Methylene-linked bis-phenylbenzimidazoles - a new scaffold to target telomeric DNA/RNA hybrid duplex.

We report a series of novel methylene-linked bis-phenylbenzimidazoles intercalators that stabilize telomeric DNA/RNA hybrid (tDRH) structures by up to 7.2 °C at a 1 µM ligand concentration while having negligible affinity for DNA/DNA duplexes, although with a low affinity for quadruplex DNA. We have used molecular modelling studies to rationalize this selectivity, concluding that the methylene spacer between the terminal benzimidazole and phenylene moieties plays a key role in facilitating the bis-intercalating process. This scaffold may be used to develop chemical tools or new therapeutics to selectively target the telomeric DNA/RNA duplex without affecting normal genomic DNA.

ABCB1 genetic polymorphism influences the pharmacology of the new pyrrolobenzodiazepine derivative SJG-136.

ATP-binding cassette transporter P-glycoprotein (ABCB1) is responsible for the multidrug resistance (MDR1) phenotype observed in cancer cells. SJG-136, a new pyrrolobenzodiazepine dimer, is a sequence-dependent DNA crosslinking agent and substrate of ABCB1. We previously showed that colon cancer cell lines expressing high levels of ABCB1 showed a lower sensitivity to SJG-136. Here, we show that in 3T3 isogenic fibroblasts, ABCB1 genetic polymorphism differentially affects ABCB1 gene expression and transport function. However, this genotype-phenotype relationship was not observed in immortalized lymphocytes, which expressed 10- to 1000-fold less ABCB1 than colon cancer cell lines. Consistent with this, the cytotoxicity of SJG-136 in 3T3 fibroblasts was affected by ABCB1 genetic polymorphism but not in immortalized lymphocytes. ABCB1 genetic polymorphism is therefore likely to affect drug sensitivity in tissues expressing high levels of the transporter and in which significant variability is observed.

Fludarabine-mediated suppression of the excision repair enzyme ERCC1 contributes to the cytotoxic synergy with the DNA minor groove crosslinking agent SJG-136 (NSC 694501) in chronic lymphocytic leukaemia cells.

In this study, we set out to establish whether fludarabine could enhance the DNA interstrand crosslinking capacity of SJG-136 in primary human chronic lymphocytic leukaemia (CLL) cells and thereby offer a rationale for its clinical use in combination with SJG-136. SJG-136 rapidly induced DNA crosslinking in primary CLL cells which was concentration-dependent. Further, the level of crosslinking correlated with sensitivity to SJG-136-induced apoptosis (P=0.001) and higher levels of crosslinking were induced by the combination of SJG-136 and fludarabine (P=0.002). All of the samples tested (n=40) demonstrated synergy between SJG-136 and fludarabine (mean combination index (CI)=0.54+/-0.2) and this was even retained in samples derived from patients with fludarabine resistance (mean CI=0.62+/-0.3). Transcription of the excision repair enzyme, ERCC1, was consistently increased (20/20) in response to SJG-136 (P<0.0001). In contrast, fludarabine suppressed ERCC1 transcription (P=0.04) and inhibited SJG-136-induced ERCC1 transcription when used in combination (P=0.001). Importantly, the ability of fludarabine to suppress ERCC1 transcription correlated with the degree of synergy observed between SJG-136 and fludarabine (r(2)=0.28; P=0.017) offering a mechanistic rationale for the synergistic interaction. The data presented here provides a clear indication that this combination of drugs may have clinical utility as salvage therapy in drug-resistant CLL.

Preclinical pharmacology of the pyrrolobenzodiazepine (PBD) monomer DRH-417 (NSC 709119).

The pyrrolobenzodiazepine monomer DRH-417 is a member of the anthramycin group of anti-tumor antibiotics that bind covalently to the N2 of guanine within the minor groove of DNA. DRH-417 emerged from the EORTC-Drug Discovery Committee and NCI 60 cell line in vitro screening programs as a potent antiproliferative agent with differential sensitivity towards certain cancer types such as melanoma, breast and renal cell carcinoma (mean IC(50) = 3 nM). DRH-417 was therefore tested for in vivo activity. The maximum tolerated dose (MTD) was established as 0.5 mg/kg given i.p. Marked anti-tumor activity was seen in two human renal cell cancers, one breast cancer and a murine colon tumor model (p<0.01). A selective HPLC (LC/MS) analytical method was developed and plasma pharmacokinetics determined. At a dose of 0.5 mg kg(-1), the plasma AUC was 540 nM h (197.1 ng h ml(-1)) and the peak plasma concentration (171 nM [62.4 ng ml(-1)]) occurred at 30 min., reaching doses levels well above those needed for in vitro antiproliferative activity. Genomic profiling of in vivo sensitive tumors revealed that the latter have an activated insulin-like growth factor signaling pathway.

Influence of P-glycoprotein expression on in vitro cytotoxicity and in vivo antitumour activity of the novel pyrrolobenzodiazepine dimer SJG-136.

SJG-136 is a novel pyrrolobenzodiazepine dimer analogue that acts as a minor-groove interstrand DNA cross-linking agent. The present study investigated the impact of ABCB1 (mdr-1) expression on the activity of SJG-136 using both in vitro and in vivo systems. SJG-136 was highly potent in the colon cancer cell lines HCT-116, HT-29 and SW620 (IC50 0.1-0.3 nM). However, HCT-8 and HCT-15 cells expressing significant levels of mdr-1 were less sensitive (IC50 2.3 and 3.7 nM, respectively) using a SRB assay. The cytotoxicity was increased in HCT-15 and A2780(AD) in presence of 5 microg/ml verapamil. Mdr-1 mRNA expression was determined by qRT-PCR and correlated to SJG-136 IC50s (r2=0.86, P=0.0001). Isogenic 3T3 cells expressing mdr-1 cDNA (3T3 pHamdr-1) were less sensitive to SJG-136 than the parental 3T3 cells (IC50 208 and 6.3 nM, respectively). Finally, SJG-136 (120 microg/kg/d dx5) was highly active against A2780 xenografts (SGD=275) but not A2780(AD) xenografts (SGD=67).

Synthesis of the first example of a C2-C3/C2'-C3'-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepine dimer.

We report the first example of a C2-C3/C2'-C3'-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer 16 synthesised through a new and efficient route, thus establishing that C2-C3-endo unsaturation enhances both cytotoxicity and DNA-binding affinity in A-Ring-linked PBD dimers but to a lesser extent than C2/C2'-exo-unsaturation. This new route has allowed the preparation of multi-gram quantities of the related clinical candidate 1 and should lead to more structurally diverse PBD dimer analogues.

Design, synthesis, and evaluation of a novel pyrrolobenzodiazepine DNA-interactive agent with highly efficient cross-linking ability and potent cytotoxicity.

A novel sequence-selective pyrrolobenzodiazepine (PBD) dimer 5 (SJG-136) has been developed that comprises two C2-exo-methylene-substituted DC-81 (3) subunits tethered through their C8 positions via an inert propanedioxy linker. This symmetric molecule is a highly efficient minor groove interstrand DNA cross-linking agent (XL(50) = 0.045 microM) that is 440-fold more potent than melphalan. Thermal denaturation studies show that, after 18 h incubation with calf thymus DNA at a 5:1 DNA/ligand ratio, it increases the T(m) value by 33.6 degrees C, the highest value so far recorded in this assay. The analogous dimer 4 (DSB-120) that lacks substitution/unsaturation at the C2 position elevates melting by only 15.1 degrees C under the same conditions, illustrating the effect of introducing C2-exo-unsaturation which serves to flatten the C-rings and achieve a superior isohelical fit within the DNA minor groove. This behavior is supported by molecular modeling studies which indicate that (i) the PBD units are covalently bonded to guanines on opposite strands to form a cross-link, (ii) 5 has a greater binding energy compared to 4, and (iii) 4 and 5 have equivalent binding sites that span six base pairs. Dimer 5 is significantly more cytotoxic than 4 in a number of human ovarian cancer cell lines (e.g., IC(50) values of 0.0225 nM vs 7.2 nM, respectively, in A2780 cells). Furthermore, it retains full potency in the cisplatin-resistant cell line A2780cisR (0.024 nM), whereas 4 loses activity (0.21 microM) with a resistance factor of 29.2. This may be due to a lower level of inactivation of 5 by intracellular thiol-containing molecules. A dilactam analogue (21) of 5 that lacks the electrophilic N10-C11/N10'-C11' imine moieties has also been synthesized and evaluated. Although unable to interact covalently with DNA, 21 still stabilizes the helix (Delta T(m) = 0.78 degrees C) and has significant cytotoxicity in some cell lines (i.e., IC(50) = 0.57 microM in CH1 cells), presumably exerting its effect through noncovalent interaction with DNA.

Design and synthesis of novel pyrrolobenzodiazepine (PBD) prodrugs for ADEPT and GDEPT.

Three N10-(4-nitrobenzyl)carbamate-protected PBD prodrugs (9a, 9b and 15) have been synthesized and evaluated for potential use in nitroreductase-based ADEPT and GDEPT therapies. An approximately 100-fold activation was observed for the DC-81 prodrug 9a.

Effect of C2-exo unsaturation on the cytotoxicity and DNA-binding reactivity of pyrrolo[2,1-c][1,4]benzodiazepines.

A series of novel C2-exo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) has been synthesised via a versatile pro-C2 ketone precursor. C2-exo-unsaturation enhances both DNA-binding reactivity and in vitro cytotoxic potency.

Effect of C2/C3-endo unsaturation on the cytotoxicity and DNA-binding reactivity of pyrrolo[2,1-c][1,4]benzodiazepines.

A series of novel C2,C3-endo unsaturated pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) has been synthesised via cleavage of the N10-Alloc protecting group from appropriate precursors. Biophysical and biological evaluations show that the presence of C2/C3-endo unsaturation in the PBD C-ring enhances both DNA-binding reactivity and in vitro cytotoxic potency.

Synthesis, in vitro antiproliferative activity, and DNA-binding properties of hybrid molecules containing pyrrolo[2,1-c][1, 4]benzodiazepine and minor-groove-binding oligopyrrole carriers.

The synthesis, biological activity, and DNA-binding properties of a series of four hybrids prepared by combining polypyrrole minor groove binders and pyrrolo[2,1-c][1,4]benzodiazepine (PBD) 13, related to the naturally occurring anthramycin (3) and DC-81 (4), have been described, and structure-activity relationships have been discussed. These hybrids 22-25 contain from one to four pyrrole units, respectively. To investigate sequence selectivity and stability of drug/DNA complexes, DNase I footprinting and arrested polymerase chain reaction (PCR) were performed on human c-myc oncogene, estrogen receptor gene, and human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR) gene sequences. The antiproliferative activity of the hybrids has been tested in vitro on human myeloid leukemia K562 and T-lymphoid Jurkat cell lines and compared to antiproliferative effects of the natural product distamycin A 1, its tetrapyrrole homologue 17, DC 81 (4), and the PBD methyl ester 12. The results obtained demonstrate that the hybrids 22-25 exhibit different DNA-binding activity with respect to both distamycin A 1 and PBD 12. In addition, a direct relationship was found between number of pyrrole rings present in the hybrids 22-25 and stability of drug/DNA complexes. With respect to antiproliferative effects, it was found that the increase in the length of the polypyrrole backbone leads to an increase of in vitro antiproliferative effects, i.e., the hybrid 25 containing the four pyrroles is more active than 22, 23, and 24 both against K562 and Jurkat cell lines.

Design, synthesis, and evaluation of a novel sequence-selective epoxide-containing DNA cross-linking agent based on the pyrrolo[2, 1-c][1,4]benzodiazepine system.

Synthetic routes have been investigated to prepare a novel C8-epoxide-functionalized pyrrolo[2,1-c][1,4]benzodiazepine 6 as a potential sequence-selective DNA cross-linking agent (Wilson et al. Tetrahedron Lett. 1995, 36, 6333-6336). A successful synthesis was accomplished via a 10-step route involving a pro-N10-Fmoc cleavage method that should have general applicability to other pyrrolobenzodiazepine (PBD) molecules containing acid- or nucleophile-sensitive groups. During the course of this work, a one-pot reductive cyclization procedure for the synthesis of PBD N10-C11 imines from nitro dimethyl acetals was also discovered, although this method results in C11a racemization which can reduce DNA binding affinity and cytotoxicity. The target epoxide 6 was shown by thermal denaturation studies to have a significantly higher DNA-binding affinity than the parent DC-81 (3) or the C8-propenoxy-PBD (15), which is structurally similar but lacks the epoxide moiety. The time course of effects upon thermal denaturation indicated a rapid initial binding phase followed by a slower phase consistent with the stepwise cross-linking of DNA observed for a difunctional agent. This was confirmed by an electrophoretic assay which demonstrated efficient induction of interstrand cross-links in plasmid DNA at concentrations >1 microM. Higher levels of interstrand cross-linking were observed at 24 h compared to 6 h incubation. A Taq polymerase stop assay indicated a preference for binding to guanine-rich sequences as predicted for bis-alkylation in the minor groove of DNA by epoxide and imine moieties. The pattern of stop sites could be partly rationalized by molecular modeling studies which suggested low-energy models to account for the observed binding behavior. The epoxide PBD 6 was shown to have significant cytotoxicity (45-60 nM) in the A2780, CH1, and CH1cis(R) human ovarian carcinoma cell lines and an IC(50) of 0.2 microM in A2780cis(R). The significant activity of 6 in the cisplatin-resistant CH1cis(R) cell line (IC(50) = 47 nM) gave a resistance factor of 0.8 compared to the parent cell line, demonstrating no cross-resistance with the major groove cross-linking agent cisplatin.

Effect of A-ring modifications on the DNA-binding behavior and cytotoxicity of pyrrolo[2,1-c][1,4]benzodiazepines.

Several A-ring-modified analogues of the DNA-binding antitumor agent DC-81 (5) have been synthesized in order to study structure-reactivity/cytotoxicity relationships. For two molecules (23 and 30) the modifications required the addition of a fourth ring to give the novel dioxolo[4,5-h]- and dioxano[5,6-h]pyrrolo[2,1-c][1, 4]benzodiazepin-11-one (PBD) ring systems, respectively. Another three analogues (34, 38, and 48) have the native benzenoid A-ring replaced with pyridine, diazine, or pyrimidine rings to give the novel pyrrolo[2,1-c][1,4]pyridodiazepine, pyrrolo[2,1-c][1, 4]diazinodiazepine, and pyrrolo[2,1-c][1,4]pyrimidinodiazepine systems, respectively. The other new analogues (16a,b) have extended chains at the C8-position of the DC-81 structure. During the synthesis of these compounds, a novel tin-mediated regiospecific cleavage reaction of the dioxole intermediate 18 was discovered, leading to the previously unknown iso-DC-81 (20). In addition, an unusual simultaneous nitration-oxidation reaction of 4-(3-hydroxypropoxy)-3-methoxybenzoic acid (8) was found to produce 3-(4-carboxy-2-methoxy-5-nitrophenoxy)propanoic acid (9), a key intermediate, in high yield. In general, the results of cytotoxicity and DNA-binding studies indicated that none of the changes made to the A-ring of the PBD system significantly improved either binding affinity or cytotoxicity in comparison to DC-81. This result suggests that the superior potency of natural products such as anthramycin (1), tomaymycin (2), and sibiromycin (3) is due entirely to differences in C-ring structure, and in particular exo or endo unsaturation at the C2-position and C2-substituents containing unsaturation. This study also provided information regarding the influence of A-ring substitution pattern on the relative stability of the interconvertible N10-C11 carbinolamine, carbinolamine methyl ether, and imine forms of PBDs.

Synthesis of novel C7-aryl substituted pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) via pro-N10-Troc protection and Suzuki coupling.

Novel C7-aryl pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) have been synthesized via Suzuki coupling between a 7-Iodo N10-Troc-protected PBD carbinolamine and commercially available boronic acids.

Design, synthesis and biological activity of a pyrrolo [2,1-c][1,4]benzodiazepine (PBD)-distamycin hybrid.

We report the synthesis of a new hybrid 13 which is a combination of the naturally occurring antitumor agent distamycin A 1 and the pyrrolo[2,1-c][1,4]benzodiazepine 11, related to the naturally occurring anthramycin 2. The antitumor activity of the hybrid 13 was tested in vitro and compared to the natural product distamycin 1 and the PBD 11.

Inhibition of bacteriophage T7 RNA polymerase in vitro transcription by DNA-binding pyrrolo[2,1-c][1,4]benzodiazepines.

The interactions of several pyrrolo[2, 1-c][1,4]benzodiazepine (PBD) antitumor antibiotics with linearized plasmid pGEM-2-N-ras DNA have been analyzed by quantitative in vitro transcription (QIVT) and in vitro transcription footprinting (IVTF) methods. A concentration-dependent inhibitory effect of the PBDs on transcription is observed using both techniques. The rank order for overall inhibition of transcription by the QIVT method is found to be: sibiromycin > tomaymycin > anthramycin > DC-81 > neothramycin, whereas the IVTF experiments show a different ranking: sibiromycin > anthramycin > neothramycin > tomaymycin. In addition, stimulation of transcription was observed at low PBD concentrations in both the QIVT and IVTF experiments. These results demonstrate unequivocally that the formation of PBD-DNA adducts at AGA-5' base sequences on the transcribed strand results in transcription blockage for all PBDs examined. Furthermore, the sequence of flanking base pairs appears to influence the degree of blocking, with the sequences ACAGAAA-5', AAAGATG-5', AGAGATA-5', and CAAGAAC-5' providing the most pronounced blocks for all PBDs studied in this system. Neothramycin and tomaymycin cause additional blocks at some GGA-5' and TGA-5' sequences. Parallel MPE-Fe(II) footprinting studies have revealed PBD binding sites on both the transcribing and nontranscribing strands, although all transcription blocks determined from the IVTF assays are due to drug bound on the transcribing DNA template strand.

A novel series of DNA triple helix-binding ligands.

We have examined the effect of a series of substituted imidazothioxanthones on the stability of an intermolecular DNA triple helix by DNase I footprinting. We find that several of these compounds promote the formation of a complex between T5C5 and the target site A6G6.C6T6, suggesting that they bind specifically to triplex DNA. The only inactive derivative lacked a protonatable function in the side chain, suggesting that this is an essential feature for triplex stabilization. These compounds, which are amongst the first triplex-binding ligands which possess an uncharged chromophore, are selective for the T.AT rather than the C+.GC triplet.

Preclinical pharmacology and antitumour activity of the novel sequence-selective DNA minor-groove cross-linking agent DSB-120.

We examined the in vitro cytotoxicity, antitumour activity and preclinical pharmacokinetics of the novel sequence-selective, bifunctional alkylating agent DSB-120, a synthetic pyrrolo[1,4][2,1-c]benzodiazepine dimer. DSB-120 was shown to be a potent cytotoxic agent in vitro against a panel of human colon carcinomas [50% growth-inhibitory concentration (IC50) 42 +/- 7.9 nM, mean +/- SE, n = 7] and two rodent tumours (L1210 and ADJ/PC6). Antitumour activity was assessed in the bifunctional alkylating-agent-sensitive murine plasmacytoma ADJ/PC6 using a variety of administration protocols. The maximal antitumour effects were observed following a single i.v. dose but the therapeutic index was only 2.6. DSB-120 was less effective when given i.p. either singly or by a daily x 5 schedule. After a single i.v. dose at the maximum tolerated dose (MTD, 5 mgkg-1) the plasma elimination was biphasic, with a short distribution phase (t1/2 alpha 4 min) being followed by a longer elimination phase (t1/2 beta 38 min). Peak plasma concentrations were 25 micrograms ml-1, the clearance was 1.3 ml g-1 h-1 and the AUC0-infinity was 230 micrograms ml-1 min. Concentrations of DSB-120 in ADJ/PC6 tumours were very low, showing a peak of 0.4 micrograms g-1 at 5 min. The steady-state tumour/plasma ratio was about 5% and the AUC was only 2.5% of that occurring in the plasma. DSB-120 appeared to be unstable in vivo, with only 1% of an administered dose being recovered unchanged in 24-h urine samples. Plasma protein binding was extensive at 96.6%. In conclusion, the poor antitumour activity of DSB-120 may be a consequence of low tumour selectivity and drug uptake as a result of high protein binding and/or extensive drug metabolism in vivo.