To generate the desired compound library, sets of suitably modified platinum complexes and 9-aminoacridine derivatives, the two building blocks of the hybrid agents, were synthesized. We then used highly efficient amine addition to platinum-coordinated nitrile ligand, a classical metal-catalyzed reaction for forming CN bonds,16
to generate the amidine-linked hybrids (based on the prototype with X = NH in ). The clean conversion to hybrid agent without the formation of stoichiometric by-products, which is reminiscent of organic click chemistry,17
provides an ideal platform for generating diversity within the library of platinum–acridines (for details see the Experimental Section).
A total of 6 nitrile-modified platinum complexes and 10 acridines were synthesized as building blocks for the modular click assembly of 60 hybrids (). Structural diversity in this small library was achieved by varying the nonleaving group(s) (R1
) and nitrile ligands (R2
) in the platinum moieties, as well as the geometry of the acridine side chain (R3
). Several of the unmodified parent compounds studied previously were also generated. Conjugatable functional groups were placed strategically within the platinum and acridine moieties as attachment points for targeted carriers. These include hydroxyl (in P5, P6, A3
, and A4
) and carboxylic acid (in A5–A7
) groups, which lend themselves as coupling partners in (reversible) ester, carbamate, and amide linkages.18
In addition, terminal azide groups were installed in extended side chains using amide (A8, A9
) and carbamate (A10
) coupling chemistry. These derivatives are also of interest for their use as imaging tools in bioorthogonal reactions19
with alkyne-modified fluorophores to study the subcellular distribution of the hybrid agents.
Library of Platinum (P) and Acridine (A) Building Blocks with Design Elements Highlighted.
To demonstrate the utility of this approach, 60 micro-scale reactions were assembled containing stoichiometric amounts of platinum (P
) and acridine (A
) and allowed to incubate in DMF under conditions that produce hybrid agent without any detectable side products (see experimental details and the Supporting Information
). Automated in-line high-performance liquid chromatography–electrospray mass spectrometry (LC-ESMS) was used to monitor the progress of the reactions and to calculate the yield of each hybrid. This was possible by integrating the HPLC traces recorded at an acridine-specific wavelength assuming the chromophores in the acridine percursors (A
) and in the newly formed hybrids show the same absorptivity. An example of an LC profile along with ESMS characterization of the reaction mixture is given in for hybrid P6-A1
(for a complete set of 60 LC-MS profiles see the Supporting Information
). Note the absence of side products and high yield (80%) of the conversion. Baseline separation of the two fractions corresponding to unreacted acridine and product platinum–acridines was observed for all 60 samples analysed, which greatly facilitated quantification and mass spectrometric characterization of the hybrids.
Figure 1 High-throughput LC-ESMS analysis of “click” reaction mixtures. (A) Reverse-phase HPLC trace for the reaction of platinum precursor P6 with acridine A1. (B) ESMS spectrum of A1 (HPLC fraction with retention time 8.8 min) recorded in positive-ion (more ...)
Species in solution were identified by their molecular-ion peaks and fragments generated by in-source collision-induced dissociation (CID). The reaction mixtures were diluted with appropriate amounts of media so that each sample contained the same concentration of hybrid agent prior to incubations with cancer cells. NCI-H460 cells were then exposed to a fixed concentration of 50 nM of each platinum–acridine and the viability of the cancer cells relative to untreated control was assessed after 72 h of incubation using a colorimetric cell proliferation assay. The assay was also performed with the platinum and acridine precursors, which proved to be at least two orders of magnitude less cytotoxic than the hybrids. These control experiments were necessary to demonstrate that unreacted precursors in reactions that did not go to completion did not significantly contribute to the inhibition of cell proliferation (see the Supporting Information
for an example). Finally, to further validate the pre-screening approach, selected compounds of interest were resynthesized and their IC50
values determined in the same cell line ().
To validate our combinatorial approach as a tool for establishing SAR in platinum–acridines, the data set generated was examined by plotting cell viabilities for the 60 samples (P1-A1 to P6-A10). To gain insight into the relative importance of the fragments P and A in each compound, samples were numerically sorted into groups of hybrids containing the same platinum moiety and groups sharing the same acridine ligand (). The former alignment demonstrates that for hybrids containing the same platinum moiety (P1–P6) the biological activity usually decreases significantly with increasing degree of modification and length of the (functionalized) 9-aminoacridine side chain (). Analysis of the global activity profiles for P1–P6 also shows that variation of the nitrile ligand (R2 = Et in P1, P3, and P5 vs. R2 = Me in P2, P4, and P6) leads to little variation in activity and produces pairs of most similar compounds. By contrast, replacement of the ethane-1,2-diaminoethane (en) non-leaving group with ammine (NH3) ligands (P1/P2 vs. P3/P4) enhances the cytotoxicity of hybrids containing acridines A5–A7. For several of the less active derivatives introduction of rac-1,3-diaminopropan-2-ol (pn2-OH) as a nonleaving group (in P5 and P6) resulted in improved cancer cell kill (average cell viabilities in were 55% for P1/P2, 45% for P3/P4, and 42% for P5/P6, relative to control cells). Likewise, a plot of cell viabilities for the 10 types of hybrids defined by common acridine moieties () gives important clues about SAR in this library of compounds. In general, the derivatives containing ethylene groups in the acridine side chain performed better than those containing extended propylene chains, based on average cell viabilities of 15% and 30% calculated for the pairs A1/A3 and A2/A4, respectively. Derivatives A6 and A7, which contain extended, carboxylic acid-modified side chains that only differ in the positioning of the secondary amino function () produce an average reduction in cell viability of merely 50% while showing similar activity profiles. Extension of the side chains to contain azide functionalities (A8–A10) follows the same trend and leads to a further decrease in cytotoxicity of the corresponding hybrids, which form a cluster of highly similar profiles (). On the basis of mean cell viabilities calculated across the entire set of 60 library members, hybrids containing A1, A3, and P6 resulted in the strongest cell kill effect ().
Figure 2 Biological activity profiles for the 60 compounds based on the viabilities of treated cells relative to untreated control determined in a colorimetric cell proliferation assay. The test compounds are sorted and color-coded by common platinum moieties (more ...)
Relative potencies of acridine (A) and platinum (B) fragments as building blocks in hybrid agents expressed as ± % deviations from the mean cell viability (M.V.) determined across the entire set of 60 library members.
To assess the utility of the library screen as a tool for target compound identification, a structurally diverse subset of four analogues of interest was resynthesized and tested in NCIH460 cells. IC50
values were calculated from the corresponding dose–response curves (see in the Supporting Information
) and are summarized along with data acquired previously for the parent compounds in . Of the hybrids chosen, the azide-functionalized compound P1-A8
required the highest concentration (110 ± 8 nM) to inhibit cell proliferation by 50%, consistent with its relatively poor performance in the prescreen. (It should be noted, however, that this derivative is still an order of magnitude more potent than cisplatin in this cell line.9
) Compounds that reduced cell viability to levels of ≤20% of control in the high-throughput assay resulted in significantly lower IC50
values. The hydroxy-modified hybrids P4-A3
showed the highest cell-kill potential with the latter reaching cytotoxicity levels in the low-nanomolar concentration range similar to the unmodified platinum–acridines P1-A1
Summary of structural elements and biological activity for selected library members.
The pre-screening method provides insight into the relative importance of the two functionally interdependent components, based on the similarity of activity profiles and clustering of individual library members. Because the method is based on testing reaction mixtures without purification steps at an arbitrarily fixed concentration of test compound, the cell viabilities extracted from it do not correlate with IC50
values, which have to be calculated from the corresponding drug–response curves. Potential complications in evaluating the biological activity of library members may arise in cases in which incomplete conversion of the building blocks to hybrid is observed. Under such circumstances, unreacted platinum or acridine precursors may contribute to the cell kill observed in the pre-screen. Furthermore, synergistic effects between multiple components, which might lead to enhanced cell kill, cannot be completely ruled out. Generally these mixtures resulted in poor inhibition of cancer cell proliferation, indicating that both the precursors and the hybrid agent were only marginally cytotoxic. However, in some cases, such as P3-A7
, which showed only 50% conversion but reduced cell viability by more than 70%, it had to be confirmed that the hybrid and not unreacted precursor caused the cell kill, which proved to be the case (see the Supporting Information
). Finally, in addition to demonstrating the feasibility of our library design and its validity as a prescreening tool, we have identified promising hydroxyl-modified hybrids P4-A3
as potential “warheads” for the desired application of targeted prodrug delivery.