Discovery of a Chiral DNA-Targeted Platinum–Acridine Agent with Potent Enantioselective Anticancer Activity

Abstract

A structure–activity relationship study was performed for a set of rigidified platinum–acridine anticancer agents containing linkers derived from chiral pyrrolidine and piperidine scaffolds. Screening a library of microscale reactions and selected resynthesized compounds in non-small-cell lung cancer (NSCLC) cells showed that cytotoxicities varied by more than three orders of magnitude. A potent hit compound was discovered containing a (R)-N-(piperidin-3-yl) linker (P2–6R), which killed NCI-H460 and A549 lung cancer cells 100 times more effectively than the S enantiomer (P2–6S). P2–6R accumulated in A549 cells significantly faster and produced 50-fold higher DNA adduct levels than P2–6S. Ligand similarity analysis suggests that only module 6R may be compatible with strainless monofunctional intercalative binding. NCI-60 screening and COMPARE analysis highlights the spectrum of activity and potential utility of P2–6R for treating NSCLC and other solid tumors.

Metal-based anticancer agents that cause DNA damage patterns and cellular responses different from the bifunctional adducts (cross-links) induced by cisplatin and its derivatives have the potential to elicit unique mechanisms of cell death, which may overcome several of the drawbacks experienced with the clinical drugs.^[1] Among these nonclassical agents are several classes of metallodrugs that exert their pharmacological effects by forming monofunctional adducts with DNA.^[2] Depending on the geometry of the complex and the ligand set, these adducts may effectively stall DNA processing enzymes, trigger DNA damage response (DDR) pathways, or indirectly produce DDR-independent stress signals that lead to cancer cell death.^[3]

Monofunctional platinum–acridines overcome the chemoresistance to conventional platinum-based drugs observed in many solid tumors.^[4] The most active hybrid agents (Figure 1a, where L is NH₃ or L₂ is an aliphatic diamine) show up to three orders of magnitude higher potencies than cisplatin in non-small-cell lung cancer (NSCLC) cell lines and maintain high activity at nanomolar concentrations in this notoriously DNA repair-proficient form of cancer.^[5] The level of cell kill produced by platinum–acridines critically depends on the rapid formation of these monofunctional intercalative hybrid adducts in alternating pyrimidine–purine dinucleotide sequences,^[6] which cause not only DNA double-strand breaks due to stalled replication forks, but also inhibit its transcription.^[7]

Figure 1.

a) Library design and synthetic strategy. b) Summary of 19 enantiomeric (E) and diastereomeric (D) relationships among library members (#= 5 or 6).

In the present study we addressed the role of the flexible N-(2-aminoethyl)ethanimidamide linker that connects the metalating and intercalating moieties in platinum–acridines (highlighted in Figure 1a). Specifically, a structure–activity relationship (SAR) study was performed to probe the stereochemical requirements for the submicromolar-to-nanomolar activity observed in this class of compounds and to assess if their potency can be further improved by incorporating the freely rotatable linker into a rigidified, heterocyclic scaffold. Introducing conformational constraints in the unbound form to generate a geometry that mimics the DNA-bound state of the agent may minimize the entropic penalty and improve its target interactions.^[8] Combinatorial screening of a small library of platinum–acridines not only demonstrates that the choice of linker geometry is critical for achieving nanomolar activity, the results also reveal an unprecedented level of enantioselective control of the cytotoxic properties in DNA adduct-forming metallodrugs.

Previous SAR studies have demonstrated that the most active platinum–acridines contained a linker module featuring a flexible ethylene moiety that allows strainless concomitant monofunctional nucleo-base platination and intercalation.^[4,9] In the new derivatives, the previously optimized spacer was incorporated into five- and six-membered ring systems. This was achieved by formal ring closure between the N-methyl group and C(2) in N¹-(acridin-9-yl)-N²-methylethane-1,2-diamine (A1), which produces a stereo-center at C(2). Two pairs of enantiomeric acridine derivatives were generated from the corresponding enantiomerically pure 3-aminopyrrolidine and 3-aminopiperidine precursors: (R)-N-(pyrrolidin-3-yl)acridin-9-amine (5R), (S)-N-(pyrrolidin-3-yl)acridin-9-amine (5S), (R)-N-(piperidin-3-yl)acridin-9-amine (6R), and (S)-N-(piperidin-3-yl)acridin-9-amine (6S) (Figure 1a, see the Supporting Information for synthetic details). A small modular library of platinum–acridines was then generated from these acridine derivatives and the platinum(II)–nitrile precursors [PtCl(L-L)(MeCN)]NO₃, P1-P5, using our previously reported Scheme of coupling reactions via amine addition to metal-activated nitrile.^[10] In P1-P5, ethane-1,2-diamine (en), propane-1,3-diamine (pn), (1R,2R)-cyclohexane-1,2-diamine (R,R-dach), (1S,2S)-cyclohexane-1,2-diamine (S,S-dach), and 2,2-dimethylpropane-1,3-diamine (Me₂pn) were introduced as bidentate nonleaving groups (L-L). Using microscale reactions, 25 library members were synthesized in this manner, encompassing 11 enantiomeric and 8 diastereomeric relationships (Figure 1b). Following previously validated methodology,^[10] the library was analyzed by LC-MS for conversion to the desired platinum–acridines (see the Supporting Information) and reactions were pre-screened directly for activity in A549 lung cancer cells.

A549 cancer cells were exposed to fixed concentrations of each of the 25 library members (10 and 100 nM, corresponding to IC₅₀ and IC₉₀ levels typically observed for the most active platinum–acridines^[5b]) in a 96-well-plate format for 72 hours. Relative cell viabilities were determined colorimetrically (Figure 2a), and the density of viable (surviving) cells treated at 100 nM hybrid agent was plotted for each derivative as a fraction of untreated control (Figure 2b,c). Two highly cytotoxic hits, P2–6R and P5–6R, were identified by simple visual inspection of the plate, based on the absence of the color change associated with the bioreduction of tetrazolium dye (MTS) to formazane by viable cells (Figure 2a). Significantly, the corresponding enantiomers, P2–6S and P5–6S, appeared to have no major effect on cell viability under the same conditions.

Figure 2.

Results of the library prescreening in A549 cells. a) Section of the assay plate after exposure of cells to MTS reagent. Wells containing the hits identified in this assay and their inactive isomers are highlighted by white boxes. b),c) Fractions of surviving cells after treatment with the 25 hybrids (100 nM) relative to an untreated control (Ctrl) sorted by common acridine-linker (b) and platinum moieties (c), respectively. Columns for the two hits and their inactive isomers are highlighted in green and red, respectively. The data represents the mean and the error bars the standard deviation (SD) for one of two experiments performed in duplicate.

From a comparison of activity data across the entire range of library members several structure–activity patterns emerge (Figure 2b,c): i) Platinum–acridines derived from A1 and 6R are generally the most active, except when combined with either enantiomer of the dach-modified platinum moieties; ii) platinum–acridines containing six-membered diamine chelates as nonleaving groups (pn, Me₂pn) perform best when combined with 6R; the overall lowest fraction of viable cells after treatment with 100 nM hybrid agent are observed for P2–6R (13%) and P5–6R (21%); iii) the most active compounds show a high level of enantioselectivity, while P2–6S and P5–6S show no activity at all at the highest concentration tested; and iv) compounds derived from 6S, 5R, and 5S show only moderate activity when combined with P1 containing a five-membered en chelate as non-leaving group.

To further investigate the above trends and validate P2–6R as a true hit, pure batches of this compound and three derivatives sharing the same platinum moiety, P2–6S, P2–5R, and P2–5S, were synthesized and their IC₅₀ values determined in A549 and NCI-H460 lung cancer cells. Similar dose–response patterns are observed in both cell lines, which critically depend on the geometry and stereochemistry of the linker modules (Figure 3a,b). The activities across each set of the four compounds vary by more than 5000-fold in A549 and by 120-fold in NCI-H460 (Table 1). The piperidine-based derivative, P2–6R, is by far the most cytotoxic compound in both cell lines where it performs equally well with IC₅₀ values of 17 ± 1 nM and 16 ± 1 nM, respectively. In A549 cells, P2–6R proved to be more active than the achiral prototype P2-A1, which contains an acyclic linker (Table 1). By contrast, the enantiomer, P2–6S, shows significantly decreased cytotoxicity by two orders of magnitude, resulting in IC₅₀ values in the low micromolar range (Table 1), which confirms the prescreening results. The opposite scenario is observed for the derivatives containing a pyrrolidine linker. Here, P2–5R was significantly less active than P2–5S and showed the poorest activity of all compounds tested in both cell lines. Unlike P2–6R and P2–6S, which performed equally well in both cell lines, P2–5R and P2–5S showed significantly enhanced cytotoxicity in NCI-H460 compared to A549.

Figure 3.

Dose-response curves for new compounds screened in a) A549 and b) NCI-H460 lung cancer cells.

Table 1:

Antiproliferative activity (IC₅₀ ± SEM, μM^[a]) of test compounds in lung cancer cells.

Comp.	A549	NCI-H460
P2–5R	> 100	2.3 ± 1.5
P2–5S	2.6 ± 1.7	0.27 ± 0.02
P2–6R	0.017 ± 0.001	0.016 ± 0.001
P2–6S	1.5 ± 0.1	1.6 ± 0.1
P2-A1	0.029 ± 0.003[b]	0.008 ± 0.002[b]
Cisplatin	26 ± 5	1.4 ± 0.2

^[a]Data represents means ± standard errors of the mean (SEM) for 72-h treatments of at least 2 independent experiments performed in triplicate.

^[b]Data previously reported in Ref. [13].

The above SAR demonstrate that linker geometry and chirality have a profound impact on the biological activity in this small set of platinum–acridines. In particular, the cell kill observed for P2–6R, which is 100-fold more cytotoxic than P2–6S, implies that one or more enantioselective steps must exist in this compound s mechanism of action, most likely at the DNA target level. This may include differences in the rate with which the hybrid adducts form in nuclear DNA, their ability to stall DNA-processing enzymes, as well as their recognition and removal by the DNA repair machinery.^[11] The effects of chirality on these processes have previously been demonstrated for the enantiomeric forms of cisplatin derivatives bearing chiral nonleaving groups, which includes the clinical drug (1R,2R)-dach-1,2-diamineoxalatoplatinum-(II) (oxaliplatin).^[12] In the case of dach-containing platinum drugs, however, comparatively minor differences in cytotoxicity between enantiomers of approximately 3–5-fold have been observed.^[11b,12a] Likewise, the enantiomers P3-A1 and P4-A1 studied previously, which both contain an achiral linker but R,R-dach and S,S-dach nonleaving groups, respectively, show essentially the same level of cell kill.^[13] These observations suggest that the correct stereochemistry of the linker is far more critical than the chirality of the classical nonleaving groups for the biological activity of monofunctional intercalative hybrid agents.

While details of the target interactions and cellular processing of the chiral platinum–acridines remain to be investigated, simple quantification of intracellular platinum and DNA adduct frequencies for P2–6R and P2–6S provide compelling evidence for a high degree of chiral discrimination at the cellular level. When A549 cells were treated with the two enantiomers, P2–6R showed a significantly higher cellular accumulation than P2–6S by 4-fold (Figure 4A). Most importantly, the DNA adduct levels achieved by the R isomer were 50 times higher relative to those observed for the S isomer under the same conditions, resulting in 50 adducts and 1 adduct per 100000 DNA base pairs, respectively (Figure 4B). These findings confirm that the major cause of the enantioselective anticancer activity lies at the genome level, consistent with the prior knowledge of the mechanism of platinum–acridines,^[6] although contributions from enantioselective interactions with other cellular components cannot be ruled out.

Figure 4.

Quantification of A) total intracellular platinum and B) platinum content in cellular DNA extracted from A549 cells treated with 1 μM P2–6R or P2–6S for 4 h at 37°C (means ± standard deviation for triplicate experiments, ***p <0.001, two-tailed t-test). C) Ligand overlay analysis for the A1-based acridine-linker moiety in PDB 1XRW (red) and the corresponding fragments derived from 6R and 6S (colored by element). Calculated root-mean-square deviations (RMSD for common non-hydrogen atoms in) between the experimental and the modeled chiral structures 6R and 6S were 0.20 and 0.59, respectively. For details of the modeling procedure and results for 5R and 5S, see the Supporting Information.

Computational ligand similarity modeling provides insight into a potential cause of the higher DNA affinity and potency of P2–6R. We performed a simple ligand overlay analysis to determine if any of the new chiral heterocyclic linkers would be able to adopt the geometry observed for the DNA adduct formed by a platinum–acridine derivative containing an ethylene-based linker (PDB ID 1XRW)^[9b] (Figure 4C). The adduct geometry in 1XRW, which allows classical intercalation of the acridine chromophore perpendicular to the B-DNA axis, proved to be a prerequisite for rapid DNA binding and, ultimately, the high potency in this class of hybrid agents.^[4] Only the fragment derived from (R)-N-(piperidin-3-yl)acridin-9-amine in P2–6R was able to faithfully mimic the linker conformation observed in the A1-based prototype. By contrast, the stereochemistries of the other derivatives showed major misalignments with the experimental structure, potentially rendering the corresponding hybrid agents incompatible with optimal monofunctional intercalative binding (see caption of Figure 4 and the Experimental Section for details).

To investigate the spectrum of activity of the hit P2–6R, the compound was screened in 60 cell lines from 9 tissues of origin by the Developmental Therapeutics Program (DTP) at the National Institutes of Health (NCI-60). Additionally, the activity data acquired for this compound was compared with the anticancer profile of cisplatin, carboplatin, and oxaliplatin (Figure 5) and statistically analyzed using the COMPARE tools.^[14] P2–6R is the most active anticancer agent with potent growth inhibition in the 20–40 nM range in several of the NSCLC cell lines (A549, NCI-H226, NCI-H460, NCI-H522), as well as cell lines representing other solid tumors, including UACC-62 (melanoma), SN12C (renal cell carcinoma), DU-145 (androgen receptor-negative prostate cancer), and T-47D (breast cancer). While P2–6R proved to be more active than cisplatin by approximately 20-fold (based on GI₅₀ endpoints) across the entire range of cell lines, a distinct advantage over the clinical drug of up to a 1000-fold is observed for the most sensitive cell lines. This cell line-specific enhancement observed for P2–6R results in a more than 2000-fold difference in activity between the most responsive and the most resistant cell line (ΔlogGI₅₀ = 3.3), while GI₅₀ values for cisplatin vary only by approximately 30-fold (ΔlogGI₅₀ = 1.5). A similar, although less pronounced trend is observed when P2–6R is compared with oxaliplatin. Most importantly, Pearson correlation analysis of the data sets demonstrates that the chemosensitivity profile of P2–6R shares no common features with those observed for cisplatin and its derivatives, confirming the unique mechanism of action of the hybrid agent (p values for pairwise comparisons were 0.169, 0.065, and 0.222 for cisplatin, carboplatin, and oxaliplatin, respectively).

Figure 5.

Heat map presentation of NCI-60 screening results (logGI₅₀ values) for P2–6R and the FDA-approved platinum drugs cisplatin, carboplatin, and oxaliplatin. Relative activities are highlighted using a two-color scale with the darkest shades of red and blue indicating the highest and lowest activities, respectively. For the corresponding five-dose mean graphs, see the Supporting Information. N.D.: no data available.

The results of the NCI-60 screening are encouraging because they not only confirm the high potential of platinum–acridines in NSCLC, but also demonstrate that the potency translates to other solid tumors. Many aggressive and metastatic forms of cancer require innovative treatment options that result in a longer-lasting response and reduced mortality rates, as therapeutic failure and disease progression owing to multifactorial tumor resistance remain common obstacles.^[15] Classical chemotherapy, most notably platinum-based regimens, continues to be a standard of care for several advanced solid tumors^[16] despite the promise of the newer molecularly targeted therapies and immuno-oncology drugs.^[17] Given the limitations of current anticancer therapies, there is a continued need for new pipelines of mechanistically unique drugs with distinct activity profiles, such as the newly discovered hybrid agent P2–6R.

In conclusion, in the current study, which was designed to further elucidate the SAR in platinum–acridine anticancer agents, a conformationally constrained, chiral derivative with high potency in NSCLC and other solid tumors was discovered. Introducing rigidified, chiral linker moieties into these agents allowed us to probe the stereochemical requirements for the nanomolar anticancer activity that can be achieved with this pharmacophore. The SAR delineated in this study provide an incentive for additional mechanistic work and further optimization of this class of agents. Additionally, the NCI-60 screening results provide a compelling argument why platinum–acridines should not be considered as just another cisplatin derivative, but as a treatment option complementary to the traditional chemotherapies.