Azide-Alkyne Cycloaddition En Route to 1H-1,2,3-Triazole-Tethered Isatin-Ferrocene, Ferrocenylmethoxy-Isatin, and Isatin-Ferrocenylchalcone Conjugates: Synthesis and Antiproliferative Evaluation.

Diverse series of isatin-ferrocene conjugates were synthesized via Cu-promoted azide-alkyne cycloaddition reaction with an aim of probing their antiproliferative structure-activity relationship against MCF-7 (estrogen receptor positive) and MDA-MB-231 (triple negative) cell lines. Among the synthesized conjugates, isatin-ferrocenes proved to be more potent against MCF-7, whereas ferrocenylmethoxy-isatins exhibited activity against MDA-MB-231 cell lines. However, the introduction of chalcone moiety among these hybrids resulted in the complete loss of activity against the tested cell lines, as evident by isatin-ferrocenylchalcones. The conjugates 5a and 9c proved to be the most potent among the series against MCF-7 and MDA-MB-213 cell lines, exhibiting IC50 values of 31.62 and 20.26 μM, respectively.

Introduction

Cancer, characterized by abnormal growth of cells, is the second most fatal disease in the world. The use of tobacco, over body weight, gene mutation, and hormone and immune conditions are few driving forces responsible for the emergence of this deadly disease.[1] Breast cancer is considered one of the most common cancers with a high rate of mortality.[2] Formation of lumps in the breast tissue is the prevalent symptom of breast cancer, while less common symptoms may include persistent changes in the breast such as thickness, swelling, distortion, and spontaneous nipple discharge.[1] In United States, 30% of women are expected to develop breast cancer over their lifetime, with an expected 40 610 breast cancer-related deaths in 2017.[3] The efficacy of current chemotherapeutics against breast cancer is low, and significant improvements are achieved only in 30–40% of the patients.[4] Worldwide, medicinal chemists are striving to discover novel, effectual, and less toxic antibreast cancer agents which can expand the success spectrum among cancer cases.

Isatin (1H-indole-2,3-dione) is a privileged scaffold present endogenously in both human and other mammalian tissues. The molecular architecture of isatin is indeed an ideal platform for carrying out structural modification and derivatization to probe its further biological potential.[5] Isatin derivatives have been reported to exhibit a range of biological properties such as anticancer,[6] antidepressant,[7] anticonvulsant,[8] antifungal,[9] antiHIV,[10] and antiinflammatory[11] activities. The Food and Drug Administration approval of SU11248 (SUTENT), a 5-fluoro-3-substituted isatin derivative for the treatment of advanced renal carcinoma and gastrointestinal stromal tumors, has established isatin as a potentially useful lead for anticancer drug development.[12] C-5- and C-6-substituted isatin analogues were selective monoamine oxidase B inhibitors, with 5-(4-phenylbutyl)isatin being 18 500-fold more potent than the parent molecule.[13] A number of reports have appeared lately on the anticancer activities of isatin conjugates, which proved them to be more efficient than cis-platin.[14] Tyrosine kinase inhibition, inhibition of cyclin-dependent kinases, and/or caspase inhibition are some pathways through which isatin has been reported to exhibit its anticancer potential.[15]

The application of bioorganometallic chemistry in medicine, especially via the inclusion of the ferrocene nucleus among organic frameworks, is a rapidly developing area of research.[15b] Numerous studies have suggested that the replacement of an aryl ring with a ferrocenyl moiety has improved the biological activities of the heterocyclic scaffolds or has fashioned new medicinal properties.[17] The most emblematic example of such a strategy could be of ferrocifen, obtained via replacement of one of the phenyl rings of tamoxifen with ferrocene, resulting in the development of the earliest organometallic selective estrogen receptor modulators. Ferrocifen displayed an uncommon feature of being antiproliferative against both hormone-dependent (MCF-7) and hormone-independent (MDA-MB-231) breast cancer cell lines. Such a dual mode of action underlines the importance of replacing the aryl ring with ferrocene because the purely organic form exhibited antiestrogenic activity only against MCF-7 cell lines.[18]

With an aim of discovering varied biologically relevant molecular conjugates,[19] the present manuscript is one of our endeavors toward targeting breast cancer which includes the synthesis of a series of 1H-1,2,3-triazole-linked isatin–ferrocene, ferrocenylmethoxy–isatin, and isatin–ferrocenyl–chalcone conjugates, as depicted in Figure . These molecules were evaluated for their antiproliferative activities against estrogen receptor positive (ER+) MCF-7 cells and triple negative (ER−) MDA-MB-231 cells. 1H-1,2,3-Triazole core has drawn considerable attention in the field of medicinal chemistry mainly because of its capability of forming H-bonds, which eventually improves its solubility and interaction with biomolecular targets. The stability toward metabolic degradation as well as varied biological activities make 1H-1,2,3-triazole a privileged scaffold which can be used as a linker in the molecular-hybridization approach.[20]

Figure 1

Designed 1H-1,2,3-triazole tethered isatin–ferrocene, ferrocenylmethoxy–isatin, and isatin–ferrocenyl–chalcone conjugates.

Results and Discussion

Synthetic methodology for obtaining the desired isatin–ferrocene conjugate 5 involved an initial base-promoted N-alkylation of 5-substituted isatin 1 with methyl iodide or benzyl bromide in dry dimethyl formamide to yield N-alkylated isatin 2 which was subsequently reacted with trimethylsilyl iodide to afford spiroindoline-oxirane-2-ones 3. Epoxide ring opening of 3 with sodium azide in an ethanol/water (90:10) mixture afforded 3-(azidomethyl)-3-hydroxy-indolin-2-one 4. Cu-promoted azide–alkyne cycloaddition of 4 with ethynyl ferrocene resulted in the formation of a crude product which was purified by column chromatography using (10:90) a methanol/chloroform mixture as the eluent to afford the desired 1H-1,2,3-triazole-tethered isatin–ferrocene conjugate 5 in good yields (Scheme ).

Scheme 1

Synthesis of 1H-1,2,3-Triazole-Linked Isatin–Ferrocene Conjugates 5a–h

Another precursor, viz., O-propargylated ferrocene methanol 8, required for ferrocenylmethoxy–isatin conjugates, was synthesized via NaBH4-promoted reduction of ferrocene carboxaldehyde 6 in dry tetrahydrofuran to afford ferrocene methanol 7 with subsequent O-propargylation using sodium hydride. Cu-promoted azide–alkyne cycloaddition reaction of 4 with O-propargylated-ferrocene methanol 8 led to the isolation of a crude solid, which was purified via column chromatography using a CHCl3/methanol (95:5) mixture as the eluent, to yield the desired conjugate 9 in good yields (Scheme ). The structure to the synthesized conjugate was assigned on the basis of spectral studies and analytical evidence. The compound, for example 9a, exhibited a molecular ion peak at 351.1457 in its high resolution mass spectrum. Its proton nuclear magnetic resonance (1H NMR) spectrum showed the presence of a singlet at δ 4.18, corresponding to 5H (cyclopentadiene ring of ferrocene), along with singlets at δ 4.33 (2H) and 4.61 (2H) because of the ferrocene ring protons. The appearance of a pair of singlets at δ 4.18 (2H) and 4.26 (2H), corresponding to O–CH2, along with a characteristic singlet at δ 7.74 (1H) because of the triazole ring proton confirmed the assigned structure. The appearance of absorption peaks at δ 68.6, 68.7, 69.6, and 75.1 due to ferrocene ring carbons along with the presence of absorption peak δ 175.3 due to the isatin ring carbonyl in the 13C NMR spectrum further corroborated the assigned structure.

Scheme 2

Synthesis of 1H-1,2,3-Triazole-Linked Ferrocenylmethoxy–Isatin Conjugates 9a–h

A precursor, viz., O-propargylated ferrocenyl chalcone 12, was prepared via an initial propargylation of 4-hydroxyacetophenone 10 to yield 11, which was subjected to aldol condensation with ferrocene–carboxaldehyde 8. Cu-promoted azide–alkyne cycloaddition reaction between 3-(azidomethyl)-3-hydroxy-indolin-2-one 4 and 12 afforded the desired 1H-1,2,3-triazole-tethered isatin–ferrocenylchalcone conjugate 13, as depicted in Scheme , the structure to which were again assigned on the basis of spectral data and analytical evidences.

Scheme 3

Synthesis of 1H-1,2,3-Triazole-Linked Isatin–Ferrocenylchalcone Conjugates 13a–h

The synthesized 1H-1,2,3-triazole-tethered isatin–ferrocene, ferrocenylmethoxy–isatin, and isatin–ferrocenylchalcone conjugates were then evaluated for their antiproliferative activities against MCF-7 (ER+) and MDA-MB-231 (ER−) human breast cancer cell lines using 3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The percentages of growth inhibition of MCF-7 and MDA-MB-231 cells were recorded at varying concentrations of the synthesized conjugates, and the results are depicted in Figures S13–S19 (Supporting Information), using plumbagin as the positive control. IC50 values, which is the concentration required to inhibit the growth of 50% of the cells by the test compounds, is summarized in Table . As evident, the compounds proved to be more active or cytotoxic to MCF-7 cells compared to MDA-MB-231 cells. A closer inspection of Table revealed an interesting structure–activity relationship, with antiproliferative activities being dependent upon the basic framework of the synthesized conjugates. 1H-1,2,3-Triazole-tethered isatin–ferrocene conjugates, 5a, 5e, 5f, and 5h, proved to be potent against MCF-7 cell lines compared to MDA-MB-231 cell lines, exhibiting IC50 values of 31.62, 53.48, 57.10, and 34.99 μM, respectively. In particular, the conjugate 5a exhibited selectivity against the MCF-7 over the MDA-MB-231 cell line, where a concentration of 97.11 μM is needed to inhibit the growth. 1H-1,2,3-Triazole-tethered ferrocenylmethoxy–isatin conjugates, viz., 9b, 9c, and 9f, on the other hand, showed selectivity against the MDA-MB-231 over MCF-7 cell lines, as evident by their low IC50 values. 9b, 9c, and 9f exhibited IC50 values of 45.67, 20.26, and 38.29 μM, respectively, against the ER– cell line, whereas IC50 values of 70.31, 67.34, and 78.30 μM were observed against the ER+ cell line, with an exception being 5h, which proved to be effective against both ER+ as well as ER– cell lines, exhibiting IC50s of 34.99 and 33.73 μM, respectively. The introduction of the chalcone moiety among isatin–ferrocene conjugates, viz., 13a–h, reduced the antiproliferative activities, with conjugate 13h proving to be ineffective against both the cell lines. The loss of activity with the inclusion of the chalcone moiety among the synthesized hybrids could be attributed to its reported induction of cytoprotective enzymes.[21] The log p values for all the tested conjugates were also calculated (Table S1) and were lower than 5.0, indicative of the fact that the compounds are not lipophilic.

Table 1

IC50 Values of the Test Compounds in MCF-7 and MDA-MB-231 Cellsa

compounds	MCF-7 (μM)	MDA-MB-231 (μM)	log p	compounds	MCF-7 (μM)	MDA-MB-231 (μM)	log p
5a	31.62	97.11	–2.60	9e	ND	ND	–1.24
5b	61.86	71.19	–2.09	9f	78.30	38.29	–0.72
5c	68.91	70.26	–1.81	9g	ND	ND	ND
5d	79.23	80.12	–2.14	9h	67.02	55.27	–0.77
5e	53.48	64.67	–0.83	13a	68.73	76.88	–1.38
5f	57.10	47.51	–0.31	13b	67.84	>100	–0.87
5g	75.88	69.60	–0.04	13c	69.02	96.93	–0.59
5h	34.99	33.73	–0.36	13d	66.69	89.39	–0.92
9a	70.30	63.34	–3.02	13e	55.70	>100	0.39
9b	70.31	45.67	–2.50	13f	69.02	70.71	0.91
9c	67.34	20.26	–2.23	13g	70.71	72.10	1.18
9d	ND	ND	ND	13h	>100	>100	0.86
tamoxifen	50	75
plumbagin	3.2	4.4

ND = not determined.

Conclusions

A series of isatin–ferrocene, ferrocenylmethoxy–isatin, and isatin–ferrocenylchalcone conjugates were synthesized via a Cu-promoted azide–alkyne cycloaddition reaction along with the evaluation of their antiproliferative activities against MCF-7 (ER+) and MDA-MB-231 (ER−) cell lines. The activity data revealed the ferrocenylmethoxy–isatins to be more selective against ER– cell lines, and thus, it can open up avenues for further investigation into this type of molecular framework for developing targeted therapies against ER– breast cancer. Furthermore, isatin–ferrocene conjugates exhibited selectivity against the ER+ cell line, and further modifications may prove useful for treating about 70% of all breast cancer patients. It would also be useful to test these compounds against tamoxifen-resistant cell lines in future to examine if these compounds have the ability to inhibit the growth of tamoxifen-resistant breast cancer cells.

Experimental Section

General Information

Melting points were determined by an open capillary using a Veego precision digital melting point apparatus (MP-D) and are uncorrected. 1H NMR spectra were recorded in CDCl3 with a Bruker AVANCE II (500 MHz) spectrometer using tetramethylsilane (TMS) as the internal standard. Chemical shift values are expressed as parts per million downfield from TMS, and the J values are in hertz. Splitting patterns are indicated as s: singlet, d: doublet, t: triplet, m: multiplet, dd: double doublet, ddd: doublet of a doublet of a doublet, and br: broad peak. 13C NMR spectra were recorded in CDCl3 with a Bruker AVANCE II (125 MHz) spectrometer using TMS as the internal standard. Mass spectra were recorded on a Bruker high resolution mass spectrometer (micrOTOF-QII). Elemental analyses were performed on a Heraeus CHN-O-Rapid elemental analyzer.[20e] Column chromatography was performed on a silica gel (60–120 mesh) using an ethyl chloroform/hexane mixture as the eluent.

Procedure for the Synthesis of Isatin–Ferrocene Conjugates (5a–h)

Copper sulfate (0.05 mmol) and sodium ascorbate (0.13 mmol) were added to a well-stirred solution of ethynyl ferrocene (1 mmol) and N-alkylazido-isatin 4 (1 mmol) in an ethanol/water (90:10) mixture.[20e] The reaction mixture was stirred at room temperature for 10–12 h, and the progress was monitored via thin-layer chromatography (TLC). On completion, water (20 mL) was added, and the extractions were carried out with dichloromethane (2 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under vacuum to yield a crude product 5, which was purified via column chromatography using a 10:90 (methanol/chloroform) mixture.

Procedure for the Synthesis of Ferrocenylmethoxy–Isatin Conjugates (9a–h)

Copper sulfate (0.05 mmol) and sodium ascorbate (0.13 mmol) were added to a stirred solution of O-propargylated ferrocenyl-methanol 8 (1 mmol) and N-alkylazido-isatin 4 (1 mmol) in an ethanol/water (95:5) mixture. The reaction mixture was allowed to stir for 6–8 h at room temperature, and the progress was monitored via TLC. Water (20 mL) was added, and the reaction mixture was extracted with dichloromethane (2 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield a crude product 9, which was purified via column chromatography using a 5:95 (methanol/chloroform) mixture.

Procedure for the Synthesis of Isatin–Ferreocenylchalcone Conjugates (13a–h)

To a stirred solution of ferrocenyl chalcone 12 (1 mmol) and N-alkylated-azido-isatin 4 (1 mmol) in an ethanol/water mixture, were added copper sulfate (0.05 mmol) and sodium ascorbate (0.13 mmol). The reaction mixture was allowed to stir at room temperature for 8–10 h. After completion, as evident by TLC, water (20 mL) was added, and the extraction was carried out with dichloromethane (2 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield a crude product 13, which was purified via column chromatography using a 5:95 (methanol/chloroform) mixture.

Materials and Methods

The cytotoxicity of conjugates, viz., 5a–h, 9a–h, and 13a–h, was tested on two different breast cancer cell lines, namely, ER+ MCF-7 cells and triple negative MDA-MB-231 (ER−) using MTT assay.[22]

Cell Culturing

MCF-7 cells were cultured in complete Dulbecco’s media Eagle’s medium (DMEM) with 5% fetal bovine serum (FBS) and 1% penicillin–streptomycin, and MDA-MB-231 cells were cultured in 3:1 DMEM and Ham’s F12 with 10% FBS and 1% penicillin–streptomycin; both cell lines were incubated at 37 °C and 5% carbon dioxide.

Methods

Cells were seeded in 96-well plates at a density of 5000 cells per well in triplicate in media. After 24 h, the test compounds [dissolved in dimethyl sulfoxide with concentration <0.2%] diluted in DMEM were added to each well. Cells were treated with a range of different concentrations of the drug (1, 5, 10, 20, 50, and 100 μM). Following a 24 h incubation with the test compounds, sterile 5 mg/mL MTT (Sigma-Aldrich) dissolved in phosphate-buffered saline was added to each well and incubated with cells for 2 h. Solubilization solution (10% sodium dodecyl sulfate and 10 mM HCl) of equal volume to the wells was then added to each well, which was incubated with cells for 16 h at 37 °C. The optical density of each well at 570 nm was determined using a microtiter plate reader (Tecan Sunrise microplate reader, Magellan software).

Statistical Analysis

The statistical analysis was performed using Excel, and IC50 values were estimated using GraphPad Prism 5 software (Hearne Scientific Software). The experiments were performed in duplicate, and the statistical significance was calculated using Student’s t-test. A p-value of less than 0.05 was used to estimate the significance of the observations. A Z-factor[23] was calculated for each 96-well plate, and assays having the Z-factor >0.5 were included in the statistical analysis.[24]