Incorporation of Estrogen with chemo-therapeutic agents

Pioneering work of the following groups describing steroid-linked platinum(II) complexes are reported in 2009, Essigmann et al. from MIT reported modification of DNA damaging [Pt(ethylenediamine)Cl2] through a carbamate containing linker from the ethylenediamine to the steroid residue. (Figure 49) The ligand proved to have 28 % relative binding affinity (RBA) for the ER as compared to 17β-Estradiol. After covalent binding to a synthetic DNA duplex 16-mer, the affinity for the ER is still retained. [(Est-en)PtCl2] showed enhanced toxicity against the ER positive ovarian cancer cell line (CAOV3) than did the control compound. [(Est-en)PtCl2] was also more toxic to a ER positive breast cancer line (MCF-7) than to an ER negative line (MDA-MB231). These results indicate that both the presence of the estradiol moiety and the expression of the ER in target cells contribute to the enhanced activity.^[117]

Figure 49. Estrogen conjugated DNA damaging [(Est-en)PtCl2].^[117]

Bérubé and co-workers from Université du Québec reported the synthesis of three estradiol-linked platinum(II) complex analogs to cisplatin via 6-14 carbon atom chains. (Figure 50) The biological activity was evaluated against ER(+) MCF-7 and ER (-) MDA-MB-231 cell lines. Interestingly, their bioacitivity are more potent than cisplatin. The complex with n = 4 exhibited a promising cytotoxicity towards all the tested cancer cells, complex with n = 2 shows the least cytotoxicity. This may be due to the short linkage which causes additional steric hindrance around the 17-hydroxyl group of the steroid, which is necessary for binding to the ER.^[118]

Figure 50. Estradiol-linked Pt(II) complex via 6-14 carbon atom chains.^[118]

In 2007, Schobert from the University of Bayreuth reported several di-chloro(6-amino methyl nicotinate) platinum(II) estrogen conjugates. Their cellular accumulation and inhibitory effect have been investigated on ER positive and ER negative MCF-7 and MDA-MB-231 breast cancer cell lines.

The complex with 3-O-linked estrogen, distinctly inhibits the growth of MCF-7 cells. While the complex with 17-O-linked androgen against ER positive and ER negative breast cancer cell lines is less effective. Both complexes bind strongly to sex hormone-binding globulin. The results suggest that the accumulation of both complexes in ER(+) breast cancer cells is a receptor-mediated process.^[119] (Figure 51)

Figure 51. 3-O and 17-O linked Pt(II) estrogen conjugates.^[119] 

In 2006, Rodger from the University of Warwick reported an ethinylestradiol linked cationic platinum complexes, where the estrogen as the effective delivery group is linked to a terpyridine ligand via the ethinyl functionality. (Figure 52) Studies on ER positive MCF-7 breast cancer cells confirmed its binding ability to the ER.^[120]

Figure 52. Cationic Pt(II) ethinylestradiol conjugate. ^[120] 

3.3.3 Description of proposed research

We propose a synthetic plan as shown in Scheme 3. Starting with commercially available ethinylestradiol, its CuAAC reaction with azido functionalized salan Ti(IV)-bis-cheates will give the corresponding Ti(IV) complexes containing an estrogen molecule as a targeting group.

Scheme 3. Intended approach towards estrogen functionalized salan Ti(IV)-bis-chelates.

Work in this chapter is carried out in the following directions:

(1) Synthesis of azido functionalized “dipic” with ethylene glycol spacers of different lengths and their corresponding salan Ti(IV)-bis-chelates of type 19a-d.

(2) CuAAC reaction between azido salan Ti(IV)-bis-chelates with ethinylestradiol to give conjugates of type 59a-d.

(3) Stability study and bioevaluation.

3.3.4 Experiment and results

3.3.4.1 Synthesis of azido-PEG-dipic derivatives

The synthesis of glycol linkers begins with 2-chloroethan-1-ol 57a, by reaction with NaN3

according to the reported method.^[121] we obtained 2-azidoethan-1-ol 14a in 80 % yield. We then applied this method to diethylene glycol 57b, triethylene glycol 57c and tetraethylene glycol 57d, and the corresponding products 14b-d were obtained in good yields. (Table 12)

Table 12. Synthesis of azido-PEG linkers14a-d.

Entry n Product Yield^a (%)

1 1 2-azidoethan-1-ol 14a 80

2 2 2-(2-azidoethoxy)ethan-1-ol 14b 90

3 3 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol 14c 83

4 4 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethan-1-ol 14d 88

a Isolated yield.

Tosylation of 14a-14d afforded corresponding tosylated azido-PEG linkers 15a-15d. By reaction with 4-hydroxy dipic dimethyl ester 11, 4-azido-PEG dipic-dimethyl esters 16a-16d were obtained..

These compounds were directly converted to the corresponding acids 17a-d by hydrolysis in the presence of KOH in ethanol at 80 ^oC. (Table 13)

Table 13. Synthesis of "α-ω" azido-PEG-dipic derivatives 17a-d.

Entry n “dipic” derivatives Yield^a (%)

16 17

1 1 4-(2-azidoethoxy)-dipic a 67 a 83

2 2 4-(2-(2-azidoethoxy)ethoxy)-dipic b 66 b 95

3 3 4-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-dipic c 67 c 82 4 4 4-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethoxy)-dipic d 72 d 78

a Isolated yield.

3.3.4.2 Synthesis of the corresponding azido-PEG salan Ti(IV)-bis-chelates

Having those four dipic derivatives 17a-d at hand, we then synthesized the corresponding salan Ti(IV)-bis-chelates through ligand exchange reaction with salan Ti(IV)-isopropoxide intermediate 6f in anhydrous THF under N2 protection. Four azido functionalized salan Ti(IV)-bis-chelates 19a-d were obtained. (Table 14)

Table 14. Synthesis of azido-PEG salan Ti(IV)-bis-chelates 19a-d.

Entry n 19 Yield^a (%)

1 1 a 92

2 2 b 44

3 3 c 68

4 4 d 26

a Isolated yield.

3.3.4.3 CuAAC reaction studies

CuAAC reactions between 19a-d and ethinylestradiol 58 were carried out under the following conditions: CuSO4 (50 mol %), TBTA (5 mol %), L-ascorbic acid (1 equiv.), DCM/MeOH/H2O (6:

10: 3). The reaction was heated to 80 ^oC under N2 protection by conventional heating in an oil bath.

Reaction of Ti(IV) complex 19a proceeded smoothly and gave the estrogen functionalized Ti(IV) complex 59a in 40 % yield. Ti(IV) complexes with PEG linkers 19b and 19c also reacted with ethinylestradiol 58 efficiently and afforded corresponding Ti(IV) complexes 59b and 59c in moderate yields (Table 15, entry 2 and 3). However, the reaction between Ti(IV) complex containing tetra-ethyleneglycol linker 19d and 58 proceeded rather slow. After the reaction kept stirring for 5 days, complex 59d was isolated in 7 % yield. (Table 15)

Table 15. Synthesis of estrogen functionalized salan Ti(IV)-bis-chelates 59a-d via CuAAC approach.

a All reaction performed on a 0.05 mmol scale. ^b Isolated yield.

To further study the CuAAC approach of azido-PEG Ti(IV) complex 19d with ethinylestradiol 58, we optimized the reaction conditions by using microwave heating. The reaction was run under microwave irradiation for 4 h at 80 ^oC. Complex 59d was obtained in 24 % yield, whereas starting material 19d was recycled in 70 % yield. Prolongation of the reaction time resulted in the formation of degradation product, which complicate the work-up procedure. (Figure 53)

N

Figure 53. Synthesis of estrogen-PEG salan Ti(IV)-bis-chelate 59d under microwave irradiation.

The obtained complexes 59a-d were characterized by ¹H NMR, ¹³C NMR, IR, MS, HRMS and UV-Vis spectra. From the NMR spectra, the AB pattern signals of the salan “backbone” (NCH2CH2N and CarCH2) are visible by two doublets, besides, characteristic proton signals of the triazole were observed as singlets at around 8 ppm for both 59a-d (59a: 8.02 ppm, 59b: 7.95 ppm, 59c: 7.85 ppm, 59d: 7.82 ppm). In the ¹³C NMR, all carbon signals were observed. (For detailed data, see experimental section 5).

3.3.4.4 Stability studies

Stability test were carried out by time-resolved ¹H NMR spectroscopy under the following conditions: Ti(IV) complex 59a (15 µmol), 1,4-dinitrobenzene as internal reference (1 mg, 7.5 µmol, 0.5 equiv), D2O (0.24 ml, 1000 equiv), [D8]-THF (0.4 ml). ¹H NMR sample was measured at 37 ^oC and incubated at 37 ^oC. Data gathered by monitoring the decrease in isolated signals of the titanium-bound salan backbone and increase of new signals, eg. free salan. Integrals are normalized against the internal standard.

From the ¹H NMR spectra, complex 59a maintained its stability for at leatst 24 h at 37 ^oC under tested condition. (Figure 54) Even after two weeks, no degradation was detected. In the salan Ti(IV)-bis-chelate 59a-59d containing 2,4-dimethyl substitutions on the salan, the 2-methyl group efficiently shields the Ti(IV) center from attack by nucleophiles. This has already been proved by our previous report.^[5] The estrogen motif on the dipic does not have significant influence towards the complex stability.

Figure 54. Example of time resolved ¹H NMR spectra recorded during the hydrolysis study of 59a showing no sign of degradation even after two weeks of continues incubation. Signals marked A, B, C, D, E are Har of 1,4-dinitrobenzene, Har of complex, CarCH2 of the complex, D2O and [D8]-THF.

3.3.4.5 Bioevaluation

Complexes 59a-d were tested for their cytotoxicity against Hela S3 and Hep G2 cell lines, which are known to be ER negative. All complexes showed no cytotoxicities against these two cell lines which might result from the insufficient estrogen receptor expression of the cell lines. We are currently running the bioevaluation against two ER positive cell lines, i.e. human breast adenocarcinoma cell line MCF-7 and human colon adenocarcinoma grade II cell line HT-29.

3.3.5 Summary and outlook

In this part, we synthesized four estrogen functionalized salan Ti(IV)-bis-chelates linked via four different ethylene glycols spacers. Their structures were characterized by ¹H, ¹³C NMR, HRMS, MS, IR and UV-Vis spectroscopy (For detailed data, see experimental part). Hydrolysis studies in presence of 1000 equiv. D2O revealed that complex 59a exhibit excellent stability in aqueous media.

Preliminary bio-evaluation showed that all four estrogen functionalized Ti(IV)-bis-chelates 59a-d are nontoxic against Hela S3 and Hep G2 cell lines. Further bio tests against ER positive cell lines are currently under investigation. 

3.4 Cytotoxic salan Ti(IV)-bis-chelates modified by Sonogashira reaction