Description:FORM 2
The Patents Act 1970
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The Patent Rules 3003
COMPLETE SPECIFICATION
(See Section 10 and rule 13)
1. TITLE: TRIAZOLE BASED SPIRO-OXINDOLES AND DERIVATIVES THEREOF AND METHOD FOR SYNTHESIS THEREOF
2. APPLICANT (s):
Name in Full Nationality Address of the Applicant
UNIVERSITY OF DELHI INDIA House No. Dr. B. R. Ambedkar Center for Biomedical Research, faculty of science, University of Delhi, North Campus, Delhi- 110007, India
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City
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3. PREAMBLE OF THE DESCRIPTION: The following COMPLETE specification particularly describes the disclosure and the manner in which it is performed.

FIELD OF THE DISCLOSURE
The present disclosure generally relates to anti-cancer compounds. In particular, the present disclosure relates to triazole-based spiro-oxindoles and derivatives thereof and a method for preparation of the triazole-based spiro-oxindoles and derivatives thereof. The present disclosure also relates to a composition based on triazole-based spiro-oxindoles and derivatives thereof to treat cancer.
BACKGROUND
One of the leading causes of cancer deaths among women is breast cancer, which is one of the most frequently diagnosed life-threatening cancer found in women (ref.). Breast cancer accounts for ~25% of all women's cancer cases and ~12% of all gender-specific cancer cases. The breast cancer originates from the breast tissue and is mostly found in the inner lining of milk ducts and lobules. Approximately 10.4% of all cancer cases among women worldwide are breast cancer, making it the second most common non-skin cancer after lung cancer. Women are 200 times more likely than men to develop breast cancer. Worldwide, 2.3 million women were diagnosed with breast cancer in 2020, and 6,85,000 women died from it. Breast cancer was diagnosed in 7.8 million women in the past 5 years by the end of 2020, making it the most prevalent cancer in the world. 70,000 cases and 90,408 deaths were recorded in India according to the global cancer observatory (maintained by WHO) in 2020, representing the highest percentage (13.5%) of cancer cases out of all other cancers recorded in India. The Indian subcontinent is diverse in terms of ethnicities, cultures, lifestyles, and economics. Several areas of the Indian healthcare program have not yet reached their full potential and are highly heterogeneous. Seen as the most common type in urban women and the second most common in rural women. Often, women do not seek medical care early because of literacy issues and economic concerns. About 60-75% of all breast cancers are invasive ductal carcinomas of no particular type. The special types of breast cancer account for up to 25% of all cases and there are now at least 17 such entities recognized by the WHO.
It is commonly recognized that cancer is a complex multifactorial disease and, therefore, may not be treated with single-drug therapy. Accordingly, new agents combining diverse pharmacophores in a single hybrid molecule might represent a goal for the treatment of cancer and indeed a big effort has been put into the identification of anticancer multitargeted hybrid agents. Therefore, synthetic chemists targeted two or more biological receptors to generate bifunctional pharmaceutical molecules. This could be achieved by modifying the most popular pharmacophores as multi-targeting agents; the hybrid molecules, obtained by the bio-conjugation of a broad spectrum of heterocyclic derivatives for multi-drug therapy.
A number of therapeutic approaches are available to control breast cancer, including chemotherapy, radiotherapy, surgery (mastectomy), and combination therapy, none of which is without side effects (Drugs approved for Breast cancer, National Cancer Institutes, 2021: (https://www.who.int/news-room/fact-sheets/detail/breast-cancer)). New breast cancer drugs are approved by the U.S. Food and Drug Administration a majority of the time. Exemplary known compounds have been shown in Figure 1. The majority of them are financial burdens for low-income and middle-class families, which is of concern from India's perspective. Some of the known compounds to treat cancer may be required in higher dosages depending upon condition of a patient having cancer. The higher dosages of such synthetic compounds have many side effects which such patients may fail to fight due to low immunity thereof. In another instance, some compounds at initial stage of cancer may be administered in low dosages. However, such compounds may not be that much effective to treat the particular stage hence may be administered in multiple dosages. Beyond certain level of dosages, if such compounds administered, the compounds may be toxic to blood cell lines.
Therefore, there exists a need for synthesizing compounds or the compounds-based compositions for treatment of breast cancer, which bear low cost in synthesizing thereof, higher efficacy in case of low dosage too, and fewer side effects, along being non-toxic irrespective of higher and lower dosages as compared to that of the existing drugs.
OBJECTS OF THE EMBODIMENT
One object of the present disclosure is to provide triazole based spiro-oxindole compounds and derivatives thereof.
Another object of the present disclosure is to provide a low-cost method for synthesis of triazole based spiro-oxindole compounds and derivatives thereof.
Another object of the present disclosure is to provide a composition derived from triazole based spiro-oxindole compounds and derivatives thereof to treat cancer, particularly breast cancer.
Another object of the present disclosure is to provide the aforementioned composition which is non-toxic at higher dosage of administration of even up to 120 µM dose in HEK 293 Cell lines.
Another object of the present disclosure is to provide the aforementioned composition which shows efficacy to treat cancer cell lines even at lower dosage of ~62 µM of administration of even up to 120 µM dose in HEK 293 Cell lines.
In this respect, before explaining at least one embodiment of the present disclosure in detail, it is to be understood that the disclosure is not limited to its application to the details of processing and to the arrangements of the components set forth in the following description or illustrated in the drawings. The disclosure is capable of embodiments in addition to those described and of being practised and carried out in various ways. Also, it is to be understood that the phraseology terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.
SUMMARY
In an embodiment, the present disclosure discloses a triazole-based spiro-oxindoles based chemical compound (200) and derivatives thereof. The compound (200) is as shown in Figure 2A.
In another aspect, a method (400) for the synthesis of triazole-based spiro-oxindoles based chemical compound (200) and derivatives thereof is disclosed. The method (400) involves condensation of alkyne derivative of isatin (2), malononitrile (3), and 4-chloroethyl acetoacetate (4), in the presence of the catalytic amount of DBU in water under reflux condition for 30 minutes to obtain the compound (200).
In yet another embodiment, a composition comprising compound (6) nomenclated as ethyl (R)-2'-amino-5-chloro-3'-cyano-2-oxo-6'-((prop-2-yn-1-yloxy)methyl)spiro[indoline-3,4'-pyran]-5'-carboxylate in the range of 0.00119 wt% to 0.00479 wt %, 0.07813 wt% of DMSO, and remaining 99.9% of excipient is disclosed.
BRIEF DESCRIPTION OF THE DRAWINGS
Other objects, features, and advantages of the embodiment will be apparent from the following description when read with reference to the accompanying drawings. In the drawings, wherein like reference numerals denote corresponding parts throughout the several views:
Referring to Figure 1, shows a list of exemplary known (Prior Art) compounds for the treatment of breast cancer;
Referring to Figure 2A, shows a schematic chemical structure of a triazole based spiro-oxindole compound (200), in accordance with an embodiment of a present disclosure;
Referring to Figure 2B1-2B12, shows schematic chemical structures of twelve potential derivatives of the triazole based spiro-oxindole compound (200), in accordance with the embodiment of the present disclosure;
Referring to Figure 3, shows a reaction mechanism during synthesis of the exemplary derivative compound (6), in accordance with another embodiment of the present disclosure;
Referring to Figures 5A-5C, show bar graphs depicting cell death after treating MCF7 cells with the compounds shown in Figures 2B1-2B9: (Figure 5A) MCF7 Cells, (Figure 5B) MDA-MB-231, and (Figure 5C) HEK293 cells, in accordance with the embodiment of the present disclosure;
Referring to Figures 6A-6B, illustrate IC50 plots of the anti-breast cancer compound (6) evaluated against MCF7 and MDA-MB-231 cells, in accordance with the embodiment of the present disclosure;
Referring to Figures 7A-7B, illustrate images of cancer cells before treatment with the aforementioned compound/composition, (Figure 7A) MCF-7 (breast-cancer cell line), (Figure 7B) HEK-29 (Human embryonic kidney cells, control: non-cancerous cell line), in accordance with the illustrative embodiment of the preset disclosure;
Referring to Figures 8A-8J, shows representative images of MDA-MB-231 cells before treatment with the compound/composition, in accordance with the illustrative embodiment of the preset disclosure;
Referring to Figures 9A-9J, shows compounds inducing the breast cancer cell selective cytotoxicity, in accordance with the illustrative embodiment of the preset disclosure;
Referring to Figure 10, shows a graph depicting binding energies based on docking scores against Topoisomerase II, in accordance with the illustrative embodiment of the preset disclosure.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
Many modifications will be apparent to those skilled in the art without departing from the scope of the present invention as hereinbefore described with reference to the accompanying drawings.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
As used herein, the singular forms “a”, “an”, “the” include plural referents unless the context clearly dictates otherwise. Further, the terms “like”, “as such”, “for example”, “including” are meant to introduce examples which further clarify more general subject matter, and should be contemplated for the persons skilled in the art to understand the subject matter.
Spiro compounds have long been preferred in organic synthesis due to a variety of significant biological actions, including anti-tumor, anti-inflammatory, antimicrobial, antitubercular, and acetyl cholinesterase-inhibitory properties. Because of their widespread distribution in several natural products and bioactive compounds, spirocyclic oxindoles have emerged as intriguing synthetic targets. An important class of physiologically active natural and synthetic compounds having antifungal, antibacterial, anticancer properties are pyrans and fused pyrans. In addition, pyrans serve as the fundamental components of a number of natural products. 2-amino-Pyrans are employed as pigments, photoactive compounds, and potentially biodegradable agrochemicals. The triazole moiety is known to be hydrophilic in nature due to its dipolar character and ability to form H-bonds and thus plays an important role in bioorganic chemistry. Moreover, its synthetic versatility, stable nature, anchoring property, and being a bio-isostere make triazole an attractive scaffold for medicinal chemists. Furthermore, isatin can be used as a raw material for the synthesis of a variety of polyfunctional heterocyclic compounds such as quinolones and indoles.
Thus, the present disclosure discloses a triazole based spiro-oxindole compound (200), chemical structure thereof as shown in Figure 2A. Following Table 1 and Figures 2B1-2B12 show list of all the twelve derivatives of the compound (200). Out of the twelve derivatives, compound (6) shown in Figure 2B7 shows best results for the treatment of cancer.
S. No. Chemical Structure Name of the compounds Chemical name of the Compounds
1
5

ethyl (R)-2'-amino-3'-cyano-2-oxo-6'-((prop-2-yn-1-yloxy)methyl)spiro[indoline-3,4'-pyran]-5'-carboxylate
2
8a ethyl (R)-2'-amino-3'-cyano-6'-(((1-(2-(2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-2-oxospiro[indoline-3,4'-pyran]-5'-carboxylate
3
8b ethyl (R)-2'-amino-6'-(((1-(2-(5-chloro-2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-3'-cyano-2-oxospiro[indoline-3,4'-pyran]-5'-carboxylate
4
8c ethyl (R)-2'-amino-6'-(((1-(2-(5-bromo-2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-3'-cyano-2-oxospiro[indoline-3,4'-pyran]-5'-carboxylate
5
8d 5-(acetoxymethyl)-6-(4-((((R)-2'-amino-3'-cyano-5'-(ethoxycarbonyl)-2-oxospiro[indoline-3,4'-pyran]-6'-yl)methoxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-2,3,4-triyl triacetate
6
8e ethyl (R)-2'-amino-3'-cyano-6'-(((1-(2-(5-fluoro-2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-2-oxospiro[indoline-3,4'-pyran]-5'-carboxylate
7
6 ethyl (R)-2'-amino-5-chloro-3'-cyano-2-oxo-6'-((prop-2-yn-1-yloxy)methyl)spiro[indoline-3,4'-pyran]-5'-carboxylate
8
9a ethyl (R)-2'-amino-5-chloro-3'-cyano-6'-(((1-(2-(2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-2-oxospiro[indoline-3,4'-pyran]-5'-carboxylate
9
9b ethyl (R)-2'-amino-5-chloro-6'-(((1-(2-(5-chloro-2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-3'-cyano-2-oxospiro[indoline-3,4'-pyran]-5'-carboxylate
10
9c ethyl (R)-2'-amino-6'-(((1-(2-(5-bromo-2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-5-chloro-3'-cyano-2-oxospiro[indoline-3,4'-pyran]-5'-carboxylate
11
9d 5-(acetoxymethyl)-6-(4-((((R)-2'-amino-5-chloro-3'-cyano-5'-(ethoxycarbonyl)-2-oxospiro[indoline-3,4'-pyran]-6'-yl)methoxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-2,3,4-triyl triacetate oxospiro[indoline-3,4'-pyran]-5'-carboxylate
12
9e ethyl (R)-2'-amino-5-chloro-3'-cyano-6'-(((1-(2-(5-fluoro-2,3-dioxoindolin-1-yl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)-2-oxospiro[indoline-3,4'-pyran]-5'-carboxylate
Following Table 2 enlists synthesized triazole based spirooxindole derivatives of the compound (200) having different R and R1 group.
S. No. Compounds R1 R Yields (%)
1. 5 H - 98
2. 8a H H 92
3. 8b H Cl 85
4. 8c H Br 86
5. 8e H Acyl protected d-glucose 88
6. 8d H F 77
7. 6 H - 96
8. 9a Cl H 93
9. 9b Cl Cl 87
10. 9c Cl Br 78
11. 9e Cl Acyl protected d-glucose 80
12. 9d Cl F 84

Figure 3 shows a reaction mechanism for the synthesis of the compound (6). The compound (6) nomenclated as ethyl (R)-2'-amino-5-chloro-3'-cyano-2-oxo-6'-((prop-2-yn-1-yloxy)methyl)spiro[indoline-3,4'-pyran]-5'-carboxylate is synthesized by three-component condensation of isatin, malononitrile, and alkyne derivative of ethyl acetoacetate, in water using DBU 1,8-diazabicyclo [5.4.0] undec-7-ene as catalyst. The standard reaction conditions are optimized by initial condensation of alkyne derivative of 1 mmol (0.00119 wt% to 0.00479 wt%) of compound (2) nomenclated as isatin, compound (0.00119 wt% to 0.00479 wt%) (3) nomenclated as malononitrile, and compound (4) nomenclated as 4- chloroethyl acetoacetate, in the presence of the catalytic amount of 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) in water under reflux condition for 30 min to yield 98% of the compound (6).
Furthermore, the compounds (5) and (6) are treated with azide derivative of isatin compounds (7a-d) to get the compounds (8a, 8b, 8c, 8d). The reaction conditions for such a reaction are optimized by utilization of alkyne derivative compounds (5 and 6) with azides (7a-d) in the presence of CuSO4 and sodium ascorbate in DMF for 20 min, as shown in Figure 3B.
Similarly compounds 8e and 9e were obtained by the click reaction of the compounds (5, 6) with azide of acyl protected sugar compound (7e), respectively as shown in Figure 3C.
In another embodiment of the present disclosure, a triazole based spiro-oxindoles based composition for the treatment of breast cancer is disclosed. The composition includes the compound (6) in the weight range of 0.00119 wt% to 0.00479 wt% in 0.07813 wt% of DMS, and remaining 99.9 wt% of cell culture media/ PBS/ Saline as excipient. The composition is nontoxic even up to 120 µM dose in HEK 293 Cell lines. The composition has fewer side effects at higher dosage of administration of even up to 120 µM dose in HEK 293 Cell lines. The composition shows efficacy of treating cancer cell lines even at lower dosage of administration of even up to dose (~62 µM) in MCF-7 and MDA-MB-231 Cell lines.
Materials and Methods:
The MCF-7, MDA-MB-231, and HEK293 cell lines were cultivated in DMEM (Dulbecco's Modified Eagle's Medium, Gibco, St. Louis, USA) supplemented with 10% FBS (Fetal Bovine Serum, Gibco, USA) and 1% Pen-Strep (penicillin-streptomycin, Gibco, USA) in a humidified CO2 (5%) incubator at 37°C. To maintain cell growth, the cells were cultured as a monolayer in T-25 (25 cm2) culture flasks for four passages prior to commencing the compound treatment. In the experiments, DMSO (Sigma Aldrich, USA) served as the vehicle control, while PBS (Sigma Aldrich, USA) was utilized as the solvent control. Tamoxifen (GLR Innovations, India; Cat No. GLR19.143958), a known compound with anti-breast cancer activity, was employed as the positive control.
Ex-vivo anti-breast cancer activity screening against MCF-7 and MDA-MB-231 cells by MTT cell viability assay:
Two series of synthesized compounds, namely series 1 and series 2, were screened for their potential anticancer activity against breast cancer cells, MCF-7 and MDA-MB-231. To assess any nonspecific effects and potential toxicity, a non-breast cancer human cell line, HEK293, was included in the screening process. The MCF-7, MDA-MB-231, and HEK293 cells were revived and maintained for a minimum of four passages to ensure an adequate number of cells for the experiments. To initiate the screening, adhesive cells were detached from the matrix using a cell scraper/0.25% trypsin, and 20,000 cells were seeded per well in 24-well flat-bottom plates. The plates were then placed in a CO2 incubator at 37°C for 20-24 hours to allow cell attachment. All the compounds were dissolved in 200% DMSO to create a 200 mM stock solution for each compound (Table 1). Subsequently, the compounds were diluted in DMEM complete media to achieve a final concentration of 200 µM. The cells were treated with the compounds at a concentration of 200 µM, dissolved in DMEM media, in triplicates. Additionally, positive control drug (tamoxifen), vehicle control (1% DMSO), and solvent control (PBS) were included in the experiment. The cells were incubated with the treatment for 48 hours. After the incubation period, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) from Sigma-Merck, USA, was dissolved in DMEM growth media to prepare a stock solution with a concentration of 5 mg/mL. The cells were treated with a final concentration of 0.2 mg/mL MTT solution and incubated for 1 hour at 37°C in the dark. Following the removal of the MTT-containing media, formazan granules were dissolved in 1 mL of 200% DMSO with gentle shaking. The cell viability was assessed by measuring the absorbance at 570 nm using a Tecan Multiplate Reader (SpectraMax® iD3 multi-mode microplate reader). For the titration of the top four compounds and the calculation of IC50 values against MCF-7 and MDA-MB-231 cells, further dilutions of the compounds (200 µM, 10 µM, and 1 µM) were prepared in DMSO and treated in triplicates
The cells were subjected to microscopic examination using an Eclipse Ni-E microscope from Nikon. Prior to the addition of any compounds, drugs, or control solutions, the cells were observed under the microscope using a 10X objective lens to establish a baseline. Subsequently, the cells were treated with various compounds for a duration of 48 hours. The synthesized compounds were used at a concentration of 200 µM, while the positive control drug (tamoxifen) was employed at a concentration of 20 µM. As for the control solutions, 1% of vehicle (DMSO) and solvent (PBS) were utilized. Observations of the cells were made again using the microscope after the compound treatment period. The purpose of this observation was to assess any visible changes or effects induced by the compounds on the cells compared to the baseline observations. The microscope's 10X objective lens was used for this evaluation.
In order to identify potential targets for breast cancer treatment, a small in-house chemical library consisting of 12 derivative compounds as aforementioned was created. Such compounds were designed based on known moieties from FDA-approved anti-cancer and anti-breast cancer medications. To determine a suitable target protein, a literature search was conducted, leading to the selection of Human Topoisomerase II alpha (PDB ID: 5GWK) as a promising candidate. Topoisomerase II alpha is an enzyme that plays a crucial role in controlling and modifying the topological structure of DNA during transcription. It is involved in various processes such as chromosome condensation, chromatid separation, and the relief of torsional stress during DNA transcription and replication. The enzyme catalyzes the temporary breaking and rejoining of two strands of DNA, allowing them to pass through each other and altering the DNA's topology. There are two forms of this enzyme, alpha and beta, with the alpha form being the target for several anticancer agents. Mutations in the gene encoding this enzyme have been associated with drug resistance development. To investigate the molecular interaction between the target protein and the compounds in the in-house library, docking analysis was performed. Each compound, including Tamoxifen as a negative control (which should not bind tightly to the topoisomerase protein as its actual target is the estrogen receptor, although it is a known anti-cancer drug), was individually docked to the Topoisomerase II alpha protein using the SwissDock online server (http://www.swissdock.ch/docking). The protein structure (PDB ID: 5GWK) was downloaded in .pdb format, and the ligands were prepared using ChemDraw software to draw their 2-dimensional structures and convert them to 3-dimensional structures in .sdf and .mol2 formats. The protein and ligand files were then uploaded to the SwissDock server for docking simulations. Upon completion of the docking process, the docked ligands were scored based on their delta G values (in kcal/mol). The binding energy scores were used to rank and analyze the compounds, with a focus on identifying the top hits against Topoisomerase II alpha. Visualization software such as UCSF Chimera and PyMOL were utilized to inspect the results. To establish a correlation between the in-silico screening and in vitro binding, ex vivo experiments were conducted using the MCF-7 cell line. Both the negative control (Tamoxifen) and the top hits from the in-house compound library (as shown in Table 2) were further evaluated in these experiments. The methodology used for the ex vivo experiments was described in the previous section.
Results:
Table 3: Molecular weight and dilutions of test compounds and control drugs.
Drug name Solvent
Molecular mass
(g/mol) or Dalton Chemical formula Working concentration
(µM) Volume of DMSO (in µL) for 200 mM stock Color of solution after mixing compounds with DMSO
5 DMSO 379.37 C20H17N3O5 200 84.3 Yellow

8a DMSO 595.57 C30H25N7O7 200 50.3 Orange

8b DMSO 630.01 C30H24 Cl N7O7 200 47.6 Orange

8c DMSO 674.47 C30H24 Br N7O7 200
44.4 Orange

8e DMSO 752.69 C34H36N6O14
200
38.5 Yellow

8d DMSO 613.56 C30H24FN7O7 200
52.1 Reddish orange

6 DMSO 413.81 C20H16ClN3O5 200
77.3 Colorless

9a DMSO 630.01 C30H24ClN7O7 200
53.9 Pale orange

9b DMSO 664.46 C30H23Cl2 N7O7 200
42.1 Orange

9c DMSO 708.91 C30H23ClBr N7O7 200
47.9 Pale orange

9e DMSO 787.13 C34H35ClN6O14 200 36.8 Bright yellow
9d DMSO 648.00 C30H23ClFN7O7 200
46.2 Orange

Control-1
Tamoxifen DMSO 371.51 C26H29NO 200 94.2 Colorless

The synthesized compounds were subjected to evaluation for their potential anti-breast cancer activity using the MTT cell viability assay. Specifically, MCF-7 and MDA-MB-231 cell lines were utilized to assess the effects of the compounds on breast cancer cells, while the HEK293 cell line was employed to examine any non-specific effects. The results depicted in Figure 5A, obtained from the MTT viability assay conducted with the MCF-7 cell line, were analyzed. The viability of the cells treated with PBS (control) was considered as 200%, representing the maximum viability observed. Subsequently, the viability of the cells treated with the compounds was calculated and normalized relative to the viability of the PBS-treated cells. Among the tested compounds, namely 6, 9a, and 9c, notable anti-breast cancer activity was observed. At a concentration of 200 µM, these compounds induced significant cell death in MCF-7 cells, resulting in viabilities of 46.35%, 60.16%, and 67.94%, respectively. This indicates a corresponding cell death of 53.65%, 39.84%, and 32.06%. Similarly, when evaluated on MDA-MB-231 cells, these compounds exhibited cell viabilities of 47.98%, 70.60%, and 66.26%, corresponding to cell death rates of 52.02%, 29.4%, and 33.74%, respectively.
Cancer cell-specific cytotoxicity
To further examine the cancer cell-specific cytotoxicity of these compounds in comparison to the existing drug tamoxifen, their effects on the HEK293 cells were investigated (Figure 5B). The compounds (6, 9a, and 9c) demonstrated selective cytotoxicity towards MCF-7 and MDA-MB-231 cells, while exhibiting minimal impact on the non-cancerous HEK293 cells. In contrast, the compound (5) displays cytotoxic effects in both cell lines and did not exhibit specific anti-breast cancer activity. Such findings highlight the potential of the synthesized compounds, particularly the compounds (6, 9a, and 9c), as promising and selective anti-breast cancer agents. Such compounds demonstrated significant cytotoxicity in breast cancer cells while sparing the non-cancerous HEK293 cells as shown in Figure 5C.
In order to determine the IC50 values, three compounds (6, 9a, and 9c) were treated with MDA-MB-231 and MCF-7 cells with varying doses of the compounds. However, the results for the compounds (9a and 9c) were not that promising as that of the compound (6). The compound (6) exhibits the highest potential for anti-breast cancer efficacy among the tested in-house compounds, with an approximate IC50 of 62µM against both MCF7 and MDA-MB-231 cell lines. Figures 6A-6B show IC50 plots of the anti-breast cancer compound (6) evaluated against MCF7 (Figure 6A) and MDA-MB-231 cells (Figure 6B). Each experiment was repeated thrice and the plots are showing the corresponding SD values.
Screening experiments on twelve in-house compounds were performed to evaluate their inhibitory activity against three cell lines: MDA-mB-231, MCF7, and HEK293. Among these compounds, one particular compound, named the compound (6), exhibited promising results in terms of its inhibitory potency against the MCF7 cell line. The IC50 value of the compound (6) in MCF7 cells was determined to be approximately 62 µM. Such a finding suggests that the compound (6) has the potential to be a highly effective compound for targeting MCF7 cells, highlighting its significance as a potential therapeutic agent in the context of breast cancer research.
The microscopic analysis was performed before and after compound/drug treatment using Nikon microscope at 10X magnification. MCF-7 cells treated with some of the compounds namely, 6, 9a, 9b, 9c, 9d, and 9e and tamoxifen for 48 h have shown significant changes in their morphology which can clearly be seen in photomicrographs shown in Figures 8A-8J. Cellular shrinkage (Figure 8C) and dead cells (Figure 8F) were also seen after treatment in comparison to untreated cells. The untreated MCF7 cells (Figure 7A) and HEK293 (Figure 7B) were appeared healthy under the microscope whereas, the 6, 9a, 9b, 9c, 9d, and 9e (Figure 8A-F) treated MCF-7 cells are mostly dead and morphologically very different too from normal MCF7 cells. The cells treated with vehicle control DMSO and solvent control PBS had no dead cells and cells were growing and flourishing as normal in these wells (Figure 8G, H). 8e is non-anti-breast cancerous which is not affecting cells much (Figure 8K). Whereas the cells treated with positive control tamoxifen was mostly dead as expected (Figure 8I, J).
Hence, these six compounds namely, 6, 9a, 9b, 9c, 9d, and 9e have the highest anti-breast cancer activity out of all the compounds and have better cancer selective effect. These compounds inducing the significant cell death and morphological changes in comparison to controls.
Triazole-appended Ethyl 6'-amino-6-chloro-5'-cyano-2'-methyl-2-oxo-1,2-dihydrospiro[indole-3,4'-pyran]-3'-carboxylate and Isatin derivatives are working better than alkyne derivative of Ethyl 6'-amino-6-chloro-5'-cyano-2'-methyl-2-oxo-1,2-dihydrospiro[indole-3,4'-pyran]-3'-carboxylate. Which indicates Triazole addition or modification increase the anti-breast cancer activity of series 2 compounds whereas alkyne derivative is unable to do so.
Molecular interaction studies were conducted to evaluate the ability of the in-house compounds to target topoisomerase, a critical protein involved in cancer cell survival. The compounds were designed based on a known backbone that specifically interacts with topoisomerase, and modifications were made to potentially enhance their efficacy. Targeting topoisomerase is a promising strategy as it can disrupt DNA replication, induce DNA damage, and ultimately lead to cell death or inhibition of cancer cell growth. Due to the elevated activity of topoisomerases in cancer cells, they serve as attractive targets for therapeutic interventions. Docking scores were calculated for multiple compounds, including 5, 8a, 8e, 9a, and 9b, indicating their efficacy in binding to topoisomerase. These compounds exhibited binding energies ranging from -7.31 to -8.42 kcal/mol, suggesting lower efficacy in targeting the protein. However, seven compounds (8b, 8c, 8e, 6, 9c, 9d, and 9e) demonstrated high efficacy in binding to topoisomerase, as evidenced by their binding energies ranging from approximately -8.424 to -10.93 kcal/mol. Among these top-performing compounds, four (8b, 9c, 9d, and 9e) exhibited the lowest binding energies (-9.54, -10.41, -10.39, and -10.93 kcal/mol, respectively), indicating strong interactions with topoisomerase II alpha. The docking conformations of the protein structures (PDB ID 5GWK) with the compounds revealed that specific amino acids played a vital role in the binding process. Notably, amino acids such as GLN-1095, THR-825, PHE-828, LYS-287, GLN-1049, ALA-588, ILE-574, LYS-821, and LEU-826 were involved in the binding interactions with the docked compounds. Topoisomerase II alpha consists of various domains, including TOPORIM, WHD, TOWER, ATPase, and CTD, each serving different functions such as metal binding, DNA cleavage, and dimerization. In particular, the WHD (Winged Helix Domain) and CTD domains are primarily responsible for DNA intercalation and DNA cleavage processes. These findings from the molecular interaction studies provide valuable insights into the interactions between the developed compounds and topoisomerase II alpha. The compounds demonstrating strong binding and low binding energies hold promise as potential agents for targeting topoisomerase and interfering with its functions in breast cancer cells.
Table 4: Binding energies of best compounds against Topoisomerase II alpha (PDB ID: 5GWK).
S. No. Name of the compounds Docking Score (kcal/mol) No. of Hydrogen Bonds Interacting residues through H-Bond Interacting domain of Topoisomerase II (PDB ID: 5GWK)
1 5 -7.31 0 0 None
2 8a -8.42 1 GLN1095 CTD domain
3 8b -10.49 1 GLN1095 CTD domain
4 8c -9.82 2 THR825 and PHE 828 WHD (Winged helix domain): DNA cleavage and intercalation
5 8e -9.54 0 0 None
6 8d -8.15 2 PHE828 WHD
7 6 -10.42 1 LYS287 ATPase domain
8 9a -8.34 1 GLN1049 Dimerization C-Gate
9 9b -8.21 2 ALA588 and ILE574 TOPRIM
(Metal binding)
10 9c -10.41 1 PHE828 WHD
11 9d -10.39 1 LYS821 WHD
12 9e -10.93 1 LEU826 WHD
13 Temoxifen -7.46 0 0 None
Table 4: Protein-ligand interacting amino acid residue details of compounds with Topoisomerase II alpha using UCSF Chimera software.
S. No. Name of the compounds Interacting residues
1 5 HSD824, LEU826, PHE828
2 8a GLN1094, GLN1095, HSD824, LYS735
3 8b LYS735. PHE731, HSD824, GLN1095
4 8c HSD824, THR825, LYS827, LEU826, PHE828
5 8e HSD824, GLU1092, LYS1091
6 8d LEU829, GLN1095
7 6 LEU826, THR825, HSD824, GLN1094, GLN1095, LYS287
8 9a LYS827, ASP823, GLN1049
9 9b MET587, PHE573, GLU572, TYR686, THR689, THR691, TYR692, THR690, ALA588 and ILE574
10 9c LYS827, LEU826, HSD824, GLN1095, PHE828
11 9e GLN1095, LYS735, PHE734, PRO820, ASP823, LYS821
12 9d THR825, HSD824, LYS827, PHE828, GLN1095, LEU826
13 Temoxifen ARG835, TRP1036, TYR830, PHE828

The foregoing descriptions of exemplary embodiments of the present disclosure have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to best explain the principles of the disclosure and its practical application, to thereby enable others skilled in the art to best utilize the disclosure and various embodiments with various modifications as are suited to the particular use contemplated.
, Claims:We Claim
1. A triazole-based spiro-oxindoles based chemical compound (200) and derivatives thereof comprising:

wherein
R1= H, CH3, F, Cl, Br, NO2, OCH3;
R2= CN, COOC2H5;
R3= CH3, C2H5, C3H8; and
R4= CH3, C2H5, COC6H5, CH2CH2Br, CH2CH2N3, CH2CH2CH2Br, CH2CH2CH2N3.
2. A method (400) for the synthesis of triazole-based spiro-oxindoles based chemical compound (200) and derivatives thereof, the method (400) comprising condensation of alkyne derivative of isatin (2), malononitrile (3), and 4-chloroethyl acetoacetate (4), in the presence of the catalytic amount of DBU in water under reflux condition for 30 minutes to obtain the compound (200).
3. The method (400) as claimed in claim 2, wherein the method (400) further comprising treating compounds (5) and (6) with azide derivative of isatin compounds (7a-d) to get derivative compounds (8a, 8b, 8c, 8d).
4. The method (400) as claimed in claim 2, wherein the method (400) comprising optimizing the reactions by utilization of alkyne (1 mmol) with azides (1.2 mmol) in presence of CuSO4 (0.2 mmol) and Sodium ascorbate (0.4 mmol) in DMF for 20 min.
5. The method (400) as claimed in claim 2, wherein the method (400) comprising click reaction of the compounds (5, 6) with azide of acyl protected sugar compound (7e), respectively to obtain derivative compounds (8e, 9e).
6. The method (400) as claimed in claim 4, wherein the method (400) yields 98% of the compound (6).
7. A composition comprising compound (6) nomenclated as ethyl (R)-2'-amino-5-chloro-3'-cyano-2-oxo-6'-((prop-2-yn-1-yloxy)methyl)spiro[indoline-3,4'-pyran]-5'-carboxylate in the range of 0.00119 wt% to 0.00479 wt %, 0.07813 wt% of DMSO, and remaining 99.9% of excipient.
8. The composition as claimed in claim 7, wherein the excipient comprising one or more of selected from cell culture media, PBS, Saline as excipient.
Date: August 06, 2024

Neha Goyal
Agent of the Applicant
IN/PA-4398