4-Amino-1,8-naphthalimide–ferrocene conjugates as potential multi-targeted anticancer and fluorescent cellular imaging agents

Abstract

Herein we present eight ferrocenyl 4-amino-1,8-naphthalimides. Designed as fluorescent logic gates for acidity and oxidisability, the molecules have been repurposed as anti-proliferation and cellular imaging agents. The compounds were studied in vitro against MCF-7 and K562 cancer cell lines by the MTT method. Compounds with protonable secondary amines tended to exhibit greater cytotoxicity than those with tertiary amines. Compounds with no measurable GI₅₀ values within a 24 hour time window, as well as at shorter exposure times, may be suitable as fluorescent cellular imaging probes.

Eight ferrocenyl 4-amino-1,8-naphthalimide logic gates for acidity and oxidisability are repurposed as anti-proliferation and cellular imaging agents against MCF-7 and K562 cancer cell lines.

Introduction

Cancer is a disease resulting from abnormal cellular proliferation and metastasis.¹ The disease is second only to cardiovascular disease in terms of the annual worldwide mortality rate, which is currently in excess of 10 million deaths per year,² and it is anticipated that the rate of new cases will only continue to rise.³ Hence, the need for further investment in the research and development of more effective and less toxic anticancer drugs. The prevailing wisdom today for complex diseases, such as cancer, is the innovation of medicines with multiple modes of action for various biological targets, rather than drugs selectivity for a single, specific target.⁴ One way of achieving this aim is the hybridisation of two or more structurally and/or mechanistically distinct pharmacophores into a single molecule.⁵ The combination of an organometallic unit, such as ferrocene, with a bioactive organic drug scaffold has been a promising strategy.⁶ The poster example is the antimalarial and anticancer drug, ferroquine (FQ, SSR97193, Fig. 1), a chloroquine–ferrocene conjugate with a basic alkylamine.⁷ Ferroquine was recently demonstrated to reduce the viability of prostate, pancreatic and breast cancer cells with IC₅₀ values in the low micromolar range.⁸ The molecule retains the chemical composite of chloroquine, but incorporates ferrocene within the alkyl side chain. Besides the reversible redox character of ferrocene, the intrinsic three-dimensional shape of the organometallic unit is hypothesised to facilitate binding in hydrophobic regions of biological receptors and enzymes.⁸ Ferrocifen (Fig. 1), a derivative of tamoxifen (Nolvadex™), is a promising drug candidate in preclinical trials for the treatment of hormone dependent breast cancers, such as MCF-7.⁹ It beneficially exhibits negligible side effects on non-cancerous cells contrary to classic Pt-based organometallic agents such as cisplatin, carboplatin and oxaliplatin, thus offering a better therapeutic margin of safety.¹⁰ The mechanism of action is believed to involve binding of estrogen receptors ER_α and ER_β.

Fig. 1. The chemical structures of amonafide, ferroquine and ferrocifen.

Naphthalimides (benz[de]isoquinolin-1,3-diones) are another class of molecule with proven anti-proliferation activity.¹¹ Two promising cancer drug candidates, mitonafide and amonafide (Fig. 1) progressed to phase II breast cancer clinical trials, but were halted due to unacceptable toxicity risks (i.e. the nitro NO₂ and amino NH₂ groups undergo biochemistry to give an acetylated product).¹² Amonafide (Fig. 1) is a topoisomerase (Topo) II inhibitor and DNA intercalator and induces apoptosis by blocking the binding of the Topo enzyme to double strand DNA.¹³ Another hopeful candidate, elinafide (LU79553), a symmetrical dimeric bis-naphthalimide with a cationic linker¹⁴ also acts as a topoisomerase inhibitor, and progressed to phase II for the treatment of ovarian cancer, but was discontinued due to neuromuscular dose-limiting toxicity.¹⁵ An advantageous property of naphthalimides is resistance to P-glycoproteins, a trans-membrane enzyme that binds and transports drugs to the outside of cancer cells.¹⁶ Most antineoplastics such as doxorubicin, vinblastine, gemcitabine, etoposide and paclitaxel are pumped out of the cancer cell by glycoproteins.

Gellerman has proposed molecular chimera of amonafide with other pharmacophores, such as quinones (a reductive moiety).¹⁷ We hypothesised that the combination of a 4-amino-1,8-naphthalimide with ferrocene (an oxidative moiety) may similarly yield a synergetic effect. We postulated that the electron-rich ferrocene unit could preferentially increase free radical production, and contribute to greater cytotoxicity to cancer cells (and ideally not to healthy cells) by redox-promoted DNA cleavage while the 4-amino-1,8-naphthalimide intercalates with DNA and/or interferes with the Topo enzyme. Within the literature there are several recent reviews on naphthalimide-based anticancer agents,¹¹ but only studies by Huang focus on ferrocene-appended naphthalimides.¹⁸

We have been engineering so-called Pourbaix sensors as fluorescent logic gates for sensing acidic and oxidising conditioning (Fig. 2).¹⁹ The premise for their design is based on the principles of photoinduced electron transfer (PET) systems.²⁰ The original model compromised of a fluorophore–spacer–receptor format.²¹ We have extended it to a fluorophore–spacer–electron-donor format, and amalgamated these two complementary designs.¹⁹ Ferrocene is our favorite electron-donor, and various secondary and tertiary amines as proton receptors, for quenching the emission excited state in the absence of oxidant and/or acid.

Fig. 2. The chemical structures of eight ferrocenyl–naphthalimide conjugates tested in vitro with K562 and MCF-7 cells.

Herein we have investigated the possibility of using compounds 1–8 in oncology. We report the in vitro antiproliferation activity of these eight ferrocenyl 4-amino-1,8-naphthalimide conjugates against two human cancer cells lines, K562 (a myelogenous leukemia) and MCF-7 (human breast cancer) as well as preliminary fluorescence cellular imaging. In this new application, the amine receptor sites are amendable to ensuring water solubility on protonation and supramolecular electrostatic interactions via intercalation with DNA.

Results and discussion

Compounds 3 and 4 are new chemical entities, while the other six molecules have been reported by us.^22–25 Compounds 1 and 2 were synthesised using a multi-step procedure by condensation of methylamine with the naphthalimide anhydride precursor followed by amination with piperazine or N-methylethylenediamine^22,23 and reductive amination with ferrocenecarboxaldehyde using sodium triacetoxyborohydride. Compounds 4–8 were prepared by condensation of ferrocenylmethylamine with 4-bromo-1,8-naphthalic anhydride to yield N-ferrocenyl–4-bromo-1,8-naphthalimide, and subsequently reacted with the commercially available amine by nucleophilic aromatic substitution.^24,25 Compound 3 was prepared from 4-bromo-1,8-naphthalic anhydride in the presence of excess amine followed by reductive amination with ferrocenecarboxaldehyde. The products were purified by chromatography on silica gel and/or recrystallised and charactersied by ¹H and ¹³C NMR, IR and HRMS.^22–25 Specific details for compounds 3 and 4 are available in the ESI† (Fig. S1–S5).

The compounds were screened for their bioactivity on human cancer cell lines K562 and MCF-7 at exposure time intervals of 24, 48 and 72 hours using a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay²⁶ (Table S1, ESI†). Optical density readings at 562 nm were obtained from a microplate reader as a measure of the amount of formazan formed, which is equivalent to the amount of mitochondrial dehydrogenase activity and cell viability. The optical density was plotted against the concentration of each compound as a percentage of the control, and from the dose–response curves, the 50% growth inhibition (GI₅₀) of the cell population was determined conditional to the curve decreasing below the 50% threshold, otherwise no quantitative result is given in Table 1.

In vitro average GI₅₀ (50% growth inhibition) values ± SD for 1–10 in K562 and MCF-7 human cancer cells^a,b.

Compound	Clog P	K562 GI50/μM			MCF-7 GI50/μM
		24 h	48 h	72 h	24 h	48 h	72 h
1	4.30	—	—	68 ± 3	—	40 ± 3	15 ± 1
2	4.31	—	80 ± 5	30 ± 2	—	73 ± 5	32 ± 2
3	5.93	98 ± 4	40 ± 3	30 ± 3	90 ± 5	40 ± 3	28 ± 1
4	3.63	62 ± 3	35 ± 2	18 ± 1	—	—	75 ± 5
5	4.35	—	100 ± 8	60 ± 2	—	40 ± 4	9.0 ± 0.4
6	4.09	75 ± 5	70 ± 2	58 ± 4	13 ± 1	8.0 ± 0.6	4.8 ± 0.5
7	4.22	65 ± 6	50 ± 4	45 ± 3	52 ± 3	42 ± 3	39 ± 1
8	4.23	60 ± 3	46 ± 3	38 ± 1	—	60 ± 5	55 ± 3
9	2.21	15 ± 1	9.8 ± 0.9	7.8 ± 0.8	97 ± 7	48 ± 2	2.2 ± 1.8
10	2.68	60 ± 3	38 ± 1	18 ± 1	65 ± 5	51 ± 4	46 ± 3

^aValues denoted with (−) signify the GI₅₀ was not reached within the incubation period or concentration range tested.

^bGI₅₀ values are based on a MTT assay by measuring the absorbance at 562 nm from three independent trials. Details given in ref. 26. Standard deviation (SD) error as a range ± 10%. Log P values of 9 and 10 are 1.10 and 1.65. Amonafide, ferroquine and ferrocifen molecular weights and Clog P values for 283, 433, 424 and 1.95, 4.19, 5.11. Clog P and log P values from Chemdraw version 12.0.2.1076.

With the K562 cell line, the most cytotoxic compound of the series is 4. It has a methylethylenediamine ligand at the 4-amino-1,8-naphthalimide position. Compounds 2, 3, 7, 8 are characterised by a flexible ethylene linker either ending with a ferrocene moiety, as with 2 and 3, or a tertiary amine, as with 7 and 8. The two compounds with a flexible alkyl chain attached to ferrocene 2 and 3 have comparable GI₅₀ values. The candidates with the piperazine moiety 1, 5 and 6 are the least cytotoxic suggesting conformational mobility of the ligand at the 4-position is advantageous. For 1, 2 and 5 it was not possible to calculate a GI₅₀ after 24 hours of incubation, although results were obtained after 72 hours. Compound 5 is the least cytotoxic.²⁵

With the MCF-7 cell line we observe a contrasting trend. The most cytotoxic compound of the series after 24 hours is 6 with a GI₅₀ of 13 μM, which is comparable to Amonafide (LC₅₀ ∼ 12 μM).²⁷ After 72 hours the GI₅₀ of 5 and 6 are 9 μM and 5 μM, respectively. These molecules are endowed with a piperazine ring at the 4-amino position. Compounds 1 and 3 also have GI₅₀ values below the 30 μM cytotoxicity threshold.²⁸ Compounds 2 and 7 with the flexible dimethylethylenediamine functionality are less toxic.

Compounds 2 and 3 demonstrate anti-proliferation for both cells lines with a GI₅₀ of 30 μM after 72 hours of exposure. In comparison, model compounds 9 and 10 with a methyl substituent at the imide nitrogen atom exhibited GI₅₀ of 15 μM after 24 hours and 7 μM after 72 hours of exposure in K562 cells (Fig. 3). The 4-fold difference in the GI values indicates there is clearly a difference in the cytotoxicity, which could be due to either the absence of the ferrocene moieties, and/or the higher basicity of the secondary amine groups and improved aqueous solubility properties.

Fig. 3. The chemical structures of model 4-aminonaphthalimide compounds tested in vitro with K562 and MCF-7 cells.

A comparison of 6 with model 10 (devoid of the ferrocene moiety) reveals an interesting contrast in the cytotoxicity properties towards MCF-7. Ferrocene-containing 6 has a GI₅₀ of 13 μM after 24 h to 4.8 μM at 72 h, while 10 has a GI₅₀ of 65 μM to 46 μM with a 5-fold and 10-fold cytotoxicity effect. In contrast in K562 cells, 6 has a GI₅₀ of 75 μM and 58 μM versus 60 μM and 18 μM for the model, the later a 3-fold enhancement. Clearly, the cytotoxicity increased due to the presence of the ferrocene moiety.

With cytotoxicity data from the MTT assay in hand, we investigated the possibility of using our Pourbaix sensors as cellular imaging stains. The data in Table 1 clearly shows that compounds 1, 2 and 5 are not cytotoxic to either K562 or MCF-7 cells within a 24 hour incubation period as a GI₅₀ was not reached over the examined concentration range. The best imaging results were obtained with MCF-7 cells. As depicted in Fig. 4, a vibrant green emission was observed in the nucleus of the live cells within minutes, confirming the internalisation and uptake of 2 into the MCF-7 cells.

Fig. 4. Fluorescent images of 2 (11 μM) in MCF-7 cells after 24 hours incubation period. Scale bar of 50 μM. Images were captured on an EVOS FL fluorescent microscope.

Conclusions

In summary, eight ferrocene-containing 4-amino-1,8-naphthalimide compounds were tested in vitro for anticancer activity against two prominent human cell cancer lines. Those compounds that did not reach GI₅₀ values within a 24 hour time window, as well as those used at shorter exposure times, may be suitable as fluorescent cellular imaging probes. A notably exception is 6 with a piperazine moiety, which is a potential lead drug candidate for human breast cancer. The greatest anti-proliferation activity with K562 cells was observed with 4 and non-ferrocene models 9 and 10, which possess an easily protonatable piperazine or methylethylenediamine ligand.

Over thirty years ago de Silva formalised the fluorophore–spacer–receptor format as the design basis for fluorescent pH indicators.²¹ Commercially available fluorescent probes such as LysoSensor Green DND-189²⁹ and LysoSensor Blue DND-167³⁰ are readily available as a result.³¹ Now we have explored the possibility of using ferrocene-appended naphthalimides,³² originally designed as fluorescent PET-based logic gates for acidity (pH) and oxidisability (pE), as oncology and living cell imaging tools.³³ As such, this study is the first report of the practical utilisation of Pourbaix sensors in the field of medicinal chemistry. The potential impact of ferrocene–aminonaphthalimides as anticancer drug candidates remains unknown and merits further exploration. Studies are currently underway investigating the intracellular mode of action.

Author contributions

Conceptualization (DCM), investigation (ADJ), methodology (JAB), visualization (ADJ), supervision (DCM, JAB), writing original drafts (DCM, ADJ), writing & editing (DCM, JAB).

Conflicts of interest

There are no conflicts of interest to declare.