Pyrazole amide compounds and their use against breast cancer

Abstract

The present disclosure provides new derivatives of pyrazolamide represented by the following formula (Formula I): where R=-CH2, R1 can be one of benzene, 4-methylbenzene, 4-methoxybenzene, 4-chlorobenzene, 4-fluorobenzene, 2-chlorobenzene, 3,4-dichlorobenzene, 3,5-trifluoromethylbenzene, and where R=-SO2, R1 can be one of benzene, 4-methoxybenzene, 4-chlorobenzene, 4-bromobenzene, 4-trifluoromethylbenzene, 3-trifluoromethylbenzene, 2-bromobenzene, 2-chlorothiophene, or 2,3-dichlorothiophene substituents. The present disclosure also relates to compounds of formula (I) for use as anticancer drugs in triple negative breast cancer (TNBC) cells. The present disclosure has demonstrated the efficacy of pyrazolamide derivatives of formula I (I-8) against TNBC cells by inducing cell death by various mechanisms through interaction with diverse cellular pathways. JPEG2024537811000046.jpg41170

Description

[Technical Field]

[0001] The present invention relates to compounds of formula I that are useful as anti-cancer agents in triple-negative breast cancer (TNBC) cells.

[0002] [ka]

[0003] In particular, the present invention relates to small pyrazole amide-based compounds of formula I that function as anticancer drugs in triple-negative breast cancer (TNBC) cells. More particularly, the present invention demonstrated the efficacy of one of the pyrazole amide compounds of formula I (I-8) against TNBC cells by inducing cell death by various mechanisms through interactions with diverse cellular pathways. [Background technology]

[0004] Cancer burden ranks as a leading cause of death worldwide. A recent report by GLOBOCAN 2020 estimated 19.3 million new cancer cases and 10 million cancer deaths worldwide in 2020. Breast cancer in women has surpassed lung cancer as the most commonly diagnosed cancer. 2.3 million new breast cancer cases were reported in 2020 (11.7% of total diagnoses), with a reported mortality rate of 6.9%. This represents 685,000 deaths worldwide, making it the fifth leading cause of cancer deaths. According to the national population-based and hospital-based cancer registries, the estimated number of cancer cases in India in 2020 is 1,392,179, with the most common sites being breast, lung, oral cavity, cervix, and tongue. Deaths from female breast and cervical cancer are significantly higher in developing countries compared to developed countries. The total burden of cancer is projected to rise by 47% in 2040 compared to 2020, with the rise higher in developing countries (64-95%) compared to developed countries (32-56%). Therefore, developing countries should build on their efforts to expand prevention efforts and provide adequate cancer care, which is needed to maximize global cancer control (Sung et al., Global Cancer Statistics 2020: GLOBOCAN Estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021, 71(3):209-249. doi: 10.3322 / caac.21660; Mathur et al., Cancer Statistics, 2020: Report from National Cancer Registry Programme, India. JCO Glob Oncol. 2020 6:1063-1075. doi: 10.1200 / GO.20.00122).

[0005] Breast cancer is the second leading cause of cancer death in women. It is broadly classified into three major subtypes based on the presence or absence of molecular markers for estrogen or progesterone receptors, human epidermal growth factor 2 (HER2), and triple-negative, which is the absence of three standard molecular markers. Advances in diagnostic and treatment options have dramatically increased the survival rate of breast cancer patients. Adjuvant, neoadjuvant, and targeted chemotherapy (e.g., CDK4 / 6 inhibitors, kinase inhibitors, mTOR inhibitors, PI3K inhibitors), and immunotherapy (PD-1 inhibitors, PDL-1 inhibitors) are commonly used to treat breast cancer. Doxorubicin, epirubicin, paclitaxel, docetaxel, 5-FU, cyclophosphamide, and carboplatin are commonly used chemical agents for early-stage adjuvant or neoadjuvant therapy. Paclitaxel, docetaxel, abraxane, cisplatin, doxorubicin, eribulin, ixabepilone, vinorelbine, etc. are used to treat advanced breast cancer. Drug combinations are often used to treat early stage breast cancer, but in advanced breast cancer, single chemical agents are often used (information taken from cancer.net site). Given the importance of early diagnosis of breast cancer, cooperation between public and private actors and education is essential for the success of breast cancer treatment (Al-Mahmood et al., Metastatic and triple-negative breast cancer: challenges and treatment options. Drug Deliv Transl Res. 2018, 8(5):1483-1507. doi: 10.1007 / s13346-018-0551-3, Mutebi et al., Breast cancer treatment: A phased approach to implementation. Cancer. 2020 May 15;126 Suppl 10:2365-2378. doi: 10.1002 / cncr.32910).

[0006] Triple-negative breast cancer (TNBC) accounts for 15–20% of all diagnosed cases and lacks all hormone receptors. Therefore, HER2-targeted hormone therapy and drugs are ineffective, and chemotherapy is the primary systemic treatment option. Metastatic triple-negative TNBC is the most aggressive form, and there is interest in discovering new drugs to treat it. Patients with TNBC have a high rate of distant recurrence and poor prognosis, with nearly all patients dying even with adjuvant chemotherapy. Various subtypes of TNBC have been identified using omics-based methods and expression analysis of limited gene sets from TNBC patient samples (WO2019 / 112966). This further complicates TNBC treatment. Several gene-effective predictive biomarkers have been discovered in TNBC, primarily for diagnostic and prognostic purposes (WO2016 / 037009 A1). Various chemical drugs are used to treat TNBC: doxorubicin and cyclophosphamide, doxorubicin and cyclophosphamide and 5-FU, paclitaxel, and docetaxel. Anthracycline-taxane chemotherapy is the first-line treatment, while carboplatin is considered for BRACA-positive TNBC (Pandy et al., Triple-negative breast cancer and platinum-based systemic treatment: a meta-analysis and systematic review. BMC Cancer 19, 1065 (2019). https: / / doi.org / 10.1186 / s12885-019-6253-5). The identification of immune-related targets in TNBC allows for the development of promising immunotherapeutic strategies using inhibitors of PD-1 and PDL-1 (Oualla et al., Immunotherapeutic Approaches in Triple-Negative Breast Cancer: State of the Art and Future Perspectives. Int J Breast Cancer. 2020 Nov 4;2020:8209173. doi: 10.1155 / 2020 / 8209173).In March 2019, the FDA granted accelerated approval to the immunotherapy atezolizumab (a PDL1 inhibitor) in combination with the chemotherapy drug Abraxane for metastatic triple-negative breast cancer. The addition of the PD-1 monoclonal antibody pemlolizumab to platinum-containing neoadjuvant chemotherapy resulted in a significant increase in patient responses (Schmid et al., Atezolizumab plus nab-paclitaxel as first-line treatment for unresectable, locally advanced, or metastatic triple-negative breast cancer (IMpassion130): updated efficacy results from a randomized, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol. 2020, 21(1):44–59. doi: 10.1016 / S1470-2045(19)30689-8). In April 2020, the FDA approved sacituzumab govitecan (an antibody coupled to the chemical drug irinotecan) for some patients with TNBC. Administration of CDK19 inhibitors in TNBC patients has been associated with reduced cachexia, increased survival, prolonged time to tumor progression, reduced tumor mass, time to tumor metastasis, prolonged time to tumor recurrence, tumor response, complete response, partial response, stable disease, progressive disease, or progression-free survival (WO2019 / 055977 A1). Kidney-associated antigen 1 (KAAG1) is an antigen expressed in TNBC, and high-affinity antibodies can be used to target these cells (WO2013 / 104050 A3).

[0007] Thienotriazolodiazepine compounds act against TNBC in the presence of other mitotic or mTOR inhibitors (WO2015 / 169951 A1). Multikinase inhibitor pharmacophores act against TNBC (WO2015 / 181201 A1). The important vitamin D analog AMPI-109 has remarkable cancer-specific properties, inhibiting TNBC cell proliferation and inducing apoptosis (U.S. Patent Application Publication No. 2015 / 0202173 A1).

[0008] Chemotherapy using platinum-based drugs is effective in treating TNBC, however, its use may be limited by over 40 specific side effects. Long-term chemotherapy has serious side effects and often leads to multidrug resistance. Furthermore, we do not currently have a specific anticancer drug, and each drug is more or less toxic to normal cells. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2019 / 112966 [Patent Document 2] WO2016 / 037009 Al [Patent Document 3] WO2019 / 055977 A1 [Patent Document 4] WO2013 / 104050 A3 [Patent Document 5] WO2015 / 169951 Al [Patent Document 6] WO2015 / 181201 Al [Patent Document 7] U.S. Patent Application Publication No. 2015 / 0202173 A1 [Non-patent literature]

[0010] [Non-Patent Document 1] Sung et al., Global Cancer Statistics 2020: GLOBOCAN Estimates of incidence and mortality worldwide for 36 Cancers in 185 countries. CA Cancer J Clin. 2021, 71(3):209-249. doi: 10.3322 / caac.21660 [Non-Patent Document 2] Mathur et al., Cancer Statistics, 2020: Report from National Cancer Registry Programme, India. JCO Glob Oncol. 2020 6:1063-1075. doi: 10.1200 / GO.20.00122 [Non-Patent Document 3] Al-Mahmood et al., Metastatic and triple-negative breast cancer: challenges and treatment options. Drug Deliv Transl Res. 2018, 8(5):1483-1507. doi: 10.1007 / s13346-018-0551-3 [Non-Patent Document 4] Mutebi et al., Breast cancer treatment: A phased approach to implementation. Cancer. 2020 May 15;126 Suppl 10:2365-2378. doi: 10.1002 / cncr.32910 [Non-Patent Document 5] Pandy et al., Triple negative breast cancer and platinum-based systemic treatment: a meta-analysis and systematic review. BMC Cancer 19, 1065 (2019). https: / / doi.org / 10.1186 / s12885-019-6253-5 [Non-patent document 6] Oualla et al., Immunotherapeutic Approaches in Triple-Negative Breast Cancer: State of the Art and Future Perspectives. Int J Breast Cancer. 2020 Nov 4;2020:8209173. doi: 10.1155 / 2020 / 8209173 [Non-Patent Document 7] Schmid et al., Atezolizumab plus nab-paclitaxel as first-line treatment for unresectable, locally advanced or metastatic triple-negative breast cancer (IMpassion130): updated efficacy results from a randomized, double-blind, placebo-controlled, phase 3 trial. Lancet Oncol. 2020, 21(1):44~59. doi: 10.1016 / S1470-2045(19)30689-8 [Non-patent document 8] A. Kamal, AB Shaik, BB Rao, I. Khan, GB Kumara and N. Jain, Org. Biomol. Chem., 2015, 13, 10162~10178. Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, it is necessary to identify new drugs that are more selective for cancer cells, and it is important to identify their molecular mechanisms. [Means for solving the problem]

[0012] The primary object of the present invention is to provide pyrazole amide compounds of formula I.

[0013] Another object of the present invention is to provide a process for preparing the pyrazole amide compounds of formula I.

[0014] Another object of the present invention is to evaluate the cytotoxicity of pyrazole amide compounds of formula I in TNBC cells.

[0015] Another object of the present invention is to evaluate the apoptosis induction and associated mechanisms of potential pyrazole amide derivatives of formula I in MDA MB231 cells.

[0016] Another object of the present invention is to evaluate the detachment-induced cell death (anoikis) in MDA MB231 cells by potential pyrazole amide derivatives of formula I.

[0017] Another object of the present invention is to evaluate the histone deacetylase 1 (HDAC-1) inhibitory potency of potential pyrazole amide derivatives of formula I in MDA MB231 cells.

[0018] Another object of the present invention is to evaluate the cell cycle inhibitory potency of potential pyrazole amide derivatives of formula I in MDA MB231 cells.

[0019] Another object of the present invention is to evaluate the anti-metastatic effect of potential pyrazole amide derivatives of formula I in MDA MB231 cells.

[0020] Another object of the present invention is to evaluate the effect on metabolic inhibition exerted by potential pyrazole amide derivatives of formula I in MDA MB231 cells.

[0021] Another object of the present invention is to evaluate the ability of potential pyrazole amide derivatives of formula I to interact with the EGF receptor in order to exert their effects.

[0022] Another object of the present invention is to study the main pathways affected by potential pyrazole amide derivatives of formula I through transcriptome analysis.

[0023] Another object of the present invention is to evaluate the combination of potential pyrazole amide derivatives of Formula I with paclitaxel to enhance overall efficacy.

[0024] SUMMARY OF THE INVENTION Thus, the present invention provides compounds of formula I

[0025] [ka]

[0026] [In the formula, When R=-CH2, R1 is selected from the group consisting of benzene, 4-methylbenzene, 4-methoxybenzene, 4-chlorobenzene, 4-fluorobenzene, 2-chlorobenzene, 3,4-dichlorobenzene, 3,5-trifluoromethylbenzene; When R=-SO2, R1 is selected from the group consisting of benzene, 4-methoxybenzene, 4-chlorobenzene, 4-bromobenzene, 4-trifluoromethylbenzene, 3-trifluoromethylbenzene, 2-bromobenzene, 2-chlorothiophene, or 2,3-dichlorothiophene. The present invention provides pyrazole amide compounds of the formula:

[0027] In one embodiment of the present invention, the compounds are useful as anti-cancer agents in triple-negative breast cancer (TNBC) cells.

[0028] In another embodiment of the invention, the compound of formula I is i. N-benzyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-1);

[0029] [ka]

[0030] ii. N-(4-methylbenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-2);

[0031] [ka]

[0032] iii. N-(4-methoxybenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-3);

[0033] [ka]

[0034] iv. N-(4-chlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-4);

[0035] [ka]

[0036] v. N-(4-fluorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-5);

[0037] [ka]

[0038] vi. N-(2-chlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-6);

[0039] [ka]

[0040] vii. N-(3,4-dichlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-7);

[0041] [ka]

[0042] viii. N-(3,5-bis(trifluoromethyl)benzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-8);

[0043] [ka]

[0044] ix. N-(phenylsulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-9);

[0045] [ka]

[0046] x. N-((4-methoxyphenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-10);

[0047] [ka]

[0048] xi. N-((4-chlorophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-11);

[0049] [ka]

[0050] xii. N-((4-bromophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-12);

[0051] [ka]

[0052] xiii. N-((4-(trifluoromethyl)phenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-13);

[0053] [ka]

[0054] xiv. N-((3-(trifluoromethyl)phenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-14);

[0055] [ka]

[0056] xv. N-((2-bromophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-15);

[0057] [ka]

[0058] xvi. N-((5-chlorothiophen-2-yl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-16);

[0059] [ka]

[0060] xvii. N-((4,5-dichlorothiophen-2-yl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-17).

[0061] [ka]

[0062] is selected from the group consisting of:

[0063] In another embodiment, the present invention provides a method for preparing a compound of formula I, comprising: i. condensing a compound of formula 1 with diethyl oxalate to obtain ethyl 2,4-dioxo-4-(3,4,5-trimethoxyphenyl)butanoate of formula 2;

[0064] [ka]

[0065] ii. Cyclocondensing compound 2 as obtained in step (i) with hydrazine hydrate hydrochloride to form a pyrazole ester compound of formula 3;

[0066] [ka]

[0067] iii. Base-mediated hydrolysis of the compound of formula 3 as obtained in step (ii) to give the compound of formula 4

[0068] [ka]

[0069] followed by amide coupling with an amine of formula 5 [R1-R-NH2] to give a compound of formula I; The present invention provides a method comprising:

[0070] In another embodiment of the present invention, compound I-8 has a GI of 15.08 μM with a 50% growth inhibition value. 50 It induces cytotoxicity in TNBC cells (MDA MB231) without cytotoxicity in normal fibroblasts.

[0071] In another embodiment of the present invention, compound I-8 induces apoptosis-like morphological changes in MDA MB231 cells as evidenced by phase contrast and electron microscopy images.

[0072] In yet another embodiment of the invention, the compound induces apoptosis (by activating both the intrinsic and extrinsic pathways) in MDA MB231 cells, which are TNBC cells.

[0073] In another embodiment of the present invention, compound I-8 induces cell cycle arrest at S phase in MDA MB231 cells, which are TNBC cells.

[0074] In another embodiment of the present invention, compound I-8 induces detachment-induced cell death (anoikis) in MDA MB231 cells, which are TNBC cells.

[0075] In another embodiment of the present invention, compound I-8 has substantial histone deacetylase 1 (HDAC-1) inhibitory activity in TNBC cells (MDA MB231).

[0076] In another embodiment of the present invention, compound I-8 has an anti-metastatic effect in MDA MB231 cells, which are TNBC cells.

[0077] In another embodiment of the present invention, compound I-8 has metabolic inhibition in MDA MB231 cells, which are TNBC cells.

[0078] In another embodiment of the present invention, compound I-8 interacts with the EGF receptor to exert its effect in TNBC cells (MDA MB231).

[0079] In yet another embodiment of the present invention, compound I-8 affects the signaling pathways responsible for ER stress-induced apoptosis, anoikis and cell cycle arrest as revealed by transcriptome analysis.

[0080] In another embodiment of the present invention, compound I-8 results in the clustering of up-regulated and down-regulated genes into six major clusters for regulating stress-induced apoptosis, UPR response, autophagy, integrin-mediated signaling and cell cycle regulation, respectively.

[0081] In yet another embodiment of the present invention, compound I-8 enhances the efficacy of paclitaxel such that a ten-fold lower concentration of paclitaxel is required.

[0082] In another embodiment of the present invention, compound I-8 induces nuclear fragmentation and phosphatidylserine translocation in TNBC cells (MDA MB231).

[0083] In another embodiment of the present invention, compound I-8 induced caspase-3 and caspase-9 activity in TNBC cells (MDA MB231).

[0084] In another embodiment of the present invention, compound I-8 induced reactive oxygen species generation and a decrease in mitochondrial membrane potential in TNBC cells (MDA MB231).

[0085] In another embodiment of the present invention, compound I-8 upregulated 1337 genes and downregulated 1642 genes in TNBC cells (MDA MB231) as revealed by transcriptome analysis.

[0086] In another embodiment of the present invention, compound I-8 upregulated genes involved in the endoplasmic reticulum stress response, p53 transcriptional gene network, oxidative stress, autophagy, IRE-1 alpha activating chaperone, HATS acetylating histones, ATR activating programmed cell death, and gene response in response to ER stress in TNBC cells (MDA MB231).

[0087] In another embodiment of the present invention, compound I-8 downregulated genes involved in inflammatory pathways, glycolysis-gluconeogenesis, ATR pathway, Wnt signaling pathway, Hedgehog pathway, G1-S specific transcription, FAK-mediated PI3K / AKT / mTOR pathway, ECM-receptor interaction, TGF-beta signaling, Aurora B pathway, PLK-1 pathway, cholesterol biosynthesis pathway, GPCR signaling, oxidative phosphorylation, focal adhesion, ALK-1 pathway, α5β3 and α4β1 integrin pathway, extracellular matrix organization, angiogenesis, and syndecan-1 pathway in TNBC cells (MDA MB231).

[0088] In yet another embodiment of the present invention, compound I-8 upregulated genes that are organized as six protein clusters to carry out functions in TNBC cells (MDA MB231).

[0089] In yet another embodiment of the present invention, compound I-8 downregulated genes that are organized as six distinct protein clusters to carry out functions in TNBC cells (MDA MB231).

[0090] In yet another embodiment of the present disclosure, compound I-8 enhances the efficacy of the standard drug paclitaxel (10-fold lower concentrations of paclitaxel are required in the presence of compound I-8). [Brief explanation of the drawings]

[0091] [Figure 1]1 shows a schematic diagram for synthesizing pyrazole amide derivatives of Formula I in accordance with an embodiment of the present invention. [Figure 2A] 1 shows phase contrast microscopy images of MDA MB231 cells treated with various concentrations of I-8 and comparison with the standard drug paclitaxel (50 nM) according to an embodiment of the present invention. [Figure 2B] 1 shows scanning electron microscopy (SEM) images of MDA MB231 cells treated with I-8 (25 μM) and paclitaxel (50 nM) according to an embodiment of the present invention. [Figure 3] 1 shows phase contrast images of normal fibroblast (WI30) cells treated with various concentrations of I-8 and comparison with the standard drug paclitaxel (50 nM) according to an embodiment of the present invention. [Figure 4A] 1 shows fluorescent images showing nuclear fragmentation in MDA MB231 cells treated with various concentrations of I-8 and paclitaxel (50 nM), according to an embodiment of the present invention. [Figure 4B] 1 shows fluorescent images showing phosphatidylserine translocation in MDA MB231 cells treated with various concentrations of I-8 and paclitaxel (50 nM), according to an embodiment of the present invention. [Figure 5] 1 shows a graph of caspase-3 and caspase-9 activation in MDA MB231 cells treated with various concentrations of I-8 and paclitaxel (50 nM), according to an embodiment of the present invention. [Figure 6A] 1 depicts a FACS analysis showing upregulation of reactive oxygen species (ROS) production in MDA MB231 cells treated with various concentrations of I-8 and paclitaxel (50 nM), according to an embodiment of the present invention. [Figure 6B] 1 shows fluorescence images showing a decrease in mitochondrial membrane potential in MDA MB231 cells treated with various concentrations of I-8 and paclitaxel (50 nM), according to an embodiment of the present invention. [Figure 7]1 depicts a protein array showing upregulation of pro-apoptotic proteins and downregulation of anti-apoptotic processes upon treatment with I-8, according to an embodiment of the present invention. [Figure 8] 1 shows the anoikis-inducing effects of I-8 and paclitaxel (50 nM) at various concentrations, according to an embodiment of the present invention. [Figure 9] 1 shows FACS images demonstrating cell cycle arrest at S phase associated with treatment with I-8 and Western blot analysis demonstrating downregulation of cell cycle regulatory proteins, according to an embodiment of the present invention. [Figure 10] 1 shows a graph illustrating the HDAC inhibitory effect of I-8, in accordance with an embodiment of the present invention. [Figure 11] In accordance with an embodiment of the present invention, the anti-metastatic effect of I-8 as evidenced by the inhibition of cell migration, colony formation, and MMP-9 production is shown. [Figure 12] 1 shows FACS images demonstrating downregulation of glucose uptake upon treatment with various concentrations of I-8 and paclitaxel (50 nM), according to an embodiment of the present invention. [Figure 13] 1 shows fluorescent images demonstrating downregulation of EGFR expression upon treatment with various concentrations of I-8 and paclitaxel (50 nM), according to an embodiment of the present invention. [Figure 14] 1 depicts a volcano plot and clustered heatmap showing differential expression of genes upon treatment with I-8, according to an embodiment of the present invention. [Figure 15] 1 depicts gene set enrichment analysis (genes upregulated in transcriptome analysis) for pathways upregulated upon treatment with I-8, according to an embodiment of the present invention. [Figure 16] 1 depicts gene set enrichment analysis (genes downregulated in transcriptome analysis) for pathways downregulated upon treatment with I-8, according to an embodiment of the present invention. [Figure 17]1 depicts protein clusters and functional analysis of genes upregulated upon treatment with I-8, according to an embodiment of the present invention. [Figure 18] 1 depicts protein clusters and functional analysis of genes downregulated upon treatment with I-8, according to an embodiment of the present invention. [Figure 19] 1 depicts phase contrast images demonstrating the chemosensitizing ability of I-8 in combination with paclitaxel, in accordance with an embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0092] Those skilled in the art will recognize that the present disclosure is directed to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all steps, features, compositions, and compounds individually or collectively as referred to or indicated herein, and any and all combinations of any or more of such steps or features.

[0093] definition For convenience, before further describing this disclosure, certain terms used in the specification and examples will be reviewed here. These definitions should be read and understood by one of ordinary skill in the art in light of the remainder of the disclosure. Terms used herein have meanings that are recognized and known to those of ordinary skill in the art; however, for convenience and completeness, certain terms and their meanings are described below.

[0094] The articles "a", "an" and "the" are used to refer to one or to more than one (ie to at least one) of the grammatical object of the article.

[0095] The terms "comprise" and "comprising" are used in an inclusive and open sense, meaning that additional elements may be included. They are not intended to be interpreted as "consists of only."

[0096] Throughout this specification, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of the stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0097] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference.

[0099] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the disclosure as described herein.

[0100] As discussed in the background of this disclosure, chemotherapy using platinum-based drugs is effective in treating TNBC, but its use may be limited by more than 40 specific side effects. Long-term use of chemical drugs has serious side effects and often leads to multidrug resistance. In addition, no specific anticancer drug has been discovered to date, and each drug is more or less toxic to normal cells. Therefore, there is an unmet need to identify new drugs that are more selective for cancer cells, and identifying their molecular mechanisms is important.

[0101] In one embodiment of the present invention, the compound of formula I is more specific for triple-negative breast cancer cells and has chemosensitizing properties.

[0102] [ka]

[0103] wherein when R=-CH2, R1 is selected from the group consisting of benzene, 4-methylbenzene, 4-methoxybenzene, 4-chlorobenzene, 4-fluorobenzene, 2-chlorobenzene, 3,4-dichlorobenzene, and 3,5-trifluoromethylbenzene; and when R=-SO2, R1 is selected from the group consisting of benzene, 4-methoxybenzene, 4-chlorobenzene, 4-bromobenzene, 4-trifluoromethylbenzene, 3-trifluoromethylbenzene, 2-bromobenzene, 2-chlorothiophene, or 2,3-dichlorothiophene. The present invention provides pyrazole amide compounds of the formula:

[0104] The compounds of formula I, I-1 to I-17, were evaluated for their anticancer potential in triple-negative breast cancer cells (MDA MB231). Preliminary cytotoxicity studies were performed for all synthesized compounds (I-1 to I-17) by MTT assay. The GI of compound I-8 [N-(3,5-bis(trifluoromethyl)benzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide] was 50The value was found to be 15.08 μM. The compound caused significant morphological changes in the cells similar to those of apoptosis, which were studied by phase contrast and electron microscopy images (FIG. 2A and B).

[0105] The effect of I-8 on the toxicity of normal fibroblasts was evaluated and found to be non-toxic (Fig. 3).

[0106] I-8 showed significant nuclear fragmentation and phosphatidylserine translocation, which are key features of apoptosis, as studied by DAPI and Annexin V staining (Fig. 4A and B). I-8 upregulated caspase activity. Cells undergoing apoptosis release the enzyme caspases to execute the process. Caspase 3 and caspase 9 activity was measured in I-8-treated cells, and the results were significant (Figure 5).

[0107] I-8 upregulated ROS production and a decrease in mitochondrial membrane potential. The intrinsic pathway of apoptosis involves mitochondria, and a decrease in mitochondrial membrane potential and excessive ROS production are key features of this pathway. Significant ROS production and a decrease in mitochondrial membrane potential were observed in I-8-treated cells (Figure 6A and B).

[0108] I-8 upregulated proteins involved in the intrinsic and extrinsic pathways. Protein array experiments by Western blotting showed the upregulation of key proteins involved in apoptosis regulation. Proteins upregulated in the extrinsic and intrinsic pathways were Bad, Bax, Bid, Bim, caspase 3, caspase 8, cytochrome c, Fas, Fas ligand, HSP60, HSP70, HTRA, p21, p27, p53, SMAC, STNFR1, and XIAP (Figure 7).

[0109] I-8 induced detachment-induced cell death (anoikis). The percentage of growth inhibition in control and anoikis chambers was favorably reduced by I-8 treatment (FIG. 8).

[0110] I-8 induced cell cycle arrest at S phase. After labeling with PI, the distribution of cells in different phases was monitored by FACS. Western blot analysis showed that cell cycle regulatory proteins at the G1-S transition, such as cyclin B1, cyclin A2, and CDK, were downregulated (Figure 9).

[0111] I-8 exhibited HDAC inhibition. HDAC inhibitory activity measured by enzyme assay showed significant inhibition (Figure 10). I-8 exhibited anti-metastatic effects, as evidenced by the inhibition of cell migration in scratch wound assays, the inhibition of colony formation, and the reduction of MMP-9 activity (Figure 11).

[0112] I-8 induced metabolic inhibition. Glucose uptake studies by FACS analysis showed a significant decrease in glucose uptake (FIG. 12). I-8 showed a decrease in EGFR expression. Fluorescence images showed a decrease in EGFR labeling (Figure 13).

[0113] I-8 showed upregulation of 1337 genes and downregulation of 1642 genes in the whole transcriptome analysis. Isolation of RNA from control and I-8 treated cells and their transcriptome sequencing using Illumina technology resulted in the extraction of differentially expressed genes (Figure 14).

[0114] I-8 treatment showed upregulation of signaling pathways such as ER stress-induced apoptosis, UPR response, autophagy, and HDAC acetylation. Gene set enrichment analysis was performed by comparing the upregulated genes with a canonical set of target genes to identify the upregulated pathways (Figure 15).

[0115] I-8 treatment downregulated many signaling pathways that are important for the cell death process. Gene set enrichment analysis was performed to identify downregulated pathways by comparing the downregulated genes with a canonical set of target genes. The main downregulated pathways identified by I-8 treatment included inflammation, glycolysis / gluconeogenesis, Wnt signaling, TGF-beta signaling, integrin signaling, Hedgehog pathway, angiogenesis, syndecan-1 pathway, and FAK-mediated PI3K / Akt / mTOR signaling pathway (Figure 16).

[0116] I-8 treatment resulted in a clustering of upregulated targeted genes (six major clusters) encoding proteins required for tumor suppression and stress-induced apoptosis and autophagy (Figure 17).

[0117] I-8 treatment resulted in a clustering of downregulated target genes (six major clusters) that encode proteins required for the downregulation of integrin-mediated signaling and cell cycle arrest (Figure 18).

[0118] I-8 enhances the chemotherapeutic potential of the standard drug paclitaxel.

[0119] Growth inhibition studies revealed that the use of 5 μM I-8 reduced the concentration of paclitaxel required (for 50% growth inhibition) by 10-fold compared to the original concentration (Figure 19).

[0120] While the inventive subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternative embodiments of the inventive subject matter, will become apparent to those skilled in the art upon reference to the description of the inventive subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the inventive subject matter as defined. [Example]

[0121] The present disclosure will now be illustrated by examples, which are intended to illustrate the practice of the present disclosure and are not intended to be limiting in any way to the scope of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods, devices, and materials are described herein. Where such methods and conditions may apply, it should be understood that the present disclosure is not limited to the specific methods and experimental conditions described.

[0122] The following examples are provided as illustrations of the practice of the present disclosure in actual practice and therefore should not be construed as limiting the scope of the disclosure.

[0123] Example 1 N-Benzyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-1) One equivalent of benzylamine (26.79 mg, 0.027 mL, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), HOBt (40.53 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at 25 °C for 6–7 h. After completion of the reaction, the reaction mixture was extracted with DCM. The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 50% ethyl acetate and hexane as eluents to obtain the product. The compound N-benzyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 78 mg (84%). 1 H NMR (500 MHz, CDCl3) δ 7.29 - 7.28 (m, 3H), 7.26 (s, 1H), 6.96 (s, 1H), 6.82 (s, 2H), 4.60 (d, J = 6.0 Hz, 2H), 3.84 (s, 3H), 3.82 (s, 6H). 13 C NMR (125 MHz, CDCl3) δ 161.9, 153.6, 138.5, 137.8, 128.6, 127.6, 127.5, 125.1, 102.9, 96.1, 60.8, 55.9, 43.3. HRMS (ESI) (m / z): C 20 H 21 N3O4, (M+H) + Calculated value: 368.16103; Measured value: 368.16069.

[0124] Example 2 N-(4-methylbenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-2) One equivalent of 4-methylbenzylamine (30.29 mg, 0.032 mL, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), HOBt (40.53 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 6–7 h. After completion of the reaction, the reaction mixture was extracted with DCM. The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 50% ethyl acetate and hexane as eluents to obtain the product. The compound N-(4-methylbenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 92 mg (96%). 1 H NMR (500 MHz, CDCl3) δ 7.20 (d, J = 8.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 2H), 6.95 (s, 1H), 6.82 (s, 2H), 4.56 (d, J = 6.0 Hz, 2H), 3.84 (s, 3H), 3.82 (s, 6H), 2.30 (s, 3H). 13 C NMR (126 MHz, CDCl3) δ 161.9, 153.6, 138.4, 137.1, 134.7, 129.3, 127.6, 102.9, 96.4, 60.8, 55.9, 43.1, 21.1.HRMS (ESI) (m / z): C 21 H 23N3O4, (M+H) + Calculated value: 382.17668; Measured value: 382.17592.

[0125] Example 3 N-(4-Methoxybenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-3) One equivalent of 4-methoxybenzylamine (34.29 mg, 0.032 mL, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), HOBt (40.53 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 6–7 h. After completion of the reaction, the reaction mixture was extracted with DCM. The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 50% ethyl acetate and hexane as eluents to obtain the product. The compound N-(4-methoxybenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 78 mg (79%). 1 H NMR (500 MHz, acetone) δ 12.87 (s, 1H), 7.95 (s, 1H), 7.33 (d, J = 7.5 Hz, 2H), 7.15 - 7.12 (m, 3H), 6.89 (d, J = 7.5 Hz, 2H), 4.54 (d, J = 5.5 Hz, 2H), 3.90 (s, 6H), 3.77 (s, 6H). 13C NMR (126 MHz, acetone) δ 158.9, 153.9, 128.9, 113.7, 102.9, 59.7, 55.6, 54.6, 41.8. HRMS (ESI) (m / z): C 21 H 23 N3O5, (M+H) + Calculated value: 398.17160; Measured value: 398.17049.

[0126] Example 4 N-(4-chlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-4) One equivalent of 4-chlorobenzylamine (35.40 mg, 0.030 mL, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), HOBt (40.53 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 6–7 h. After completion of the reaction, the reaction mixture was extracted with DCM. The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 50% ethyl acetate and hexane as eluents to obtain the product. The compound N-(4-chlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 81 mg (81%). 1H NMR (500 MHz, CDCl3) δ 7.25 - 7.24 (m, 4H), 6.96 (s, 1H), 6.80 (s, 2H), 4.57 (d, J = 6.0 Hz, 2H), 3.85 (s, 3H), 3.83 (s, 6H). 13 C NMR (126 MHz, DMSO) δ 161.8, 153.3, 147.6, 143.6, 138.9, 137.6, 131.2, 129.2, 129.1, 128.3, 128.1, 124.3, 102.9, 60.1, 56.1, 55.7, 41.3. HRMS (ESI) (m / z): C 20 H 20 ClN3O4, (M+H) + Calculated value: 402.12206; Measured value: 402.12255.

[0127] Example 5 N-(4-Fluorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-5) One equivalent of 4-fluorobenzylamine (31.28 mg, 0.028 mL, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), HOBt (40.53 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 6–7 h. After completion of the reaction, the reaction mixture was extracted with DCM. The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 50% ethyl acetate and hexane as eluents to obtain the product. The compound N-(4-fluorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 72 mg (75%). 1 H NMR (500 MHz, acetone) δ 12.70 (s, 1H), 7.96 (s, 1H), 7.29 (dd, J = 8.5, 5.5 Hz, 2H), 7.00 (s, 3H), 6.94 (t, J = 9.0 Hz, 2H), 4.45 (d, J = 6.5 Hz, 2H), 3.75 (s, 6H), 3.61 (s, 3H). 13 C NMR (126 MHz, acetone) δ 162.8, 160.9, 153.9, 129.5, 129.5, 114.9, 114.8, 103.0, 59.7, 55.6, 41.6.HRMS (ESI) (m / z): C 20 H 20 FN3O4, (M+H) + Calculated value: 386.15161; Measured value: 386.15124.

[0128] Example 6 N-(2-chlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-6) One equivalent of 2-chlorobenzylamine (35.40 mg, 0.030 mL, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), HOBt (40.53 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 6–7 h. After completion of the reaction, the reaction mixture was extracted with DCM. The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 50% ethyl acetate and hexane as eluents to obtain the product. The compound N-(2-chlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 91 mg (91%). 1 H NMR (500 MHz, acetone) δ 12.82 (s, 1H), 8.02 (s, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.29 - 7.27 (m, 1H), 7.19 - 7.13 (m, 2H), 7.01 (s, 3H), 4.56 (d, J = 6.0 Hz, 2H), 3.77 (s, 6H), 3.62 (s, 3H). 13C NMR (125 MHz, acetone) δ 153.9, 138.6, 136.7, 132.6, 129.2, 128.9, 128.6, 127.1, 103.0, 102.5, 59.8, 55.7, 40.3.HRMS (ESI) (m / z): C 20 H 20 ClN3O4, (M+H) + Calculated value: 402.12206; Measured value: 402.12154.

[0129] Example 7 N-(3,4-Dichlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-7) One equivalent of 3,4-dichlorobenzylamine (44.01 mg, 0.033 mL, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), HOBt (40.53 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, A. B. Shaik, B. B. Rao, I. Khan, G. B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 6–7 h. After completion of the reaction, the reaction mixture was extracted with DCM. The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 50% ethyl acetate and hexane as eluents to obtain the product. The compound N-(3,4-dichlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 69 mg (63%). 1H NMR (500 MHz, acetone) δ 13.16 (s, 1H), 8.40 (s, 1H), 7.58 (d, J = 2.0 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.36 (dd, J = 8.5, 2.0 Hz, 1H), 7.17 (s, 2H), 7.14 (s, 1H), 4.62 (d, J = 6.0 Hz, 2H), 3.90 (s, 6H), 3.76 (s, 3H). 13 C NMR (125 MHz, acetone) δ 153.9, 141.1, 138.7, 131.6, 130.4, 130.1, 129.6, 127.7, 103.1, 102.5, 59.7, 55.7, 41.4.HRMS (ESI) (m / z): C 20 H 19 Cl2N3O4, (M+H) + Calculated value: 436.08309; Measured value: 436.08256.

[0130] Example 8 N-(3,5-bis(trifluoromethyl)benzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-8) One equivalent of 3,5-bis(trifluoromethyl)benzylamine (60.78 mg, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), HOBt (40.53 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 6–7 h. After completion of the reaction, the reaction mixture was extracted with DCM. The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 50% ethyl acetate and hexane as eluents to obtain the product. The compound N-(3,5-bis(trifluoromethyl)benzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 108 mg (86%). 1 H NMR (500 MHz, acetone) δ 12.72 (s, 1H), 8.34 (s, 1H), 7.92 (s, 2H), 7.78 (s, 1H), 6.99 (d, J = 5.5 Hz, 3H), 4.68 (d, J = 6.0 Hz, 2H), 3.75 (s, 6H), 3.62 (s, 3H). 13 C NMR (126 MHz, acetone) δ 153.9, 143.5, 138.6, 131.2, 130.9, 128.3, 124.7, 122.6, 120.7, 120.6, 120.6, 103.1, 102.6, 59.8, 55.7, 41.8.HRMS (ESI) (m / z): C 22 H 19 Calculated for F6N3O4, (M+H)+: 504.13580; Found: 504.13430.

[0131] Example 9 N-(Phenylsulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-9) One equivalent of benzenesulfonamide (39.29 mg, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), DMAP (30.54 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 16 h. After completion of the reaction, 1 M HCl (3 mL) was added to the reaction mixture. The reaction mixture was then extracted with DCM. The organic layer was dried over anhydrous MgSO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 70% ethyl acetate and hexane as eluents to obtain the product. The compound N-(phenylsulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a pale yellow solid; 37 mg (35%). 1 H NMR (500 MHz, CDCl3) δ 11.82 (s, 1H), 9.78 (s, 1H), 8.15 (d, J = 8.0 Hz, 2H), 7.65 (t, J = 7.5 Hz, 1H), 7.55 (t, J = 8.0 Hz, 2H), 6.97 (s, 1H), 6.77 (s, 2H), 3.85 (s, 9H). 13C NMR (126 MHz, MeOD) δ 153.7, 133.4, 128.6, 127.8, 103.4, 102.9, 59.8, 55.4.HRMS (ESI) (m / z): C 19 H 19 ClN3O6S, (M+H) + Calculated value: 418.10728; Measured value: 418.10637.

[0132] Example 10 N-((4-Methoxyphenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-10) One equivalent of 4-methoxybenzenesulfonamide (46.80 mg, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), DMAP (30.54 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 16 h. After completion of the reaction, 1 M HCl (3 mL) was added to the reaction mixture. The reaction mixture was then extracted with DCM. The organic layer was dried over anhydrous MgSO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 70% ethyl acetate and hexane as eluents to obtain the product. The compound N-((4-methoxyphenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a pale yellow solid; 38 mg (34%). 1H NMR (500 MHz, DMSO) δ 13.91 (s, 1H), 13.29 (s, 1H), 7.92 (s, 2H), 7.20 - 7.09 (m, 4H), 5.75 (s, 1H), 3.84 (s, 9H), 3.67 (s, 3H). 13 C NMR (125 MHz, DMSO) δ 160.9, 159.6, 153.7, 149.9, 130.3, 118.2, 104.3, 60.5, 56.4, 56.1, 55.4.HRMS (ESI) (m / z): C 20 H 21 N3O7S, (M+H) + Calculated value: 448.11785; Measured value: 448.11839.

[0133] Example 11 N-((4-chlorophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-11) One equivalent of 4-chlorobenzenesulfonamide (47.91 mg, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), DMAP (30.54 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 16 h. After completion of the reaction, 1 M HCl (3 mL) was added to the reaction mixture. The reaction mixture was then extracted with DCM. The organic layer was dried over anhydrous MgSO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 70% ethyl acetate and hexane as eluents to obtain the product. The compound N-((4-chlorophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a pale yellow solid; 46 mg (41%). 1 H NMR (500 MHz, CDCl3) δ 9.72 (bs, 1H), 8.10 (d, J = 9.0 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.26 (s, 1H),6.98 (s, 1H), 6.76 (s, 2H), 3.87 (s, 9H). 13 C NMR (125 MHz, DMSO) δ 160.8, 153.2, 129.5, 128.8, 104.0, 102.7, 60.1, 55.9.HRMS (ESI) (m / z): C 19 H 18 ClN3O6S, (M+H) + Calculated value: 452.06831; Measured value: 452.06874.

[0134] Example 12 N-((4-bromophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-12) One equivalent of 4-bromobenzenesulfonamide (59.02 mg, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), DMAP (30.54 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 16 h. After completion of the reaction, 1 M HCl (3 mL) was added to the reaction mixture. The reaction mixture was then extracted with DCM. The organic layer was dried over anhydrous Na2SO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 70% ethyl acetate and hexane as eluents to obtain the product. The compound N-((4-bromophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a pale yellow solid; 64 mg (52%). 1 H NMR (500 MHz, DMSO) δ 13.96 (s, 1H), 13.37 (s, 1H), 7.90 - 7.83 (m, 4H), 7.24 (s, 1H), 7.10 (s, 2H), 3.83 (s, 6H), 3.67 (s, 3H). 13 C NMR (125 MHz, DMSO) δ 162.8, 161.6, 161.0, 153.7, 132.3, 130.1, 104.6, 103.2, 60.6, 56.4.HRMS (ESI) (m / z): C 19 H 18 BrN3O6S, (M+H)+ Calculated value: 496.01779; Measured value: 496.01878.

[0135] Example 13 N-((4-(trifluoromethyl)phenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-13) One equivalent of 4-(trifluoromethyl)benzenesulfonamide (56.29 mg, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), DMAP (30.54 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, A. B. Shaik, B. B. Rao, I. Khan, G. B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 16 h. After completion of the reaction, 1M HCl (3 ml) was added to the reaction mixture. The reaction mixture was then extracted with DCM. The organic layer was dried over anhydrous MgSO4, and the solvent was evaporated in vacuo. The product was obtained from the residue on silica gel (100-200) column chromatography using 70% ethyl acetate and hexane as eluents. The compound N-((4-(trifluoromethyl)phenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a pale yellow solid; 59 mg (49%). 1 H NMR (500 MHz, DMSO) δ 13.29 (s, 1H), 8.17 (d, J = 7.0 Hz, 2H), 7.98 (s, 2H), 7.20 (s, 1H), 7.10 (s, 2H), 3.83 (s, 6H), 3.67 (s, 3H). 13C NMR (126 MHz, DMSO) δ 153.7, 128.9, 126.3, 104.6, 103.1, 60.5, 56.4.HRMS (ESI) (m / z): C 20 H 18 F3N3O6S, (M+H) + Calculated value: 486.09467; Measured value: 486.09550.

[0136] Example 14 N-((3-(trifluoromethyl)phenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-14) One equivalent of 3-(trifluoromethyl)benzenesulfonamide (56.29 mg, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), DMAP (30.54 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, A. B. Shaik, B. B. Rao, I. Khan, G. B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 16 h. After completion of the reaction, 1M HCl (3 ml) was added to the reaction mixture. The reaction mixture was then extracted with DCM. The organic layer was dried over anhydrous MgSO4, and the solvent was evaporated in vacuo. The product was obtained from the residue on silica gel (100-200) column chromatography using 70% ethyl acetate and hexane as eluents. The compound N-((3-(trifluoromethyl)phenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 32 mg (26%). 1H NMR (500 MHz, DMSO) δ 8.29 - 8.26 (m, 2H), 8.06 (s, 1H), 7.87 (s, 1H), 7.24 (s, 1H), 7.11 (s, 2H), 3.83 (s, 6H), 3.67 (s, 3H). 13 C NMR (126 MHz, DMSO) δ 153.2, 131.5, 130.3, 124.3, 104.2, 102.7, 60.0, 55.9.HRMS (ESI) (m / z): C 20 H 18 F3N3O6S, (M+H) + Calculated value: 486.09467; Measured value: 486.09547.

[0137] Example 15 N-((2-bromophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (l-15) One equivalent of 2-bromobenzenesulfonamide (59.02 mg, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), DMAP (30.54 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, AB Shaik, BB Rao, I. Khan, G.B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 16 h. After completion of the reaction, 1 M HCl (3 mL) was added to the reaction mixture. The reaction mixture was then extracted with DCM. The organic layer was dried over anhydrous MgSO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 70% ethyl acetate and hexane as eluents to obtain the product. The compound N-((2-bromophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 58 mg (47%). 1 H NMR (500 MHz, CDCl3) δ 11.63 (s, 1H), 10.14 (s, 1H), 8.45 (d, J = 7.5 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.50 (t, J = 7.0 Hz, 1H), 6.97 (s, 1H), 6.79 (s, 2H), 3.87 (s, 9H). 13 C NMR (126 MHz, CDCl3)δ 159.0, 153.9, 145.6, 145.2, 138.9, 137.4, 135.3, 135.1, 133.6, 127.9, 123.5, 120.2, 104.3, 102.9, 61.0, 56.23.HRMS (ESI) (m / z): C 19 H18 BrN3O6S, (M+H) + Calculated value: 496.01779; Measured value: 496.01787.

[0138] Example 16 N-((5-chlorothiophen-2-yl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (l-16) One equivalent of 5-chlorothiophene-2-sulfonamide (49.42 mg, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), DMAP (30.54 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, A. B. Shaik, B. B. Rao, I. Khan, G. B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 16 h. After completion of the reaction, 1M HCl (3 ml) was added to the reaction mixture. The reaction mixture was then extracted with DCM. The organic layer was dried over anhydrous MgSO4, and the solvent was evaporated in vacuo. The residue was purified by silica gel (100-200) column chromatography using 70% ethyl acetate and hexane as eluents to obtain the product. The compound N-((5-chlorothiophen-2-yl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 39 mg (34%). 1 H NMR (500 MHz, DMSO) δ 7.62 (s, 1H), 7.24 - 7.22 (m, 2H), 7.11 (s, 2H), 3.84 (s, 6H), 3.68 (s, 3H). 13C NMR (126 MHz, DMSO) δ 161.2, 153.2, 137.5, 127.1, 104.1, 102.7, 60.1, 55.9.HRMS (ESI) (m / z): C 17 H 16 ClN3O6S2, (M+H) + Calculated value: 458.02473; Measured value: 458.02565.

[0139] Example 17 N-((4,5-dichlorothiophen-2-yl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (l-17) One equivalent of 4,5-dichlorothiophene-2-sulfonamide (58.02 mg, 0.25 mmol) was added to a mixture of EDC.HCl (57.51 mg, 0.3 mmol), DMAP (30.54 mg, 0.3 mmol), and 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylic acid (70 mg, 0.25 mmol) (obtained by hydrolysis of ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate prepared by the method described by A. Kamal, A. B. Shaik, B. B. Rao, I. Khan, G. B. Kumara, and N. Jain, Org. Biomol. Chem., 2015, 13, 10162–10178) in DCM (2 mL) at 25 °C. The solution was stirred at room temperature for 16 h. After completion of the reaction, 1M HCl (3 ml) was added to the reaction mixture. The reaction mixture was then extracted with DCM. The organic layer was dried over anhydrous MgSO4, and the solvent was evaporated in vacuo. The product was obtained from the residue by silica gel (100-200) column chromatography using 70% ethyl acetate and hexane as the eluent. The compound N-((4,5-dichlorothiophen-2-yl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide was obtained as a white solid; 63 mg (64%). 1H NMR (500 MHz, DMSO) δ 7.69 (s, 1H), 7.18 (s, 1H), 7.11 (s, 2H), 3.84 (s, 6H), 3.68 (s, 3H). 13 C NMR (126 MHz, DMSO) δ 162.8, 153.7, 137.8, 130.5, 123.1, 104.5, 103.1, 60.5, 56.4.HRMS (ESI) (m / z): C 17 H 15 Cl2N3NaO6S2, (M+H) + Calculated value: 513.96770; Measured value: 513.96866.

[0140] Cell culture and treatments The cells used for this study, human breast adenocarcinoma (MDA-MB-231) and normal human lung fibroblast (WI 30), were obtained from NCCS, Pune, India, and ATCC, VA, USA, respectively. Cells were maintained under standard culture conditions in DMEM supplemented with 10% FBS and 100 units / ml penicillin / streptomycin at 37°C in a humidified incubator under 5% CO2. After 2–3 passages, cells were seeded onto cell culture dishes and used for various experiments.

[0141] For each experiment, a 10 mM stock solution of I-8 was prepared in DMSO and diluted with DMEM supplemented with 5% FBS so that the DMSO concentration was less than 0.1% in the treated cells. Paclitaxel at a concentration of 50 nM was used as a positive control.

[0142] Cytotoxicity study by MTT assay To test the cytotoxicity of NIIST-AC-1 in MDA MB231 cells, an MTT assay was used. 4 Cells / well were seeded into 96-well culture plates and treated with various concentrations of NIIST-AC-1 for 24 hours. The percentage of viable cells was monitored based on the purple color developed by MTT at 570 nm, from which the concentration of NIIST-AC-1 required for 50% inhibition of viability was calculated using the GI50 was calculated as a value.

[0143] Morphological analysis by phase contrast and electron microscopy Morphological analysis of cells treated with various concentrations of NIIST-AC-1 was monitored using a phase-contrast microscope Nikon EclipseTS100 (Nikon Instruments Inc., Melville, NY, USA, 20x magnification) and an SEM (Zeiss EVO 18 Special Edition) at 2kx and 5kx magnification.

[0144] Nuclear segmentation by DAPI staining Nuclear fragmentation upon treatment with NIIST-AC-1 was analyzed using a cell-permeable dye that binds to AT-rich sequences in the minor groove of DNA. After 24 hours of treatment in 96-well culture plates, cells were stained with DAPI for 20 minutes. The morphology of stained nuclei was observed using a spinning disk fluorescence microscope (BD Pathway™ 855; Biosciences) at 360 / 460 nm excitation / emission, and images were captured using BD AttoVision™ software version 1.6.

[0145] Phosphatidylserine translocation by Annexin V staining The translocation of phosphatidylserine to the outer leaflet of the plasma membrane during apoptosis was studied using the Annexin V-Cy3™ Apoptosis Detection Kit. After 24 hours of treatment with various concentrations of NIIST-AC-1, cells were washed and stained with Annexin V for 10 minutes. After removing the staining solution, binding buffer was added, and cells were observed under a spinning disk fluorescence microscope (BD Biosciences), and images were captured using the software BD AttoVision™ version 1.6.

[0146] Caspase-3 and caspase-9 assays The activity of initiator caspase-9 and downstream executioner caspase-3 was measured using a Fluorometric Assay Kit. Caspase activity was measured in cell lysates of control and treated cells using the fluorescent substrates DEVD-AFC (for caspase-3 activity) and LEHD-AFC (for caspase-9 activity). Samples were read on a fluorescence microplate reader (BioTek Synergy HT) using Gen5™ version 1.05.11 software with a 400 nm excitation filter and a 505 nm emission filter, and results were expressed in relative units of fluorescence.

[0147] Measurement of intracellular reactive oxygen species (ROS) The fluorescent probe CM-H2DCFDA (Sigma) was used to analyze the upregulation of ROS upon treatment with NIIST-AC-1. After treatment, cells were incubated in DMEM containing CM-H2DCFDA at a final concentration of 5 μM for 30 min at 37 °C. Trypsinized cells were then suspended in PBA and analyzed using a FACS (BD FACSAria II (BD Biosciences)) in the fluorescein isothiocyanate (FITC) range (excitation 495 nm, emission 529 nm bandpass filter). The mean fluorescence intensity of the various groups was analyzed using BD FACSDiva version 6.1.3 software and corrected for autofluorescence from unlabeled cells.

[0148] Measurement of mitochondrial membrane potential The JC-1 Mitochondrial Membrane Potential Assay Kit was used for fluorescence microscopy studies of the decrease in mitochondrial membrane potential in treated cells. The cells were then treated with various concentrations of NIIST-AC-1 for 24 hours. After removing the spent medium, 100 μl of freshly prepared JC-1 staining solution was added, and the samples were then incubated at 37°C in a CO2 incubator for 20 minutes. Immediately after incubation, the cells were observed under a spinning disk fluorescence microscope (BD Pathway 855), and images were captured using the software BD AttoVision™ version 1.6.

[0149] Protein array experiments on the expression of apoptotic proteins Using an antibody pair-based assay, we analyzed the expression of various proteins in the extrinsic and intrinsic pathways of apoptosis upon treatment with NIIST-AC-1, capturing 43 targets onto membranes. Lysates from control and treated cells were spotted onto the membrane as protein equivalents. Paired biotinylated detection antibodies and streptavidin-HRP were used for development using a chemiluminescence method. Densitometry software was used to compare membranes.

[0150] Anoikis assay The effect of NIIST-AC-1 on anoikis resistance was analyzed using the Anoikis Assay Kit according to the manufacturer's protocol. After pretreatment with various concentrations of NIIST-AC-1, cells were seeded onto ultra-low attachment plates (anoikis chambers). After 48 hours, cells were observed under a phase-contrast microscope (Nikon Eclipse TS100) to observe adhesion, and images were recorded using NIS-Elements 3.21.00 imaging software. Cell viability was also measured by MTT assay.

[0151] Cell cycle analysis The distribution of cells in different phases of the cell cycle was determined by flow cytometry after staining with propidium iodide. After treatment, cells were harvested, ethanol-fixed, and stained with propidium iodide for 30 minutes at 25°C. Cellular DNA content was measured using a BD FACS Fortessa X-20 SORP according to the signal detected in the FL2 channel (excitation, 493 nm; emission, 636 nm), and the data were analyzed using FloJo software, version 9. Approximately 10,000 cells were counted for each analysis, and the distribution of cells in each phase of the cell cycle was saved as a histogram. Western blot analysis was performed to study the expression profiles of various proteins involved in the cell cycle machinery.

[0152] HDAC inhibition assay Histone deacetylase inhibitory activity was measured in cell lysates using a colorimetric assay kit, where we used the substrate Ac-Lys(Ac)-pNA and trichostatin A as a positive control. After the reaction, the absorbance was measured at 400 nm, and the percentage of inhibition was calculated by comparing with the deacetylated standard Ac-Lys-pNA.

[0153] Measurement of anti-migratory capacity To understand the anti-migration effect of NIIST-AC-1, a wound healing assay was performed. After scratching, confluent cell monolayers were treated with various concentrations of the test compound for 24 hours. After that time, wound closure due to cell migration was observed under a phase-contrast microscope (Nikon Eclipse TS100). Images were recorded at 4x magnification at different time points (time points 0 and 24) using NIS-Elements 3.21.00 imaging software. Wound area was quantified using ImageJ 1.52p software.

[0154] Colony formation assay Cells pretreated with various concentrations of NIIST-AC-1 for 48 hours were plated in 6-well plates and allowed to grow and colonize for 14 days. After fixation with acetic acid and methanol, colonies were stained with crystal violet for 2 hours. Visible colonies were photographed, and colony area was quantified using ImageJ 1.52p software.

[0155] Gelatin zymography for measuring MMP activity MMP activity in spent medium from cell cultures was assayed with the substrate gelatin impregnated into an SDS-PAGE gel. After electrophoresis, the gel was washed with Triton and incubated with activation buffer 50 mM Tris containing 0.2 M NaCl and 5 mM CaCl2 at 37°C for 18–20 h to allow the reaction to occur. After staining and destaining with Coomassie Brilliant Blue, gelatin cleavage by MMPs was observed as a white, transparent band and imaged using a ChemiDoc™ MP System with IMAGE LAB™ software (Bio-Rad).

[0156] Glucose uptake studies The effect of NIIST-AC-1 on glucose uptake was analyzed by flow cytometry using 2-NBDG. After incubating cells with various concentrations of NIIST-AC-1, the cell culture medium was replaced with medium containing fluorescent 2-NBDG and incubated for 30 minutes. The samples were then analyzed using a BD FACSAria II (BD Biosciences) in the FITC range (excitation, 465 nm; emission, 540 nm bandpass filter). The mean fluorescence intensity of different groups was analyzed using BD FACSDiva version 6.1.3 software and corrected for autofluorescence from unlabeled cells.

[0157] Study of EGFR [epidermal growth factor receptor] expression Indirect immunofluorescence was used to evaluate EGFR expression after treatment with NIIST-AC-1. Treated cells were fixed with paraformaldehyde and incubated overnight with primary antibodies. After washing out the primary antibodies, cells were treated with Alexa Fluor-conjugated secondary antibodies for visualization. Cells were counterstained with DAPI for nuclear staining and observed under a fluorescence microscope (IX83 inverted microscope; Olympus Life Science, cell Sens Dimension ver. 3.1 software).

[0158] RNA sequencing for whole transcriptome analysis Total RNA was isolated from cells using the RNeasy Mini Kit. 15 μg of cellular RNA was quality assessed (Agilent Technologies Bioanalyzer) and used for mRNA library preparation. mRNA was fragmented, and first-strand cDNA was synthesized from the cleaved RNA using random primers, followed by second-strand cDNA synthesis. The purified cDNA template was enriched by PCR amplification to generate a cDNA library. The resulting library was submitted to an RNA sequencing facility for two rapid single-read 50 Illumina HiSeq sequencing runs. Raw reads from separate lanes of the same sample were combined for mapping. RNA sequencing was performed at Eurofins Genomics India Pvt. Ltd., Bengaluru 560048, Karnataka, India.

[0159] Identification of differentially expressed genes (DEGs) The output raw FASTQ files were analyzed using various tools on the Galaxy platform to extract differentially expressed genes, as follows: FastQC (Galaxy Version 0.72 + galaxy1) was used to analyze the quality of the reads; MultiQC (Galaxy Version 1.9 + galaxy1) was used to compile quality check reports; Cutadapt (Galaxy Version 1.16.6) was used for adapter sequence removal and filtering using a minimum read length of 20 and a minimum quality (phred score) threshold of 20. Again, quality was checked and results were compiled. HISAT2 (Galaxy Version 2.1.0 + galaxy7), a splice-aware alignment program, was used to map the cleaned reads to the reference genome (Homo_sapiens.GRCh38.dna_sm.primary_assembly.fa.gz) and annotation file (Homo_sapiens.GRCh38.104.gtf.gz). The output bam file was processed using StringTie (Galaxy Version 2.1.1) to assemble and quantify RNA-Seq alignments into potential transcripts. Later, normalized differentially expressed genes were determined from the Stringtie output count table using Deseq2 (Galaxy Version 2.11.40.6 + galaxy1). A total of 26,278 differentially expressed genes were obtained, of which 12,768 were upregulated and 13,610 were downregulated. These differentially expressed genes were filtered using Filter (Galaxy Version 1.1.1) to extract significant ones by adding filters of q-value ≤ 0.05 and log2 fold change > 1. The resulting 1,337 upregulated and 1,642 downregulated genes were used for further downstream analysis.

[0160] Functional and pathway enrichment analysis To further understand the biological functions of the up- and down-regulated DEGs, we performed enrichment analysis using the online tool ToppFun from the Toppgene suite. Functional annotations (biological processes, molecular functions, and pathways) were considered significant at a false discovery rate (FDR) of less than 0.05. Gene set enrichment analysis (GSEA) on a ranked list of total DEGs (based on log fold change) was performed on the CP: Classical Pathways (a collection of 2922 predefined gene sets) from the Molecular Signatures Database (MSigDB) using the GSEA Preranked tool in the standalone R program GSEA 4.1.0. Enriched pathways with an FDR q-value of less than 0.25 were considered significant.

[0161] Protein-protein interaction network and cluster identification Proteins and their interactions form a protein-protein interaction network, in which proteins are nodes and interactions are edges. To visualize the functional interactions of proteins encoded by the identified DEGs, a PPI network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING; version 11.0) with a high confidence score of 0.7. This network was then visualized using Cytoscape (version 3.7.1) software and clustered to identify functional modules using the MCODE plugin (v1.6.1) with default parameters (degree cutoff, 2; node score cutoff, 0.2; k-core, 2, and maximum depth, 100). Functional enrichment analysis of these clusters was performed using the online tool ToppFun from the Toppgene suite to identify significant functions.

[0162] Advantages of the Invention Breast cancer mortality continues to be a major public health issue in 2021. Early diagnosis and screening are two key strategies that can improve breast cancer outcomes and survival. Various types of treatment strategies, including radiation, surgery, chemotherapy, and targeted therapy, are used for breast cancer patients. Triple-negative breast cancer is the most devastating form, lacking expression of progesterone receptors, human epidermal growth factor receptor 2, and estrogen receptors. This type is most likely to spread beyond the breast and is more likely to recur after treatment. It is more common in young women under 50 years of age and is most commonly detected with inherited BRCA1 mutations. It is usually treated with a combination strategy using radiation, surgery, and chemotherapy. Doxorubicin, cyclophosphamide, and paclitaxel are common chemotherapy drugs for TNBC. Adding a taxane to an anthracycline drug has improved the risk of recurrence and mortality compared to other cytotoxic drugs. Poly (ADP-ribose) polymerase (PARP) is an enzyme that fixes DNA damage in both healthy and cancer cells. PARP inhibitors, such as olaparib and talazoparib, have been approved for the treatment of advanced TNBC with BRCA1 or BRCA2 mutations. The combination of an immune checkpoint (PDL1) inhibitor and the chemotherapy drug Abraxane (Tecentriq) has also been approved for the treatment of locally advanced metastatic triple-negative PDL1-positive breast cancer. Briefly, cytotoxic chemotherapy is the mainstay of TNBC treatment. However, not all of these drugs are specific and have numerous side effects. The immunosuppression associated with chemotherapy also leads to several other complications. The inclusion of taxanes can cause neurotoxicity, and the long-term use of anthracyclines can lead to cardiac toxicity. Therefore, the identification of small molecules with lower toxicity and their use alone or in combination with already known chemotherapy drugs holds promise.

[0163] The identification and validation of targeted therapies for TNBC patients is of paramount importance in breast cancer treatment. Small molecule-based anticancer drugs that are non-toxic to normal cells and can inhibit cancer cells along multiple pathways are needed.

Claims

1. Formula I 【Chemistry 1】 [In the formula, R=-CH 2 -If R 1 is selected from the group consisting of benzene, 4-methylbenzene, 4-methoxybenzene, 4-chlorobenzene, 4-fluorobenzene, 2-chlorobenzene, 3,4-dichlorobenzene, and 3,5-trifluoromethylbenzene; R=-SO 2 -If R 1 is selected from the group consisting of benzene, 4-methoxybenzene, 4-chlorobenzene, 4-bromobenzene, 4-trifluoromethylbenzene, 3-trifluoromethylbenzene, 2-bromobenzene, 5-chlorothiophen-2-yl, and 4,5-dichlorothiophen-2-yl.

2. 10. The compound of claim 1, useful as an anticancer drug in triple-negative breast cancer (TNBC) cells.

3. The compound of formula I is i. N-benzyl-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-1); 【Chemistry 2】 ii. N-(4-methylbenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-2); 【Transformation 3】 iii. N-(4-methoxybenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-3); 【Chemistry 4】 iv. N-(4-chlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-4); 【Transformation 5】 v. N-(4-fluorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-5); 【Transformation 6】 vi. N-(2-chlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-6); 【Transformation 7】 vii. N-(3,4-dichlorobenzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-7); 【Transformation 8】 viii. N-(3,5-bis(trifluoromethyl)benzyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-8); 【Chemistry 9】 ix. N-(phenylsulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-9); 【Chemistry 10】 x. N-((4-methoxyphenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-10); 【Chemistry 11】 xi. N-((4-chlorophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-11); 【Chemistry 12】 xii. N-((4-bromophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-12); 【Chemistry 13】 xiii. N-((4-(trifluoromethyl)phenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-13); 【Chemistry 14】 xiv. N-((3-(trifluoromethyl)phenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-14); 【Chemistry 15】 xv. N-((2-bromophenyl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-15); 【Chemistry 16】 xvi. N-((5-chlorothiophen-2-yl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-16); 【Chemistry 17】 xvii. N-((4,5-dichlorothiophen-2-yl)sulfonyl)-3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxamide (I-17). [Chemistry 18] 2. The compound of claim 1, selected from the group consisting of:

4. A process for preparing a compound of formula I according to claim 1, comprising: i. condensing a compound of formula 1 with diethyl oxalate to obtain ethyl 2,4-dioxo-4-(3,4,5-trimethoxyphenyl)butanoate of formula 2; 【Chemistry 19】 ii. cyclocondensing the compound of formula 2 with hydrazine hydrate hydrochloride to form a pyrazole ester compound of formula 3; 【Chemistry 20】 iii. Base-mediated hydrolysis of a compound of formula 3 to give a compound of formula 4 【Chemistry 21】 and subsequently reacting the compound of formula 5[R1-R-NH 2 ] wherein R and R 1 is as defined in claim 1] to obtain a compound of formula I.

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