, conjugates containing a single pyrrole ring demonstrated the strongest activity and broadest spectrum of inhibition against cancer cell lines. The SAR, spectroscopic and ES-MS assays are consistent with our current working hypothesis with the goal of selectively targeting G-4 sequences. In conclusion, the results presented suggest that the synthesis of a new series of anthracenyl isoxazole-lexitropsin conjugates, the AIMs, may represent a potential useful addition to the arsenal of anti-cancer molecules. In addition, we present structure–based evidence for the contention that the AIMs are unique among G4 binding agents in that they possess specific features which destabilize their intercalative interaction with B-DNA, and therefore, the expectation of selectivity is reasonable. Further clarification of the MoA and the effects of substitution at C5 of the isoxazole, and on the anthracene appears warranted. Those results will be reported in due course.
Mass spectra were obtained on a JEOL JMS-AX505 HA. The NMR spectra (1H and 13C) were obtained on a Bruker AVANCE 300 and 500 Digital NMR (300 and 500 MHz, respectively) using SGI-IRIX 6.5. Elemental analysis was performed by Desert Analytics Laboratory, PO BOX 41838, Tucson, AZ 85717. All reactions were performed under an inert atmosphere of nitrogen or argon. Tetrahydrofuran was distilled from sodium-benzophenone immediately before use. Flash chromatography was performed on silica gel (Merck 60Å, 230–400mesh) with freshly distilled solvents. Pyrrole starting materials 2 and 3 were prepared according to Nishwaki.21 3-(9′-Anthracenyl) 5-methyl 4-isoxazolecaboxylate 17, and the corresponding acridine 16, 22 Anthracenyl ring analogs 18–2026.27 and final product 3328 were prepared according to methodology previously reported by our lab.
To a solution of 3 (9.24 g, 35.88 mmol) in THF (40 mL), was added a solution of 3,3′-iminobis(N,N-dimethylpropylamine) (25.24 g, 134.74 mmol) at room temperature. The reaction mixture was heated at reflux for 3hrs. The reaction mixture was cooled to room temperature and the solvent was evaporated. The residue was then purified by chromatography on silica gel eluting with MeOH/Triethylamine (9/1) (Rf = 0.40) to give yellow oil 6 (4.82 g, 41.3%), which became a pale yellow solid after being triturated with hexane (20 mL); mp: 60–61 °C; 1H NMR (CDCl3): δ 7.69 (1H, d, J = 1.7 Hz), 7.66 (1H, d, J = 1.7 Hz), 3.96 (3H, s), 3.67 (4H, t, J = 7.5 Hz), 2.43-2.35 (16H, m), 1.94 (4H, m). 13C NMR (CDCl3): δ 162.4, 135.4, 127.2, 125.5, 106.7, 57.0, 46.0, 45.7, 37.0, 26.2. MS (CI): m/z (%) 340 (M+1, 100), 268 (8.62), 268 (8.62), 84 (12.83), 58 (18.52). Anal. Calcd. for C16H29N5O3: C, 56.62; H,8.61; N, 20.63. Found: C, 56.64; H, 8.33; N, 20.09.
To a solution of 6 (2.40 g, 7.38 mmol) in MeOH (175 mL), was added Pd/C (5%) (2.50 g). The mixture was hydrogenated at 37–40 psi at room temperature for 3hrs. The catalyst was removed by filtration and the solvent was removed in vacuo. To a solution of the above residue in THF (40 mL), was added a solution of 3 (3.05 g, 11.73 mmol) within 10 min. The reaction mixture was stirred at room temperature for 3 h. Solvent was removed in vacuo and the residue was then purified by chromatography on silica gel eluting with MeOH/Triethylamine (9/1) (Rf=0.34) to give pale yellow solid, 2.21 g (65%); mp: 125–127 °C. 1H NMR (CDCl3): δ 9.12(1H, s), 7.61 (1H, d, J = 1.8Hz), 7.47 (1H, d, J = 1.8 Hz), 7.25 (1H, d, J = 1.8 Hz), 6.36 (1H, d, J = 1.8 Hz), 4.07 (3H, s), 3.71 (3H, s), 3.54 (4H, t, J = 7.8 Hz), 2.34 (4H, t, J = 7.2 Hz), 2.28 (12H, s), 1.85 (4H, m). 13C NMR (CDCl3): δ 164.1, 157.8, 135.2, 127.1, 126.8, 123.9, 121.3, 117.0, 107.8, 103.6, 57.1, 45.8, 45.6, 38.4, 36.1, 27.0; MS (CI): m/z (%) 462 (M+1, 100), 377(19.91), 352 (14.52), 170 (67.70), 84 (28.51), 58 (17.97). To a solution of 8 (100 mg, 0.2 mmol) in absolute ethanol (5 mL), was added a solution of oxalic acid dihydrate (C2O4H2 ● 2H2O) (57 mg, 0.45 mmol). The mixture was stirred at room temperature until no more precipitate formed. The solid was filtered and washed with absolute ethanol (5 mL × 5). The oxalic acid salt of 8 was obtained after being dried in vacuo to yield a solid (109 mg, 90%), mp: 203–204 °C. Anal. Calcd. for C22H35N7O4● 1.5C2O4H2●0.5H2O (%): C, 49.58; H, 6.49; 16.19. Found: C, 49.81; H, 6.64; N, 16.05.
2-[[[2-[[[2-[[N,N-Bis[3-(N,N-dimethylamino)propyl]amino]carbonyl]-1-methyl-1H-pyrrol-4-yl]amino]carbonyl]-1-methyl-1H-pyrrol-4-yl]amino]carbonyl]-1-methyl-4-nitropyrrole (10). To a solution of 8 (1.13 g, 2.45 mmol) in MeOH (90 mL), was added Pd/C (5%) (2.00 g). The mixture was hydrogenated at 37–40 psi at room temperature for 3 h. The catalyst was removed by filtration and the solvent was removed in vacuo. To a solution of the above residue in THF (30 mL), was added a solution of 3 (1.00 g, 3.90 mmol) within 10 min. The reaction mixture was stirred at room temperature of 3 h. Solvent was removed in vacuo and the residue was then purified by chromatography on silica gel eluting with MeOH/Triethylamine (9/1) (Rf = 0.21) to give pale yellow solid, (0.74 g, 52%); mp: 178–179 °C; 1H NMR (DMSO-d6): δ 10.29 (1H, s), 9.89 (1H, s), 8.18 (1H, d, J = 1.7 Hz), 7.58 (1H, d, J = 1.7 Hz), 7.24 (2H, d, J = 1.7 Hz), 7.03 (1H, d, J = 1.7 Hz), 6.37 (1H, d, J = 1.7 Hz); 3.96 (3H, s), 3.85 (3H, s), 3.58 (3H, s), 2.49 (4H, t, J = 7.7 Hz), 2.15 (4H, t, J = 7.1 Hz), 2.10 (12H, s), 1.70-1.61 (4H, m). 13C NMR (CDCl3): δ 164.6, 159.2, 158.1, 135.4, 127.3, 126.7, 124.0, 123.6, 123.0, 121.8, 120.0, 117.6, 108.5, 102.9, 102.6, 57.1, 46.4, 45.6, 38.7, 37.2, 35.9, 27.1. MS (CI): m/z (%) 584 (M+1, 55.96), 397 (10.47), 275 (24.39), 153 (18.31), 149 (52.19), 122 (32.51), 107 (20.18), 101 (16.14), 81 (100). Anal. Calcd. for C28H41N9O5 (%): C, 57.62; H, 7.08; N, 21.60. Found: C, 57.23; H, 7.26; N, 21.24.
A mixture of ethyl 3-(10′-bromo-9′-anthracenyl)-5-methyl-4-isoxazole carboxalate (420 mg, 1.02 mmol), phenyl boronic acid (140 mg, 1.15 mmol), Pd2(dba)3 (45 mg, 5 mol%), P(t-Bu)3 (10% in hexane, 242 mg, 12 mol%) and KF (191 mg, 3.3 mmol) in THF (7 mL)was stirred at room temperature under argon atmosphere for 72 h. The reaction mixture was filtered and the solid was washed by THF (10 mL). The filtrate was concentrated and purified by chromatography on silica gel eluting with hexane/benzene (3:2) to give yellow crystal (382 mg, 92%); mp: 178–180 °C. 1H NMR (CDCl3): δ 7.62 (4H, m), 7.52 (3H, m), 7.32 (6H, m), 3.68 (2H, q, J = 7.2 Hz), 2.88 (3H, s), 0.33 (3H, t, J = 7.2 Hz). 13C NMR (CDCl3): δ 176.2, 161.5, 160.8, 139.3, 138.6, 131.2, 131.1, 130.5, 129.7, 129.0, 128.5, 128.4, 128.3, 127.6, 127.1, 125.9, 125.5, 125.1, 122.8, 111.5, 60.1, 13.5, 12.8. MS (EI): m/z (%) 407 (M+, 100), 319 (17.91), 295 (12.85), 252 (13.86). Anal. Calcd for C27H21NO3: C, 79.59; H, 5.19; N, 3.44. Found: C, 79.94; H, 5.09; N, 3.57.
To a suspension of anhydrous SmCl3 (0.21 g, 0.79 mmol) in THF (6 mL) was added ethyl 3-(9-anthracenyl)-5-methyl-4-isoxazolecarboxylate 17 (0.25 g, 0.72 mmol) in dry THF (5 mL). The mixture was stirred under nitrogen at room temperature for 5.5 h and was ready to be used in the following steps.
In a solution of 3,3′-iminobis(N,N-dimethylpropylamine) (0.222 g, 1.19 mmol) in THF (10 mL) was added (CH3)3Al (2M in hexane, 0.75 mL) at 0 °C during 30 min. The mixture turned into yellow. Warmed up the reaction to room temperature and kept the reaction going for another 1 h, until it was ready to be transferred in next step.
To the above-prepared mixture containing activated ester, was added the brown solution of the above prepared activated amino-lexitropsin solution at room temperature during 10 min. Kept the final mixture refluxing for 8 h. Na2SO4●10H2O (1.0 g) and cold methanol (20 mL) were added to the reaction mixture in order to quench the excessive (CH3)3Al. Solid was removed through centrifuge and a red solution was collected. The red solution was concentrated and purified by chromatography on silica gel (200–400mesh) eluting with methanol/30% NH4OH (95:5), to afford yellow oil (0.32 g, 87%). 1H NMR (CDCl3): δ 8.58 (1H, s), 8.06-8.00 (4H, m), 7.54-7.47 (4H, m), 3.06 (2H, t, J = 7.1 Hz), 2.74 (3H, s), 2.53 (2H, t, J = 7.1 Hz), 2.00 (6H, s), 1.87 (6H, s), 1.63 (2H, t, J = 6.9 Hz), 1.45 (2H, t, J = 6.9 Hz), 1.11-0.95 (2H, m), 0.90-0.83 (2H, m). 13C NMR (CDCl3): δ 170.8, 163.1, 157.8, 131.4, 131.1, 130.8, 130.3, 129.7, 129.3, 128.8, 128.7, 128.3, 127.1, 126.2, 125.8, 125.3, 121.9, 116.3, 56.2, 47.4, 46.4, 45.6, 45.4, 42.5, 26.7, 24.9, 11.5; MS (CI): m/z (%) 473 (M+1, 100), 188 (22.58), 58 (18.63). To a solution of 23 (94 mg, 0.2 mmol) in absolute ethanol (5 mL), was added a solution of oxalic acid dihydrate (C2O4H2 ●2H2O) (57 mg, 0.45 mmol). The mixture was stirred at room temperature until no more precipitate formed. The solid was filtered and washed with absolute ethanol (5 mL × 5). The oxalic acid salt of 23 was obtained after being dried in vacuo to yield a solid (114mg, 84%), mp: 185–186°C. Anal. Calc’d. for C29H36N4O2●2C2H2O4●1.5H2O: C, 58.31; H, 6.38; N,●8.24. Found: C, 58.50; H, 6.12; N, 8.12.
To a suspension of anhydrous SmCl3 (0.21 g, 0.79 mmol) in THF (10 mL) was added 17 (0.25 g, 0.72 mmol) in dry THF (10 mL). The mixture was stirred under nitrogen at room temperature for 5.5 hrs and was ready to be used in the following steps.
A suspension of 10 % Pd/C (0.2 g) in a solution of 6 (0.385 g, 1.19 mmol) in methanol (30 mL), was stirred for 4.5 h under H2 pressure (37 psi) at room temperature. The catalyst was removed by filtration, and the solvents removed in vacuo. The hydrogenation product was dissolved into dry THF (15 mL) and (CH3)3Al (2M in hexane, 0.75 mL) was added at 0 °C during 30 min. The mixture turned into brown. Warmed up the reaction to room temperature and kept the reaction going for another 1 hr., until it was ready to be transferred in next step.
To the above-prepared mixture containing activated ester, was added the brown solution of the above prepared activated amino-lexitropsin solution at room temperature during 10 min. The mixture was refluxed for 8 h. Na2SO4●10H2O (1.0 g) and cold methanol (20 mL) were added to the reaction mixture in order to quench the excessive (CH3)3Al. Solid was removed through centrifuge and a red solution was collected. The red solution was concentrated and chromatography was performed on silica gel (200–400mesh), using methanol/30% NH4OH (95:5), to afford a pale yellow solid (0.396 g 84%), mp: 109–111°C. 1H NMR (CDCl3): δ 8.63 (1H, s), 8.04(2H, dd, J = 0.9, 8.3 Hz), 7.65 (2H, dd, J = 0.9, 8.3 Hz), 7.52-7.23 (4H, m), 6.56 (1H, d, J = 1.7 Hz), 6.40 (1H, s), 4.90 (1H, d, J = 1.7 Hz), 3.37 (3H, s), 3.21 (4H, t, J = 6.6 Hz), 2.94 (3H, s), 2.24-1.90 (16H, m), 1.60-1.45 (4H, m), 1.52 (2H, s). 13C NMR (CDCl3): δ 176.3, 163.9, 158.0, 157.9, 157.8, 157.6, 131.5, 131.2, 130.7, 129.2, 128.9, 128.4, 128.2, 127.9, 126.5, 125.8, 125.7, 125.3, 124.3, 120.7, 120.3, 116.1, 113.2, 57.2, 46.4, 45.8, 35.6, 26.6, 14.0. MS (CI): m/z (%) 595 (M+1, 100), 510 (6.09), 336 (19.88). Anal. Calc’d for C35H42N6O3●H2O: C, 68.60; H, 7.24; N, 13.71. Found: C, 68.33; H, 6.94; N, 13.61.
By the same procedure as that described for 24, from SmCl3 (0.21 g, 0.79 mmol), 17 (0.25 g, 0.72 mmol), 10 % Pd/C (0.2 g), 8 (0.55 g, 1.19 mmol) and (CH3)3Al (2M in hexane, 0.75 mL), 25 was obtained as a pale yellow solid (0.44 g 78%); mp: 118–120°C (dec). 1H NMR (CDCl3): δ 8.72 (1H, s), 8.12 (2H, dd, J = 0.9, 8.2 Hz), 7.70 (2H, dd, J = 0.9, 8.2 Hz), 7.55-7.50 (4H, m), 7.19 (1H, s), 7.09 (1H, d, J = 1.7 Hz), 6.41 (1H, s), 6.28 (1H, d, J = 1.7Hz), 6.10 (1H, d, J = 1.7 Hz), 5.69 (1H, d, J = 1.7 Hz), 3.65 (3H, s), 3.61 (3H, s), 3.48 (4H, t, J = 7.8 Hz), 2.99 (3H, s), 2.28-2.10 (16H, m), 1.83-1.69 (4H, m), 1.52 (5H, s). 13C NMR (CDCl3): δ 176.7, 164.3, 158.7, 158.5, 157.8, 131.5, 131.3, 130.9, 129.3, 129.0, 128.6, 128.4, 128.0, 126.6, 125.9, 125.7, 125.3, 124.3, 123.6, 121.2, 120.6, 120.5, 118.8, 116.7, 113.0, 103.3, 103.2, 103.0, 57.3, 46.4, 45.7, 36.9, 35.9, 26.8, 14.1. MS (FAB): m/z (%) 717 (M+1, 71), 530, (13.19), 286 (15.85), 230 (12.43), 271 (12.37), 244 (59.35), 214 (20.21), 188 (12.72), 149 (61.28), 122 (49.88), 106 (16.22), 81 (100). Anal. Calc’d for C41H48N8O4●2.5H2O: C, 64.63; H, 7.01; N, 14.71. Found: C, 64.77; H, 6.64; N, 15.08.
By the same procedure as that described for 24, from SmCl3 (0.21 g, 0.79 mmol), 17 (0.25 g, 0.72 mmol), 10 % Pd-C (0.2 g), 10 (0.693g, 1.19 mmol) and (CH3)3Al (2M in hexane, 0.75 mL), 26 was obtained as a pale yellow solid (0.37g, 56%), mp: 131–133 °C (dec). 1H NMR (CDCl3): δ 8.56 (1H, s), 7.96 (2H, dd, J = 0.8, 8.1 Hz), 7.62 (2H, dd, J = 0.8, 8.1 Hz), 7.46-7.30 (4H, m), 7.08 (1H, d, J = 1.8Hz), 6.98 (1H, s), 6.89 (1H, d, J=1.8Hz), 6.59 (1H, s), 6.38 (1H, d, J = 1.8Hz), 6.28 (1H, d, J = 1.8 Hz), 6.23 (1H, d, J=1.8Hz), 6.20 (1H, d, J = 1.8 Hz), 5.78 (1H, s), 3.83 (3H, s), 3.58 (3H, s), 3.54 (3H, s), 3.44 (4H, t, J = 7.2 Hz), 2.91 (3H, s), 2.16-2.10 (16H, m), 1.72-1.65 (4H, m), 1.53(3H, s) 13C NMR (CDCl3): δ 179.6, 176.6, 176.0, 175.6, 170.8, 169.8, 159.6, 159.3, 158.5, 158.0, 131.4, 130.7, 129.1, 128.2, 128.1, 127.7, 126.3, 125.5, 124.9, 123.7, 123.5, 122.2, 121.9, 120.5, 120.1, 119.4, 118.9, 117.0113.3, 104.3, 103.9, 103.4, 57.0, 46.1, 45.4, 36.8, 36.7, 35.6, 13.8. MS (FAB): m/z (%) 839 (M+1, 43), 717 (11.64), 286 (29.71), 277 (15.81), 271 (25.40), 244 (100), 214 (31.05), 149 (87.91), 123 (64.75), 85 (86.34). Anal. Calc’d for C47H54N10O5●1.5H2O) C, 65.18; H, 6.63; N, 16.17. Found: C, 65.37; H, 6.56; N, 15.83.
To a suspension of anhydrous SmCl3 (393.5 g, 1.53 mmol) in THF (10 mL) was added 18 (381.3 mg, 1.04 mmol) in dry THF (10 mL). The mixture was stirred under nitrogen at room temperature for 5.5 h and was ready to be used in the following steps.
A suspension of 10 % Pd/C (310.6mg) in a solution of 12 (473.0 g, 1.86 mmol) in methanol (30 mL), was stirred for 4.5 h under H2 pressure (37 psi) at room temperature. The catalyst was removed by filtration, and the solvents were removed in vacuo. The hydrogenation product was dissolved into dry THF (15 mL) and (CH3)3Al (2M in hexane, 2 mL) was added at 0 °C during 30 min. The mixture turned into brown. Warmed up the reaction to room temperature and kept the reaction going for another 1 h, until it was ready to be transferred in next step. To the above-prepared mixture containing activated ester, was added the brown solution of the above prepared activated amino-lexitropsin solution at room temperature during 10 min. The final mixture was refluxed for 18 h. Na2SO4●10H2O (1.0 g) and cold methanol (20 mL) were added to the reaction mixture in order to quench the excessive (CH3)3Al. Solid was removed through centrifuge and a red solution was collected. The red solution was concentrated and purified by chromatography on silica gel (200–400mesh) eluting with methanol/30% NH4OH (95:5), to afford a pale yellow solid (203.7 mg, 36%), mp: 208–210 °C. 1H NMR (CDCl3): δ 8.65 (2H, dt, J = 5.4, 0.6 Hz), 7.78 (1H, br), 7.73 (2H, dt, J = 5.4, 0.6 Hz), 7.69 (2H, m), 7.57 (2H, m), 6.76 (1H, d, J = 1.2 Hz), 6.40 (1H, s), 5.19(1H, d, J = 1.2 Hz), 3.72 (3H, s), 3.31 (2H, q, J = 3.6 Hz), 3.02 (3H, s), 2.36 (2H, t, J = 3.6 Hz), 2.08 (3H, s), 1.58 (2H, m). 13C NMR (CDCl3): δ 176.5, 161.1, 157.7, 157.0, 132.8, 131.3, 128.5, 128.1, 127.6, 125.5, 125.4, 123.8, 120.2, 119.8, 118.3, 112.9, 101.5, 59.4, 45.4, 39.9, 36.5, 25.1, 13.8. MS (CI): m/z (%) 544.00 (M+, 65.88), 306.90 (58.70), 288.92 (24.86), 153.96 (100), 136.99 (90.87). Anal. Cal’d. for C30H30ClN5O3: C, 66.23; H, 5.56; N, 12. 87. Found: C, 66.07; H, 5.50; N, 12.71.
By the same procedure as that described for 30, from SmCl3 (481.5 mg, 1.87 mmol), 19 (341.6 mg, 0.83 mmol), 10 % Pd/C (490 mg), 12 (458.5 mg, 1.80 mmol) and (CH3)3Al (2M in hexane, 2 mL), 31 was obtained as a yellow solid (280mg, 61%), mp: 201.5–203.5 °C. 1H NMR (CDCl3): δ 8.70 (2H, d, J = 9 Hz), 7.71 (5H, m), 7.60 (2H, m), 7.57 (2H, m), 6.74 (1H, d, J = 1.5 Hz), 6.40 (1H, s, br), 5.24 (1H, d, J = 1.5 Hz), 3.74 (3H, s), 3.32 (2H, q, J = 3.6 Hz), 3.04 (3H, s), 2.38 (2H, t, J = 3.6 Hz), 2.11 (6H, s), 1.61 (2H, m). 13C NMR (CDCl3): δ 176.4, 161.0, 157.7, 157.0, 131.4, 130.3, 128.4, 128.0, 127.9, 127.0, 125.4, 123.8, 121.3, 119.7, 118.2, 112.9, 101.6, 59.3, 45.4, 39.7, 36.4, 25.2, 13.6. MS (CI): m/z (%) 590.01 (M+1, 100.00), 588.02 (99.87), 542.97 (19.06), 485.88 (22.84), 321.85 (31.82), 242.96 (20.71), 153.99 (50.75), 136.00 (51.68), 129.09 (65.34), 84.38 (43.93). Anal. Calc’d. for C30H30BrN5O3: C, 61.23; H, 5.14; N, 11.90. Found: C, 61.16; H, 5.29; N, 11.62.
By the same procedure as that described for 30, from SmCl3 (491.5 mg, 1.90 mmol), 19 (300.6 mg, 0.73 mmol), 10 % Pd/C (553 mg), 12 (500.5 mg, 1.97 mmol) and (CH3)3Al (2M in hexane, 2 mL), 32 was obtained as a pale yellow solid, 130 mg (30%), mp: 126–128°C. 1H NMR (CDCl3): δ 7.68 (3H, m), 7.45 (10H, m), 6.49 (1H, s), 6.47 (1H, s), 5.37(1H, s), 3.65 (3H, s), 3.25 (2H, q, 3.6Hz), 2.96 (3H, s), 2.27 (2H, t, J=3.6Hz), 2.01 (6H, s), 1.53 (2H, m). 13C NMR (CDCl3): δ 176.2, 161.5, 160.1, 157.3, 139.4, 138.8, 131.4, 131.1, 130.5, 129.7, 129.0, 128.5, 128.4, 128.3, 127.6, 127.1, 125.9, 125.5, 125.1, 123.8, 122.8, 119.8, 118.1, 111.5, 101.7, 60.1, 45.4, 39.6, 36.1, 25.0, 13.4. MS (EI): m/z (%) 587 (M+1, 39.04), 586 (M+, 100), 585 (M−1, 19.26), 484 (13.13), 320 (19.84). Anal. Calc’d. for C36H35N5O3:C, 73.82; H, 6.02; N, 11.96. Found: C, 68.39; H, 5.85; N, 11.60.
The NCI’s in vitro anti-tumor screen(Alley, Scudiero et al. 1988; Boyd 1989; Boyd and Paull 1995)13, 36, 37 consists of 60 human tumor cell lines against which compounds 22–33 are tested at a minimum of five concentrations at 10-fold dilutions. A 48 h continuous drug exposure protocol is used, and a sulforhodamine B (SRB) protein assay is used to estimate cell viability or growth.
NRN thanks the National Institute of Neurological Disease and Stroke for Grant NS38444 and P20RR015583. We would like to thank the University of Idaho Research Council, and the National Cancer Institute for financial support. Dr. Dan Zaharevitz assisted us with the COMPARE calculation and interpretation. XH, CL and KCR, acknowledge the Malcolm and Carol Renfrew Scholarship Endowment, University of Idaho. CL and KCR also thank P20RR16454.
¥While not a strict acronym, the designation AIM is in honor of the memory of Professor Albert I. Meyers.
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