Synthesis of potent MDA-MB 231 breast cancer drug molecules from single step

We have prepared novel potent breast cancer drug molecules from non-toxic and inexpensive method. Column chromatography is not necessary for purification of target molecules. The value of overall atom economy, environmental factor, environmental catalyst and product mass intensity gives additional merits for this synthetic method. Synthesized flexible dimeric imidazolium bromides showed less toxicity and gives excellent anticancer response against normal mammary epithelial cells. Novel dimeric pyridinium bromides showed excellent anticancer response against tested cancer cell lines. In cell cycle, novel flexible dimeric pyridinium bromides showed significant arrest in the G2/M phase by nearly three folds, when compared with control drug. We have studied the targeting epidermal growth factor receptor for all the synthesized flexible amino substituted and methyl substituted dimeric pyridinium bromides.

Atom Economy (AE) Atom economy of 1, 5-dibromo pentanereacts with 4-methyl pyridine is defined as how much reactant are converted into dimeric pyridinium bromide 1.
The atom economy is calculated for the following reaction (Fig. 2).

Environmental factor (EF)
The environmental factor is calculated for the following reaction by ratio of mass value of unwanted (or) wasted materials/Mass value of product molecule.
Mass of waste is calculated as follow (Fig. 3

Product mass intensity (PMI)
The ratio between total mass of (reactant + solvent) and mass of expected product.
We have calculated product mass intensity of the above reaction by using following formula (Fig. 4) Similarly, we have calculated the atom economy, environmental factors and product mass intensity of remaining reactions and presented in the Table 1.We have used non-toxic, low boiling solvent; mass value is given in the Table 1 which is used for all reaction.We have not used any expensive, toxic and environmental hazardous catalyst for the preparation of our target drug molecules.

MTT assay
Ionic liquids have rarely or never been tested for anticancer studies and our previous ionic liquid compounds showed potent anticancer activity 33 .Therefore, it is very important to evaluate the anticancer activity of newly discovered compounds against various cancer cells.
IC 50 values are greater than 20 for MCF-7 and MDA-MB-231 breast cancer cell lines, IC 50 values greater than 30 for MCF-10A breast cancer cell lines in 24 h incubation period with reference to doxorubicin which is used as a reference drug molecules 34 .IC 50 values for Phyllanthus niuri DCM extract against MCF-7 and MCF-7 ADR breast cancer cell lines showed 100 in 24 h incubation period 35 38 .
The anticancer activity of the compounds (1-4) was tested against three cancer cell lines namely human breast adenocarcinoma (MCF-7), (MDA-MB-231), and ductal carcinoma (T47D), and one human mammary epithelial (MCF-10A) cell line by MTT assay.The investigated anticancer activity of the compounds was time-dependent, i.e., cell viability decreased with increasing incubation, and their IC 50 values are listed in Table 2. IC 50 values of the compounds showed less toxicity against normal human mammary epithelial cells and more toxicity against cancer cells.All the compounds have efficiently killed the MDA-MB-231 cancer cell line compared to other tested cells (Fig. 5).Compound 1 and 2 demonstrated remarkable anticancer activity compared to other compounds against all the tested cell lines.Interestingly, the presence of an electron-donating methyl substituent in compound 2 increases lipophilicity and allows it to easily cross the blood-brain barrier and kill all cancer cells.

Apoptosis
Many researchers have demonstrated that many anticancer drugs induce apoptosis, which plays an important role in cancer drug development 33 .For this reason, we tested the morphological changes of our new compounds against MDA-MB-231 cells with acridine orange (AO) and ethidium bromide (EB) after 24 h.Morphological assessment demonstrates viable cells (control) with normal morphology and pale green nuclei.However, the MDA-MB-231 cells treated with compounds for 24 h confirmed morphological changes as a greater part of cells become shrinkage, and green apoptotic cells containing apoptotic bodies and red necrotic cells were observed (Fig. 6).Further confirming the induction of apoptosis, the MDA-MB-231 cells were also treated with compounds and stained with Hoechst 33258 for 24 h, which exhibit apoptotic highlights like chromatin fragmentation, cytoplasmic vacuolation, nuclear swelling, and cytoplasmic blebbing (Fig. 7).These data strongly suggested these compounds induced apoptosis in MDA-MB-231 cells.

Cell cycle
Cell cycle analysis was also performed to further confirm that the newly discovered compounds showed good anticancer activity against MDA-MB 231.Therefore, MDA-MB 231 cells were treated with Alkyl/aryl linker unit containing flexible methyl substituted dimeric pyridinium bromides 1 and 2 for 24 h and their cell cycle profile and induction of apoptosis were analyzed.From (Fig. 8), it is demonstrated that the compounds did not induce apoptosis as no changes occurred in SubG 0 /G 1 .Figure 7A-E also show that the compounds did not cause any change in G0/G1and S-phase compared to the control.Interestingly, both compounds significantly arrested the

Induction of cell apoptosis with annexin V-FITC and propidium iodide (PI)
The study aimed to investigate the impact of lead compounds 1 and 2, along with the positive control 5-fluorouracil (25 μM), on MDA-MB-231 cell apoptosis and necrosis.MDA-MB-231 cells were exposed to the compounds for 48 h.Following treatment, cell nuclei were stained with annexin V-FITC and propidium iodide (PI) and analyzed through flow cytometry.The findings revealed significant alterations in the apoptotic and necrotic cell populations.In the absence of treatment, only 0.09% of the cells exhibited late apoptotic characteristics, as indicated by their placement in quadrant Q2.However, when exposed to 25 μM of compounds 1 and 2, the late apoptotic cell population increased to 29.3% and 21.8%, respectively (Fig. 9).It is noteworthy that both compounds 1 and 2 exhibited lower activities while compared to the positive control, 5-fluorouracil (37.4%).
In the case of early apoptosis (quadrant Q4), 0.18% of the control cells displayed such features.This proportion significantly increased to 19.1% and 21.8% for compounds 1 and 2, respectively, post-treatment (Fig. 9).Once again, compounds 1 and 2 were less effective than 5-fluorouracil (24.6%) in inducing early apoptosis.Interestingly, compound 2 also induced necrotic cell death relative to the control (Fig. 9).The observed changes in apoptosis and necrosis were substantiated by additional staining methods, including AO/EB and Hoechst 33,258, further supported by flow cytometry analysis.

In silico pharmacokinetics
Swiss drug design tools were used to predict the bioavailability of compounds (1-4) 33 .Applications of the Swiss ADME web tool has recently contributed greatly to the design and development of anticancer, tubercular, and antimicrobial agents 33,40,41 .The bioavailability radar demonstrated six physicochemical indices such as lipophilicity (XLOGP3), size, polarity, solubility, saturation, and flexibility (Table 3; Fig. 10).The biophysical range is marked in a pink area which indicates drug-like nature.In the radar image, compounds 1 and 3 are within the limits, which is great but 2 and 4 are slightly above the saturation limit.The blue dots show that, all the compounds in egg spots lead to their efficient efflux by P-glycoprotein (Pgp +) and this confirms that these compounds easily cross the BBB.Furthermore, the skin permeability coefficient (log Kp) was calculated, which appeared to be related to lipophilicity and molecular size.A very negative value of the log Kp estimated for all compounds (− 5.26 to − 6.12 cm/s) indicates low skin penetration.www.nature.com/scientificreports/ In Table 3 values confirm that all compounds (1-4) follow Lipinski's five rules, as a result of which these compounds are expected to have excellent drug-like properties.The fact that these new compounds have molecular weights below 500 indicates that these compounds are easier to transport, diffuse and absorb than larger molecules.Compounds 1 and 2 show no hydrogen bond acceptors (nHA) whereas 3 and 4 show two hydrogen bond acceptors and none of the donor (nHD) atoms in these compounds, which are less than 10 and 5, respectively (Table 4; Fig. 10).
The number of rotatable bonds in these compounds ranges from 4 to 6 which is within the range.The TPSA values of compounds 1 and 2 were 7.76 and 3 and 4 showed the same value of 59.80, which is below the normal range and can easily cross the blood-brain barrier.From the above description, the compounds are within www.nature.com/scientificreports/ the parameter range of Lipinski's five rules (i) molecular weight ≤ 500 Da, (ii) LogP < 5, (iii) nHBD ≤ 5, (iv) nHBA ≤ 10, (v) TPSA < 140 Å 2 , which leads to adherence to criteria for oral drugs.

Targeting epidermal growth factor receptor
Epidermal growth factor receptor (EGFR) is a protein found on the surface of cells 42 .It plays an important role in cell proliferation and is often targeted by anticancer drugs 43 .EGFR has been especially studied in breast cancer, as it is an important target for the development of new treatment 44 .Research has shown that blocking EGFR can lead to decreased tumor growth and improved survival rates 45 .Many current therapies are designed to specifically target EGFR, making it a key focus for cancer research.Targeting EGFR is important for this study as dimeric pyridinium bromides show potent activity against breast cancer cells.The newly synthesized compounds were docked with EGFR, and their binding affinity and interactions were shown in Fig. 11.All four compounds interact strongly with EGFR (Table 4).1,1′Pentane-1,5-diyl)bis(4methylpyridin-1-ium)bromide 1 showed a carbon-hydrogen bond (C-H) interaction formed between the carbon atom of the CH 2 moiety and ASP831 with a distance of 3.05 Å (Fig. 11).This compound also formed 8 alkyl interactions with EGFR, with the first two interactions between the methyl moiety and LEU764 and MET769 with distances of 3.98 and 4.37 Å, respectively (Fig. 11).The second four interactions are formed between the tolyl moiety and LYS721, MET742, LEU694, and LEU820 with distances of 5.19, 5.25, 5.21, and 4.67 Å, respectively (Fig. 11).The last two interactions are formed between the CH 2 moiety and VAL702 and LEU820 with distances of 4.84 and 4.88 Å, respectively (Fig. 11).Furthermore, 1,1'pentane -1,5-diyl)bis(4-methylpyridin-1-ium)bromide 1 stabilizes the interaction of ASP831, THR830, GLU738, GLY772, LEU768, and THR766 with EGFR via vander Waals interactions.
The observed binding energy score was consistent with the experimental anticancer activity, again testifying to the potent anticancer activity of the compounds.

Conclusion
We have prepared novel potent breast cancer drug molecules from non-toxic and inexpensive method.Overall atom economy, environmental factor, environmental catalyst and product mass intensity values gives additional merits for this synthetic method.Synthesized flexible dimeric imidazolium bromides showed less toxicity against normal mammary epithelial cells.Novel dimeric pyridinium bromides showed excellent anticancer response against tested breast cancer cell lines.Alkyl substituted flexible dimeric pyridinium bromide 1 and 2 showed potential anticancer response than the amino substituted flexible dimeric pyridinium bromides 3 and 4. In cell www.nature.com/scientificreports/cycle, novel flexible dimeric pyridinium bromides showed significant arrest in the G2/M phase by nearly three folds, when compared with control drug.We have studied the targeting epidermal growth factor receptor for all the synthesized flexible amino substituted and methyl substituted dimeric pyridinium bromides.In literature, they have used expensive catalyst and involved multi step synthesis to reach their target molecules from expensive starting materials.Whereas, our current breast cancer drug molecules are obtained from easily available inexpensive starting materials.We have not used any expensive catalyst during the preparation and involves single step to reach our target molecules.We have compared the IC 50 values from available literatures, our target molecules showed least IC 50 values.So, our simple/substituted dimeric pyridinium bromides showed potent anticancer behaviour against tested breast cancer cell lines.

Experimental method
General procedure for the preparation of dimeric pyridinium bromide 1 and 3  S3).

MTT assay
5 × 10 3 cells are seeded in a 96-well plate and incubated for growth under the conditions mentioned above.Once confluency is reached, the cells are treated with different concentrations of simple/amino substituted dimeric pyridinium bromides 1-4 and incubated for 24 h.After incubation, the media is removed, and 20 μL of MTT is added and incubated for further 3 h.After 3 h, MTT is removed, and DMSO is added to dissolve the formazan crystals.The absorbance is measured at 570 nm, and the percentage of cell death is calculated using the formula

Ethical approval
This article does not contain any studies with human participants or animals performed by the authors.

Consent to participate
We comply with the ethical standards.We provide our consent to take part.
% of cell death = Absorbance of control − Absorbance of treated /Absorbance of control × 100 ): Atom economy = Molecular weight of product/ Molecular weight of reactant × 100 AE = 416.20 g/mol/(93.13+ 93.13 + 229.94) × 100 AE = 100 % Environment factor = Mass value of waste / Mass value of product N N Br Br . Sumit et al. prepared from triazole Mass of waste = Net mass of starting materials − Net mass of product obtained = 229.95g/mol + 108.14 g/mol + 108.14 g/mol 446.23 g/mol = 446.23 − 446.23 Environment factor = 0 E -factor = Mass of waste / Mass of product E -factor = 0 E -factor = 0 (Ideal value of E -factor is considered zero) PMI = (Mass of reactants + Solvent) / Mass of product

Table 1 .
Atom economy, environmental factors, product mass intensity for flexible substituted dimeric pyridinium bromides.

of steps Overall yield (%) Overall AE (%) E-factor E-solvent E-catalyst Product mass intensity (PMI) total
37thered Ospemifene-isatin conjugated drug molecules using expensive catalyst for McMurry coupling followed by additional four steps to get their triazole tethered Ospemifene-isatin conjugated drug molecules.IC 50 values of different alkyl chain length containing triazole tethered ospemifene-isatin derivatives are 53.10 and 70.71 for two and three methylene linkers respectively36.Dibiotin ester linked Ruthenium complex and cis-platin showed IC 50 values of 31.5 ± 4.7, 38 ± 1.41 against MCF 7 breast cancer cell line in 24 h incubation37.Mina and coworker reported the breast cancer effect of Metformin drug molecules on T47D breast cancer cell lines.IC 50 value for Metformin and Silibinin drug molecules are 21.20, 106.50 respectively in 24 h incubation period against T47D breast cancer cell lines