2.2. Ibuprofen
Neumann et al. designed two Pt(IV) prodrugs with two moieties of indomethacin or ibuprofen in the axial position [
21] (
Figure 3). Prodrugs
2 and
3 were synthesized by reacting the corresponding acyl chloride with oxoplatin in acetone in the presence of pyridine.
The cyclic voltammetry assay showed a great difference between the reduction potentials of prodrugs
2 and
3 (−0.36 V and −0.68 V, respectively), which Neumann et al. suggested is probably due to the strong electron-withdrawing indole system and increased steric bulk of indomethacin. Thus, a weakened bond could probably facilitate ligand dissociation, resulting in poor stability. The stability of prodrugs
2 and
3 in a reducing environment was studied in the presence of ascorbic acid over three days. The release of axial ligands was detected by
1H NMR. The release of ibuprofen and prodrug decay was observed as additional peaks in
1H NMR and appeared in the –CH
3 group region at 1.38 ppm and the –CH
2– group region at 2.9 ppm. Approximately 40% of each drug was reduced during the experiment, which was evaluated by the authors as comparable with the clearance rate of platinum-based drugs from the body [
21].
The COX-inhibiting ability of prodrugs 2 and 3 towards purified COX-1 and COX-2 isoforms demonstrated that complex 2 was a highly selective inhibitor of COX-2, while complex 3 did not demonstrate any significant inhibiting activity on either isoform.
The antiproliferative activity of prodrugs
2 and
3 with cisplatin as a control was assessed on the colon cancer cell line HCT-116, the ovarian cancer cell line OVCAR3, the triple negative breast cancer cell line MDA-MB-231, and the head and neck squamous cell carcinoma cell line 1483 HNSCC via (3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide (MTT) assay (
Table 3). Prodrugs
2 and
3 both showed antiproliferative activity surpassing that of cispaltin. It is also worth noting the extremely high activity of
3 towards MDA-MB-231 cells (cisplatin-resistant triple negative breast tumor cell line), where it was about 400-fold more toxic than cisplatin [
21].
Neumann et al. also designed oxaliplatin-based prodrugs
4 and
5 with indomethacin and ibuprofen as axial ligands [
22]. Prodrugs
4 and
5 were obtained following the procedure utilized for synthesis of coordination compounds
2 and
3, by the reaction of oxaliplatin with the corresponding acid chloride [
21] (
Figure 4)
.Cyclic voltammetry was used to evaluate how easily prodrugs
4 and
5 could be reduced, since both too high and too low reduction potentials are detrimental to the activity of the complexes [
32,
33]. The reduction potentials obtained by CV experiments were compared to those obtained for cispaltin-based prodrugs
2 and
3 [
21]. Contrary to the reduction potentials of prodrugs
2 and
3, in the case of prodrugs
4 and
5, no significant difference in reduction potential was observed (−0.52 V and −0.56 V, respectively).
The ability of prodrugs
4 and
5 to inhibit cyclooxygenases was studied on purified isoforms of ovine COX-1 and murine COX-2. Neither of the isoforms were inhibited by ibuprofen-containing complexes
3 or
5. However, while prodrug
2 with indomethacin in the axial position acts as a selective COX-2 inhibitor, its oxaliplatin analogue
4 demonstrated selectivity towards COX-1 and no activity against COX-2. Docking studies for prodrugs
2 and
4 were carried out to gain insight into the differences in binding of prodrugs
2 and
4 to COX-2. The results suggest that compound
4 cannot bind to COX-2 due to the constricted entrance in the COX-2 active site, which prevents the bulky oxaliplatin equatorial ligands of
4 from accessing the enzyme. The high inhibitory activity of prodrug
2 towards the COX-2 isoform is attributed to the additional activity of the second indomethacin moiety, while only one moiety of indomethacin in prodrug
4 interacts with the enzyme active site [
22].
The cytotoxicity of complexes
4 and
5 was evaluated by MTT assay on HCT 116 (colorectal adenocarcinoma) and MDA-MB-231 (triple negative breast adenocarcinoma) cells (
Table 4). Both prodrugs
4 and
5 showed sub-micromolar activity towards MDA-MB-23 cells, while prodrug
5 demonstrated much lower IC
50 values than prodrug
4 on HCT-116 cells (0.31 µM and 1.3 µM, respectively). However, both complexes
4 and
5 were less potent than cisplatin-based prodrug
3 with ibuprofen axial ligands. Cellular accumulation of prodrugs
2–5 was studied via ICP-MS on HCT-116 and MDA-MB-231 cell lines (
Table 5). Interestingly, no direct correlation between cytotoxicity and intracellular accumulation of prodrugs
2–5 was found, as intracellular platinum accumulation in both HCT-116 and MDA-MB-231 cells for the most potent drug (
3) was lower than for its less potent oxaliplatin analogue (
5) and the cisplatin–indomethacin complex (
2). The trend in platinum accumulation cannot be solely attributed to the lipophilicity of prodrugs
2–5; indomethacin is more lipophilic than ibuprofen (logP values are 4.27 and 3.5, respectively), however, no correlation between axial ligands and cellular uptake of prodrugs
2–5 was observed [
22].
Curci et al. designed kiteplatin-based Pt(IV) prodrug
6 with two ibuprofen moieties as axial ligands [
23]. The prodrug was obtained by the acylation of oxidized kiteplatin by the acid chloride of ibuprofen (
Figure 5). The structure of the resulting complex was confirmed by thorough analysis of multiple NMR experiments, including 2D COSY spectrum and [
1H-
13C]-HSQC 2D NMR.
195Pt NMR showed the presence a single peak, confirming the formation of only one platinum complex.
To determine the stability of prodrug
6, the CV electrochemical curves were recorded on a glassy carbon electrode. The determined reduction potential was found to be −0.93 V and is compatible to the potentials of similar platinum(IV) coordination compounds [
34]. The cytotoxicity of the compound
6 was studied on non-COX-expressing colorectal carcinoma cell lines. Prodrug
6 showed submicromolar IC
50 values, which was up to 42-fold higher than platinum(II) drugs in the assay (
Table 6).
2.4. Naproxen
Ravera et al. synthesized platinum(IV) prodrugs
8 and
9 based on cisplatin with two NSAIDs, naproxen and ketoprofen, in the axial positions (
Figure 7) [
25]. As a reference compound, asplatin
1 was chosen and synthesized as well. Asplatin
1 was prepared as described by Pathak et al. [
20]. Prodrugs
8 and
9 were prepared by the acylation of [Pt(NH
3)
2(Cl)
2(OH)(OAc)] by the corresponding acid chloride.
The lipophilicities of all three Pt(IV) prodrugs
1,
8 and
9 were evaluated using RP-HPLC (
Table 8). The logarithm of the RP-HPLC capacity factor k’ usually correlates with the octanol/water partition coefficient [
35]. The obtained log k’ values for prodrugs
1,
8 and
9 follows the same trend as for their corresponding NSAIDs. Asplatin
1 was found to be the least lipophilic, while
9 was the most lipophilic coordinating compound.
The cytotoxicity of compounds
1, 8 and
9 was evaluated via MTT assay using both cyclooxygenase (COX)-expressing and non-COX-expressing cell lines (
Table 8).
The results of the MTT assay demonstrated the significant antiproliferative activity of coordination compounds
8 and
9, which were up to 13-fold more toxic than asplatin
1 and up to 20-fold more toxic than cisplatin. No correlation between COX-2 expression and cytotoxicity was found. Based on the results of the MTT assay, lipophilicity can be assumed to be the main factor for prodrug cytotoxic activity, as the most lipophilic complex
9 is the most toxic, while the least lipophilic compound (cisplatin) has the highest IC
50 values on nearly all cell lines [
25].
In order to confirm the relationship between cytotoxicity and lipophilicity, the accumulation ratio (AR) was evaluated as the ratio between intra- and extracellular platinum concentration. Cells were incubated with 10 µM of cisplatin and prodrugs
1,
8 and
9 for 4 h. The most lipophilic prodrugs
8 and
9 demonstrated higher ARs than the less lipophilic cisplatin and asplatin
1 with AR values of 14. AR values of platinum(IV) prodrugs
8 and
9 were 14 and 12, respectively, while cisplatin and Platin-A
1 demonstrated AR values of 2 and 1, respectively. The presence of lipophilic moieties in axial positions enhances the ability to penetrate the cellular membrane, leading to increased platinum uptake and, consequently, antiproliferative activity [
36].
To confirm the assumption that the mechanism of the toxicity of prodrugs
8 and
9 is COX-independent, a RT-qPCR analysis was carried out. Two cell lines, one with high COX-2 expression (A-549) and one with low COX-2 expression (HCT-116), were chosen. Five genes were chosen to study the influence of prodrugs
8,
9, cisplatin, naproxen, and ketoprofen: Bcl-2 family genes that regulate apoptosis, COX-2, and NSAID-activated gene NAG-1. The pro-apoptotic proteins BAD (BCL2 associated agonist of cell death) and BAX (Bcl-2-associated X protein) were upregulated in the presence of all three platinum compounds in both cell lines, while the anti-apoptotic gene BCL-2 was downregulated. There is evidence that BAX upregulation promotes apoptotic activity through caspase activation [
37]. COX-2 expression was upregulated by all compounds (
1,
8,
9, cisplatin, naproxen and ketoprofen) on the low COX-expressing cells HCT-116. The NAG-1 gene, which is involved in antiproliferative activity [
38], was expressed by prodrugs
8,
9 and cisplatin on both cell lines, including HCT-116, in which little to no COX-2 activity was observed.
To sum up, the antiproliferative activity of platinum(IV) prodrugs 8 and 9 was shown to occur through a COX-independent pathway, with lipophilicity being the key factor determining the efficiency of these compounds.
In another study devoted to the synthesis of Pt(IV) prodrugs with naproxen in axial positions, Tolan et al. designed six platinum–naproxen prodrugs derived from three Food and Drug Administration (FDA)-approved platinum(II) drugs [
26] (
Figure 8). Prodrugs
10–12 with a cisplatin, carboplatin and oxaliplatin core, respectively, were obtained by the reaction of oxoplatin with the N-hydroxysuccinimide (NHS)-ester of naproxen. Platinum prodrugs
13–16 with benzoic acid, succinic acid, glutaric acid and moieties, respectively, were synthesized by the reaction of complex
10 with the anhydride of the corresponding acid. Such an approach to the design of unsymmetrical platinum(IV) prodrugs allowed these authors to finely tune the properties of the resulting complex [
39]. The second axial ligand of the platinum(IV) prodrugs
13–15 was varied to alter the resulting lipophilicities of the platinum(IV) prodrugs.
The antiproliferative activity of prodrugs
10–15 was evaluated by MTT assay on the breast cancer cell line MCF-7 and the triple negative breast adenocarcinoma cell line MDA-MB-231 (
Table 8). The cytotoxicity of all prodrugs on the MCF-7 cell line was 1.5- to 2-fold higher than that of cisplatin. The results of the MTT assay indicate an 11- to 30-fold difference between the IC
50 values of cisplatin and compounds
10–15. It is worth noting that prodrug
13, with the most lipophilic benzoate ligand in the axial position, showed the lowest IC
50 values among all prodrugs tested (
10–15) [
26].
Cell death mode was assessed by fluorescent staining of MCF-7 and MDA-MB-231 cells incubated with the most active Pt(IV) prodrug 13. The results obtained indicate that prodrug 13 induces necrosis and apoptosis (early and late, combined) of 30.56% and 22.68% cells, respectively, in MCF-7 cells, and 12.98% and 36.21%, respectively, in MDA-MB-231 cells.
Another series of naproxen-containing platinum(IV) prodrugs was obtained by Chen et al. [
27] (
Figure 9). The focus of this study was the combined action of naproxen as an inhibitor of both cyclooxygenase-2 (COX-2) and matrix metalloproteinases (MMPs) and the impact of COX and MMP inhibition on the cytotoxic activity of Pt(IV) prodrugs.
The antiproliferative activity of complexes
16–
20 was evaluated by MTT assay on four malignant cell lines: A549, A549R (cisplatin-sensitive and cisplatin-resistant lung carcinoma, respectively), SKOV-3 (ovarian cancer), and CT-26 (colon cancer) in comparison with LO-2 (a human normal liver cell line) (
Table 9). Prodrugs
19 and
20, with two naproxen moieties in axial positions, showed lesser activity than the corresponding mono-substituted complexes
17 and
18, respectively. The selectivity index (SI) was defined as the ratio of IC
50 values obtained on the normal cell line LO-2 to the average of the IC
50 values obtained on tumor cells. Cisplatin and cisplatin-based prodrug
16 showed the worst SIs of 0.5 and 0.2, respectively, while the best SI values of 1.0 and 1.5 were demonstrated by the dual-naproxen complexes
18 and
19, respectively. The most potent compound,
17, demonstrated higher activity than cisplatin on the cisplatin-resistant cell line A549R [
27].
Overexpressed levels of matrix metallopeptidase-9 (MMP-9) are associated with cancer invasion, metastasis, and inflammation In vivo [
40]. The MMP-9 inhibitory activity of prodrug
17 was evaluated by immunohistochemical staining using slices of CT-26 mice tumors in comparison with the effect of oxaliplatin and saline. Compound
17 showed significant inhibition of MMP-9 expression (6.8%), while oxaliplatin inhibition level was 8.1%, in accordance with the literature data [
41]. The observed difference in inhibitory activity is associated with the presence of the naproxen moiety in prodrug
17. However, despite high inhibitory activity towards MMP-9 expression, prodrug
17 showed low COX-inhibiting ability.
The therapeutic efficacy of prodrug 17 was assessed In vivo using a CT-26 (colon cancer) tumor model. The inhibition of tumor growth after treatment with prodrug 17 was at the level of cisplatin and oxaliplatin (317 ± 119 mm3, 390 ± 162 mm3, and 477 ± 223 mm3, respectively).
Recently, Jin et al. described two cisplatin-based naproxen-containing platinum(IV) prodrugs [
28]. Two highly potent cisplatin prodrugs with one (NP,
10) or two (DNP,
21) naproxen moieties in the axial position were obtained (
Figure 10). Prodrug
10 was already described by Tolan et al. [
26] and synthesized following a similar procedure, while compound
21 was prepared by the reaction of oxoplatin with an excess of naproxen in the presence of 2 -(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) and Et
3N in N,N-dimethylformamide (DMF).
Both prodrugs
10 and
21 demonstrated outstanding antiproliferative activity towards the breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-435 after 48 hours of incubation (
Table 10). For prodrug
21, IC
50 values varied from 0.34 to 0.17 µM, which is up to 187-fold more than that of cisplatin, while prodrug
10 showed IC
50 values from 1.11 to 0.4 µM. It is worth noting that the IC
50 values obtained by Jin et al. for prodrug
10 differ significantly from the data reported by Tolan et al.; in particular, for the MCF-7 breast cancer cell line, IC
50 values for prodrug
10 in the study by Jin et al. are 26-fold higher, and for the MDA-MB-231 triple negative breast adenocarcinoma cell line, IC
50 values are 21-fold higher than in the paper by Tolan et al. (
Table 8) [
28].
The intracellular accumulation/distribution of prodrugs 10, 21, and cisplatin was assessed by ICP-MS assay in MCF-7 cells after incubation with 0.2 µM of each complex for 24 hours. The overall platinum content was 65- and 11-fold higher for cells incubated with 21 and 10, respectively, than for cells incubated with cisplatin. However, less than 5% of platinum accumulated in the nucleus of cells incubated with both prodrugs, as most of the platinum was found in the cytosol and cell membrane.
The stability of prodrugs
10 and
21 in a reducing environment was assessed in a model solution in the presence of ascorbic acid or glutathione. While complex
10 showed complete degradation after 3 hours, complex
21 was still stable after 72 hours of incubation. An additional experiment in solution in the presence of 5′-GMP was conducted to verify the ability of prodrug
21 to form covalent adducts with DNA bases; no Pt-GMP or Pt-GMP
2 were detected by ESI-MS after 6 days of incubation. This led Jin et al. to the assumption that complex
21 acts not as a prodrug, but as a whole complex that binds to DNA in a non-covalent manner [
28].
Cell cycle arrest, as studied by flow cytometry, showed that prodrug
21 arrested the cell cycle of MCF-7 cells mostly in the S phase (87% at 0.2 µM incubation), while prodrug
10 arrested the cell cycle at the G2 and S phases. Further studies of prodrug
21’s influence on the inflammatory response showed that it can greatly suppress COX-2 and programmed death-ligand 1 (PD-L1) expression in breast cancer cells, thus leading to suppressed tumor evasion from the immune system [
42]. The expression of interleukins IL-1β and IL-6, which are crucial for the formation of an inflammatory response [
43], was also greatly reduced by prodrug
21.
The antitumor activity of both prodrugs was studied In vivo using mouse xenografts bearing MDA-MB-231 tumor cells. After therapy with 1.5 mg/kg of prodrug
21 for 15 days, the tumor volume was only 66.35 ± 26.07 mm
3 (92.8% tumor growth inhibition), while after treatment with the same dose of cisplatin and saline, this value was 660 ± 68 and 926 ± 71 mm
3, respectively. Prodrug
10 was less efficient than prodrug
21 (75.5% tumor growth inhibition), but still inhibited tumor growth better than cisplatin (227 ± 71 and 660 ± 68 mm
3 tumor volume, respectively). No significant change in body weight was observed for the mice in all series [
28].
Thus, 21 is a highly potent platinum(IV) prodrug capable of significantly suppressing tumor growth and inflammatory response.
2.5. Etodolac, Sulindac, and Carprofen
Three cisplatin-based platinum(IV) prodrugs with the FDA-approved NSAIDs etodolac, sulindac, and carprofen (
22–24) were designed by Song et al. (
Figure 11) [
29]. Compounds were synthesized by the reaction of oxoplatin with the corresponding NSAID in the presence of TBTU and triethylamine in DMF. The cytotoxicity of prodrugs
22–24 was assessed by MTT assay. IC
50 values of prodrug
22 were lower than the values of cisplatin on malignant cell lines MCF-7, A549 and HeLa, while being less toxic than cisplatin on the normal cell line MRC-5 (
Table 11). Complex
24 showed comparable results to cisplatin toxicity on MCF-7 and A549 cell lines, while being less toxic on the HeLa cell line. The most potent prodrug,
22, was 14-fold more active than cisplatin. Cellular accumulation in MCF-7 cells after 3 hours of incubation was assessed by ICP-MS assay, which indicated that the trend in antiproliferative activity correlated with the cellular uptake level, with prodrug
22 showing the highest level of platinum content.
Lipophilicity is considered the crucial factor for drug activity, so the logP values were obtained for cisplatin and the cisplatin-based prodrugs
22–24. The established optimum logP values for maximum drug bioavailability is reported to be in the range of 0–3 [
44,
45]. Among all three prodrugs (
22–
24), only one logP value was in the optimum range (prodrug
22) (
Table 11). Cisplatin and prodrug
24 showed lipophilicities lower than optimum, while prodrug
23 had a logP value higher than optimum. Thus, the obtained lipophilicity values correlate with trend in cytotoxicity and cellular accumulation.
Western blot analysis conducted on MCF-7 cells incubated with prodrug
22 showed the ability of the compound to downregulate the expression of COX-2 and MDM-2 enzymes in MCF-7 cells while inducing the expression of the pro-apoptotic genes Bax and p53. Wound healing and invasion assays were conducted to assess whether prodrug
22 is able to suppress the metastasis and invasion of tumor cells. The migration rate of MCF-7 cells incubated with complex
22 was significantly lower than the rate of control and cisplatin-treated cells (18.1%, 47.7%, and 39.9%, respectively) [
29].
The In vivo study of prodrug
22 on xenograft mice bearing MCF-7 tumors revealed the low systemic toxicity of prodrug
22, and its ability to suppress tumor growth was slightly stronger than cisplatin after 14 days of treatment (457 mm
3 and 570 mm
3, respectively). It is notable that the suppression of tumor growth in the case of prodrug
22 was accompanied with slightly reduced platinum content in the heart, liver, and lung than in the case of cisplatin, as assessed by ICP-MS analysis of harvested organs. The body weight of mice treated with prodrug
22 remained almost unchanged, while treatment with cisplatin led to a decrease in body weight by 2 g on average, from 19 to 17 g. Thus, despite suppressing tumor growth to a higher extent than cisplatin, prodrug
22 also induced significantly fewer toxic effects [
29].