E ﬀ ects of Systematic Shortening of Noncovalent C8 Side Chain on the Cytotoxicity and NF- κ B Inhibitory Capacity of Pyrrolobenzodiazepines (PBDs)

: The systematic shortening of the noncovalent element of a C8-linked pyrrolobenzodiazepine (PBD) conjugate ( 13 ) led to the synthesis of a 19-member library of C8-PBD monomers. The critical elements of 13 , which were required to render the molecule cytotoxic, were elucidated by an annexin V assay. The e ﬀ ects of shortening the noncovalent element of the molecule on transcription factor inhibitory capacity were also explored through an enzyme-linked immunosorbent assay-based measurement of nuclear NF- κ B upon exposure of JJN-3 cells to the synthesized molecules. Although shortening the noncovalent interactive element of 13 had a less than expected e ﬀ ect upon compound cytotoxicity due to reduced DNA interaction, the transcription factor inhibitory capacity of the molecule was notably altered. This study suggests that a relatively short noncovalent side chain at the C8 position of PBD is su ﬃ cient to confer cytotoxicity. The shortened PBD monomers provide a new ADC payload sca ﬀ old because of their potent cytotoxicity and drug-like properties.


■ INTRODUCTION
Since their discovery in Streptomyces bacteria over half a century ago, pyrrolo [2,1-c] [1,4]benzodiazepine (PBD) structures have been of interest as possible chemotherapeutic agents. This is due to their ability to sequence selectively bind covalently to DNA, to cause DNA strand cleavage, and to inhibit DNA-associated enzymes. 1−3 Two critical features of the PBD structure confer this covalent binding capacity on the molecule. S stereochemistry at the C11a position of the molecule bestows a right-handed longitudinal twist upon the structure, allowing an effective fit along the minor groove of the DNA helix. 4,5 Complementing this optimal fit is the presence of an interconvertible imine-carbinolamine moiety at the N10−C11 position of the molecule. This electrophilic center undergoes attack from the C2−NH 2 of guanine bases in the minor groove, forming the covalent aminal bonds responsible for their potent cytotoxicity 6−8 ( Figure 1A).
PBD structures have been found to form covalent bonds with some guanines in the DNA minor groove more frequently than others, particularly those flanked by purine bases, 5,9 although more recent data suggest kinetic preferences when guanines are flanked by pyrimidines. 10 This potential for relative selectivity of binding is of interest, given that the pathogenesis of many cancers is associated with the aberrant binding of transcription factors, such as nuclear factor kappa B (NF-κB), to sequence-specific promoter regions of DNA. 11−15 The conjugation of subunits, capable of noncovalent interactions with DNA bases, to the C8 position of the PBD structure (via a carbon linker of variable length) has led to the development of hybrid molecules that can interact selectively with longer sequences of DNA than the typical purine− guanine−purine motifs of unmodified PBD structures. Such molecules have shown a capacity to inhibit the binding of transcription factors, such as NF-Y 16 and NF-κB 17 to their respective promoter sequences. More recent C8-modified PBDs, such as KMR-28-39 ( Figure 1B) synthesized by Rahman and co-workers 18 have demonstrated the evidence of preclinical activity of such PBD conjugates. In addition to their cytotoxicity and anti-tumor activity, PBD-heterocyclic hybrids have shown significant antimicrobial activity. 19−21 More recently, PBDs are being used as cytotoxic payloads for antibody−drug conjugates, and a number of these are currently undergoing clinical evaluation. 22 Although the addition of noncovalent interactive subunits to the C8 position of PBD structures has been associated with the down-regulation of genes downstream of transcription factor activity, 17,18,23,24 the relationship between the structural requirements and the potent cytotoxicity observed for these molecules has not been fully elucidated. The extent of C8 diversification required to confer this transcription factor activity (i.e., the core noncovalent interactive pharmacophore) remains unclear. In this paper, we investigated some of these uncertainties through the systematic shortening of a C8-linked monomer (13, Figure 1C) containing a pyrrole−benzofuran motif which has been previously explored in both noncovalent distamycin 25 and covalent duocarmycin analogues 26   Synthesis of PBD Hybrids. The PBD core (N10-Alloc, C11-THP protected) of the hybrids (10) was synthesized from vanillin, as previously described in the literature 18 (see the Supporting Information for scheme). A four-carbon linker was used to connect the PBD component with the noncovalent interactive subunits, as chains of this length had proven optimal for DNA binding in previous hybrid SAR studies. The linker was located at the C8 position of the molecule to allow an isohelical fit of the noncovalent component of the hybrid along the minor groove upon covalent PBD binding. 2 After the synthesis of the PBD core, the C8 hybrid 13 was constructed using a combination of a benzofuran and an Nmethyl pyrrole moiety. This was based upon the molecular structures of the side chains of KMR-28-39 ( Figure 1B) and other molecules recently described by Basher and coworkers. 27 These benzofuran−pyrrole components were linked via Steglich amide bond formation at positions that maintained the overall fit of the hybrid for the DNA minor groove (i.e., C2/C5 for benzofuran, C1/C4 for N-methyl pyrrole components). The full synthesis of the noncovalent interactive hybrid component of 13 is summarized in the Supporting Information, with the conjugation to the PBD moiety and activation of the N10−C11 imine component of the molecule detailed in Scheme 1. A total of 19 new PBDs were synthesized by the systematic shortening of the noncovalent interactive element of 13 (Table 1). This process was analogous to the synthesis of 13, with component 11, replaced with designed building blocks to afford progressively shortened C8-PBDs.
Biological Evaluation. Cytotoxicity Screening. Given the established inhibition of the activity of the NF-κB transcription factor by previous (structurally similar) C8-linked PBD monomers, such as those described by Rahman and coworkers, 18 it was hypothesized that compound 13 and its shortened derivatives could share such properties. Accordingly, the initial screening of the compounds was carried out in primary tumor samples and a malignant cell line known to manifest aberrant activity of NF-κB. The pathogenesis of both chronic lymphocytic leukemia (CLL) and multiple myeloma has been linked to aberrant gene expression mediated, at least in part, by NF-κB. 28−33 Primary tissues were obtained from CLL patients (n = 3) with their informed consent, and the authenticated multiple myeloma cell line, JJN-3, was purchased from DSMZ (Germany). The compounds were added to cultures of the primary CLL cells and JJN-3 cells at concentrations between 1 nM and 100 μM in both primary leukemic cells and the cell line for 48 h. An annexin V binding assay was used to measure cytotoxicity, and LC 50 values were calculated from the sigmoidal dose−response curves using

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Article Prism 6.0 software. A previously synthesized C8-linked PBD hybrid featuring transcription factor inhibitory properties, GWL-78, 34 was also evaluated as a positive control. The results of this cytotoxicity evaluation are described in Table 1,  with the cytotoxicity dose−response curves obtained for each  molecule detailed in the Supporting Information. Prior to the screening of the shortened compound library in the CLL and JJN-3 cell cultures, it was expected that molecular shortening of the side chain of 13 would diminish compound activity. This was based upon the hypothesis that both the overall affinity of the PBD hybrid for DNA and the length of DNA sequences bound by the compound would be reduced because of the reduction in noncovalent interactions between the DNA minor groove and the C8 side chain of the molecule. This concept has been reflected in the cytotoxicity of previously synthesized PBD hybrids, which featured short C8-conjugated moieties. Such compounds either lacked notable cytotoxicity or were less cytotoxic than their longer side-chain counterparts. Examples of these compounds include a PBD hybrid containing a single pyrrole element synthesized by Borgatti and co-workers, as well as hybrids of similar structure by Thurston and co-workers, which were only weakly cytotoxic. 34,35 The cytotoxicity of the compounds in the primary CLL cells and the JJN-3 cell line was therefore unexpected. For several of the compounds tested, LC 50 values were obtained in the low nanomolar range, with good concordance in cytotoxicity observed between the primary CLL cells and the myeloma cell line. In total, 10 compounds maintained or improved activity with respect to 13 upon side chain shortening in primary CLL cells.
The cellular screen provided some insight into the structural features of the noncovalently interactive C8 side chain of 13 which may prove integral to its cytotoxic activity. It appeared that the presence of the terminal pyrrole moiety of 13 is not essential for its activity, as cytotoxicity was largely preserved for the benzofuran-containing compounds 14 and 15. Aromaticity of the furan component of the benzofuran structure also appeared not to be crucial for its cytotoxicity. Indeed, the dihydrobenzofuran-containing compound, 16, was one of the most cytotoxic compounds tested in primary CLL cells. Opening this furan ring also maintained activity at low nanomolar levels in both primary CLL cells and the JJN-3 cell line.
The importance of the oxygen moiety of benzofuran is less clear from this cellular evaluation. Replacement of the oxygen atom with a carbon (17) or its complete elimination (22−24) had little effect upon compound activity. This indicates that the potential hydrogen bond acceptor role of the oxygen plays little part in the overall cytotoxicity of 13. Interestingly, reduced cytotoxicity was observed for 21. In this case, the benzofuran oxygen is present as part of a phenol, rather than an ether functional group. The phenol moiety of 21 may act as a hydrogen bond donor, in contrast to its ether-containing analogues. The latter of these characteristics appeared to have an important effect on overall cytotoxicity.
Interestingly, the entire removal of the furan ring of the benzofuran side-chain component (25) failed to appreciably reduce the cytotoxicity. However, alterations to the benzene element of the benzofuran had more significant effects upon cytotoxicity. The introduction of a nitrogen atom into the ring, through the use of aminopyridine building blocks, eliminated compound activity at the dose range tested. Such a deleterious effect upon compound activity was unexpected. In a similar fashion to the introduction of the phenol group in 21, the nitrogen of the pyridine ring may ionize depending upon the pH conditions. In a mildly acidic environment, the pyridine ring is protonated (pK a of the conjugate acid is around 5.72), creating a formal positive charge. This charge may affect the ability of the molecule to penetrate the cell membrane and reach the minor groove of DNA. Moreover, the charged conjugate acid also conferred another hydrogen bond donor group on the molecule. One or both of these effects are likely to impact upon the activity of the PBD hybrids 26 and 27, which showed approximately a three-log reduction in cytotoxicity when compared to 13.
Removal of the aromaticity of this ring notably reduced the cytotoxicity. The cyclohexane and ring-opened derivatives (28−32) showed poor activity in both primary CLL cells and the JJN-3 cell line. It is possible that the spatial alteration of this portion of the side chain from a planar sp 2 hybridized structure to sp 3 derivative was responsible for this loss of activity. The removal of the delocalized electrons of the aromatic system may also have affected compound activity because of the removal of potential π−π interactions with DNA bases.
Given the surprisingly similar cytotoxicity of 13 and its shortened derivatives in the CLL primary tissue and JJN-3 cell line evaluations, it was decided to conduct an evaluation of the shortened PBD hybrids versus their parent 13 in healthy agematched B-and T-cell cultures. This would establish whether the cytotoxicity observed in the malignant cells was mirrored in non-malignant cells and whether a notional therapeutic index was apparent. Six shortened derivatives of 13, which showed potent cytotoxicity in the initial CLL and JJN-3 cellular screens, were chosen for evaluation in the healthy cells: 14, 15, 18, 19, 20, and 28. Normal peripheral blood B-cells and Tcells, derived from three donors, were incubated with the seven compounds for 48 h at concentrations ranging from 1 nM to 100 μM. Subsequently, the cells were harvested by centrifugation, and apoptosis was quantified using an annexin V assay. The cytotoxicity dose−response curves obtained are detailed in the Supporting Information, with Table 2 listing the LC 50 values obtained in both normal B-and T-cells, as well as the relative toxicity in these cells compared to primary CLL cells.
All of the compounds tested showed cytotoxicity ranging from 0.39 to 12.4 μM in both cell types. The compounds showed differential toxicity in the primary CLL cells compared to the normal B-cells and normal T-cells with notional therapeutic indices between 1 and 2 logs. The least potent

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Article compound tested, 28 showed no therapeutic index. Overall, the compounds showed better therapeutic indices against Tcells compared to B-cells with average therapeutic indices of 229.1 and 80.9, respectively. The observed therapeutic indices are large enough to consider further development of these C8linked PBDs. However, there was no correlation between therapeutic indices and the length of the molecules. Essentially, this healthy cell evaluation confirmed that extensive shortening of the noncovalent component of 13 did little to affect either the cytotoxicity or selectivity profile of the compounds. Evaluation of the Effects of 13 and Derivatives on Nuclear Levels of NF-κB. Given the lack of observable cytotoxicity differences between 13 and all but its most shortened C8-side-chain derivatives, it was decided to evaluate whether the reduction of the C8-linked noncovalent interactive side chain had any effect upon potential transcription factor inhibition by compound 13.
The potent cytotoxicity observed for 13 and its derivatives in a number of NF-κB-dependent cancer cell types provided indirect evidence for their potential as possible inhibitors of this transcription factor. Moreover, the previously reported cellular inhibition by similar PBD hybrids of NF-κB 17,18,36 provided further impetus to directly measure the potential of these novel C8-linked PBD monomers to alter the nuclear levels of this transcription factor.
As a result, the effect of 13 and five of its shortened derivatives (15,19,20,21, and 25), on nuclear levels of NF-κB in JJN-3 cells, was investigated. The derivative compounds were chosen because of their potency and to provide representative examples of the various stages of the shortening of the noncovalent interactive component of the compound. JJN-3 cell lines were chosen over the primary CLL cell samples for this analysis as the levels of nuclear NF-κB were more consistent in this cell line compared to primary tumor cells derived from different patients.
The measurement of the effects of the four compounds on nuclear levels of NF-κB involved the use of an adapted enzyme-linked immunosorbent assay (ELISA). Nuclear

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Article extracts were prepared from treated and control cells using a nuclear extraction kit (Active Motif). Activated NF-κB subunits (p65, RelB, p52 or p50) in the samples were measured by oligonucleotide-based ELISA (Active Motif). Treated and control samples were incubated in the wells of a 96-well microtiter plate that were coated with the NF-κB consensus nucleotide sequence (5′-GGGACTTTCC-3′). NF-κB from the cell samples attached to the wells and was captured by specific antibody to either p65, RelB, p50 or p52. Binding of the specific NF-κB family member was then detected by an anti-rabbit-HRP-conjugated antibody. Developing solution was then added to the plate for 5 min prior to the addition of stop solution. The intensity of the developed color was proportional to the quantity of p65, RelB, p50 or p52 in each sample, and the amount of bound NF-κB was quantified (ng/μg nuclear extract) from subunit recombinant protein standard curves. The specificity of binding to the nucleotide sequence was ascertained by premixing free consensus nucleotide to the sample before adding the nuclear extract to the well. Sample determinations were normalized by the total cellular protein, as determined by the protein assay kit (Pierce). Data were expressed as a percentage of the untreated controls.
The quantified DNA binding activity for each NF-κB subunit in cells exposed to 13, 15, 19, 20, 21 and 25 was then related to a control value obtained from cells that had not been exposed to a PBD hybrid. The experiment was repeated in triplicate, and the effect of the compounds upon DNA binding of four subunits of NF-κB is illustrated in Figures 2  and 3 below. The c-Rel subunit was not evaluated in these experiments.
The evaluation of the effects on nuclear levels of NF-κB by the shortened PBD hybrids produced some interesting results. The significant knockdown of p65 by 15 aside, the shortened compounds tested produced no significant reductions in nuclear levels of the p65 subunit activity, despite their highly potent cytotoxicity in JJN-3 cells. When placed in the context of the role of NF-κB subunit function in carcinogenesis, this was an interesting outcome. p65 overexpression has been implicated in the progression of malignancy; a previous analysis of a cohort of CLL patient samples revealed significant up-regulation of the p65 subunit 28 and this was associated with disease progression. 37

Article
The nuclear activity of p52 was unaffected or increased (13 and 19) by the PBD hybrids, whereas the RelB subunit was significantly decreased by 13, but not by the shortened derivatives. Interestingly, all three of the shortened compounds tested significantly reduced p50 levels. This was not the case for 13. However, the role of p50 in carcinogenesis is to date illdefined, 38 and in some cases, increased levels of the subunit may promote apoptotic pathways. 39 Accordingly, the differential capacity of the PBD hybrids to inhibit the p65 subunit was considered in greater detail.
An evaluation of the structures of the three compounds ( Figure 2) tested in this assay may help explain the differences in effect upon nuclear levels of NF-κB observed, especially the levels of p65. The parent 13 PBD hybrid molecule showed consistent inhibition of nuclear p65 activity. Compound 15, which also significantly inhibited the p65 subunit, shares the greatest side chain similarity with 13 of the three compounds tested. The 15 side chain retains an intact benzofuran moiety common to its predecessors. This benzofuran is disrupted in 19 and 25, with both the aromaticity and rigid cyclic structure of the furan ring of the benzofuran removed. Both compounds showed no discernible effect on nuclear p65 levels, despite their potency.
The results of this assay provided a more subtle insight into potential mechanisms of activity of PBD hybrids than a reliance on cell cytotoxicity assays alone. Although all of the compounds tested showed promising potency characteristics in a range of cancer cell lines, their NF-κB transcription factor inhibitory effects differed depending upon the side-chain composition. Compound 13, containing the longest noncovalent interactive element, displayed the most effective inhibition of NF-κB and effects upon nuclear p65, and RelB levels appeared to diminish in tandem with the reduction of this element in the derivatives 15, 19, and 25 (although some increased inhibition of p50 was observed).
The clear differences in the NF-κB inhibitory capacity observed through the systematic shortening of 13 contrasted with the relatively comparable cytotoxicity profiles of all but the most shortened library derivatives. Two possible explanations may help explain this incongruity.
First, it is possible that the effects of the noncovalent interactive elements of the PBD hybrids on nuclear NF-κB activity are unrelated to their cytotoxic mechanism of activity. Such an assessment would regard the PBD monomers primarily as traditional DNA alkylating agents, with the transcription factor inhibitory effects (such as the p65 inhibition), displayed by molecules such as 13 effects, rather than the causes of PBD cytotoxicity. In this scenario, the noncovalent element of the molecules merely serves to confer different physicochemical properties on the compound or alter the nuclear penetration rate and extent of adduct formation with DNA.
The second, more compelling explanation for differences in the transcription factor inhibitory activity between PBD hybrids with varying noncovalent interactive elements is slightly more nuanced. Although all PBD molecules feature an ability to bind covalently to DNA and act to some extent as traditional alkylating agents, some may do so in a relatively more selective manner than others. It may be argued that the shortened derivatives of 13, such as 25, which feature short, low molecular weight side chains, bind to guanines along the minor groove indiscriminately. It is unlikely that such shortened structures (which form adducts spanning c. 4−5 DNA bases) would feature either the affinity or diameter to specifically disrupt conserved 8−9 base transcription factor promotor sequences. On the other hand, the presence of longer noncovalent interactive elements (such as those found in 13) conjugated to PBD structures may subtly alter their binding distribution along the minor groove. By adding moieties capable of interacting noncovalently with DNA sequences in a relatively selective manner, the covalent adducts of PBD structures may be (to some extent) preferentially directed toward the longer, more specific sequences of DNA that are bound by transcription factors. Although the traditional effects of nonselective alkylation undoubtedly still occur, additional mechanisms of cell disruption (such as transcription factor inhibition) may also underpin the cytotoxicity of molecules such as 13.
2D QSAR Analysis. A two-dimensional quantitative QSAR study of all compounds was carried out using a multiple linear regression (MLR) method to determine if there are any correlations between the biological activity (1/LC 50 ) and physicochemical descriptors log P, molecular weight, steric factors, and electronic factors. Leave-one-out cross validation was the regression method applied in the MLR model to study the correlation of the descriptors and the LC 50 of the compounds. The results are listed in Table 3. The study showed slightly better correlation between the biological activity of the molecules in primary CLL cells (0.63) compared to JJN-3 cell lines (0.52). The correlation improved when only log P (0.74) or molecular weight (0.68) was considered in primary CLL cells. However, the correlations between biological activity in JJN-3 cell line and log P (0.58) or molecular weight (0.48) were moderate.
Molecular Modeling with DNA Sequences. As all of the compounds including the shortened compounds are expected to exert their cytotoxicity by covalent bonding with DNA and subsequent inhibition of transcription factors, a molecular modeling study was carried with four compounds (13, 20, 23, and 32) with different C8 side-chain lengths and one AT-rich and one GC-rich DNA sequences. These compounds represent a mix of longer and shorter PBD compounds synthesized with a range of cytotoxicities. The molecular docking study suggested that all four compounds are able to snugly bind with the DNA minor groove and retain the shape characteristics required for the DNA minor groove binding molecules (Figures 4, S3 and S4). The energy of binding calculation showed the highest value for compound 13 with −11 KJ/mole for AT-rich DNA and −10.7 KJ/mol for the GC-rich DNA.

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The values obtained for 20 and 23 were comparable, and the value for compound 32 was slightly lower (Table S1). The findings of the study broadly support the experimental observation as the shortened molecules retained their ability to interact with DNA sequences. However, the relative lack of activity of compound 32 cannot be explained by modeling alone as the physicochemical property of the molecule likely to have played an important role in the overall activity of the molecule.

■ CONCLUSIONS
The systemic shortening of the C8 side chain of PBD monomers show that transcription factor inhibitory effects may not necessarily translate into increased overall cytotoxicity or tolerability in vitro for PBD-type molecules. This observation may be useful in the design of monoalkylating PBD monomers that are currently being explored as standalone anticancer agents and payloads for antibody−drug conjugates. It is possible, however, that differential tolerability effects may only manifest themselves in in vivo evaluations. Indeed, the high therapeutic index of historical PBD hybrids (featuring longer noncovalent interactive elements of structure similar to that of 13) versus unsubstituted PBD cores 39 provides the rationale for in vivo investigations of the novel PBD structures described in this paper. Such evaluations are currently underway.

■ EXPERIMENTAL SECTION
Synthesis. General Material and Methods. Synthetic building blocks, reagents, solvents, and chemicals were purchased from Sigma-Aldrich, Fluorochem, Fisher Scientific, Alfa Aesar, and Bachem, respectively. All reactions carried out were monitored by thin-layer chromatography carried out on E. Merck silica gel 60 F254 plates (0.25 mm). The plates were visualized using UV light at 254 nm and potassium permanganate staining where required. Purification using flash column chromatography was carried out using standard glass columns and silica gel as a stationary phase (Merck 60, 230−400 mesh ASTM). Where freeze drying of intermediates/final compounds was carried out, this was done using a Heto-Lyolab 3000 freeze dryer. High-performance liquid chromatography coupled to mass spectrometry was carried out on reaction mixtures, intermediates, and final products. This analysis was performed on a Waters Alliance 2695 separation system using water (solvent A) and acetonitrile (solvent B) as mobile phases using a Monolithic C18 50 × 4.60 mm column by phenomenex. UV detection was performed on a diode array detector. Formic acid was used to ensure acidic conditions throughout the analysis and was used at concentrations of 0.1%.
All of the compounds tested for their biological activity are >95% pure, confirmed with two different HPLC analysis methods. A 10 min (method A) and 5 min (method B) gradient was used to analyze submitted samples. The flow rate for both was 0.5 mL/min; 200 μL was split over a zero dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV detector of the HPLC system was 220−400 nm. The function type used was a diode array (535 scans). The column type was a monolithic C18 50 × 4.60 mm column.
Method A (10 min run). 95% A 5% B for 2 min increasing to 50% B over 3 min. The gradient was then held at 50% B for 1 min and then increased to 95% B over 1.5 min. The quantity of B was then returned to 5% B over 1.5 min and held for 1 min.
Method B (5 min run). 95% A 5% B at 0 min increasing to 90% B over 3 min. The gradient was then increased to 95% B over 0.5 min

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Article and then held for 1 min. The quantity of B was then returned to 5% B over 0.5 min.
High-resolution mass spectrometry was undertaken on all final compounds synthesized. This was carried out using a Waters Micromass QTOF Global system in positive W-mode. Compounds were dissolved in DMSO at concentrations of 5 mM and introduced into the instrument using metal-coated borosilicate glass tips. 1 H NMR and 13 C NMR were carried out on a Bruker ADVANCE NMR spectrometer. The proton NMR was run at 400 Hz, whereas the carbon samples were run at 100 Hz. The temperature for all samples was 300 K. The spectra obtained for each sample were processed and analyzed using Topspin 3.0.
Synthesis of PBD Core, C8-Tails and C8-Linked PBD-Imines. PBD core 10 was synthesized following the literature procedure in nine steps in good yield. The intermediates were purified and fully characterized before progressing to the next step (Supporting Information).
Cytotoxicity screenings were carried out in triplicate with these cells at a cell density of 1 000 000 cells/mL. Cells were treated with test compounds at a range of concentrations to establish LC 50 values. Cells were incubated with the compounds for 48 h before being harvested. annexin V/PI positivity was then determined using the annexin V apoptosis detection kit (eBioscience) in accordance with the manufacturer's instructions. Following staining with FITC labeled annexin V and propidium iodide, samples were analyzed on the Accuri C6 flow cytometer to determine relative levels of cell death in the sample. Prism 6 software was then used to analyze the data to obtain dose−response curves.
Nuclear NF-κB (ELISA) Assay. 5 000 000 JJN-3 cells were treated for 4 h with test compounds alongside untreated controls before being harvested and spun down. Nuclear extracts were then produced using the nuclear extraction kit purchased from Active Motif. Nuclear extracts were stored at −80°C before use in the NF-kB family ELISA kit (Active Motif). Total protein levels in the nuclear extracts were determined using a BCA protein detection kit, which allowed the calculation of nuclear extract volume equivalent to 1 μg of total protein. This was then added in accordance with the manufacturer's instructions to the ELISA plate. An optical density reading of the plate at 450 nm was then recorded. The sample absorbance values were then normalized with the standard curve absorbance values obtained from recombinant NF-κB subunit protein purchased from Active Motif. This allowed the calculation of the concentration of each NF-κB subunit present in the nuclear extract following treatment, compared with an untreated control.
Molecular Modeling. Double-stranded DNA type B was generated by the NAB module of AMBER 16.0 package program, using the template of the sequences used in the study. The PDB file for the ligands was generated by ChemOffice 16.0. All of the DNA and ligand structures were minimized by SYBYL software before molecular docking. Molecular docking of the ligands to each of the generated DNA sequences was performed using the SMINA molecular docking program. All parameters were kept at their default values. The grid box was prepared to cover the full length of the DNA to allow the ligand molecules to explore all the possible binding sites of DNA including the major grooves and minor grooves.