Structure–activity insights and molecular modeling approaches of anti-TNBC agents: a comprehensive systematic review

Abstract

Aim

Triple-negative breast cancer (TNBC) is a highly aggressive and treatment-resistant subtype of breast cancer, characterized by the lack of hormone receptor expression. This study aims to comprehensively evaluate the structure–activity relationship (SAR), structural modification, pharmacokinetic profiles, and molecular interaction of bioactive compounds developed for TNBC treatment from 2016 to February 2025.

Methods & results

Three identified publications were complemented by a selection of 19 articles, each adhering to predefined criteria. Modification of nitrogen heterocycles, phenolic compounds, β-lactams, and halogenated derivative compounds enhanced cytotoxic potency and selectivity against key targets, including CDK9, EGFR, FOXM1, PARP1, and tubulin.

Conclusion

Strategic structural modifications significantly enhance the potency, selectivity, and pharmacokinetics of anti-TNBC agents. Future research should emphasize polypharmacology, advanced delivery strategies, and translational validation to address TNBC heterogeneity.

PLAIN LANGUAGE SUMMARY

Triple-negative breast cancer (TNBC) is an aggressive cancer type with limited treatment options. These are the reasons for the critical need for the discovery and development of novel anti-TNBC drugs, a process significantly aided by Structure–Activity Relationship (SAR) investigations. SAR studies are crucial for guiding compound modifications, ultimately aiming to identify potent compounds with exceptional anti-TNBC activity.

ARTICLE HIGHLIGHTS

• Nitrogen heterocyclic compounds effectively inhibited CDK9, Hsp90, and HDAC6 via hydrogen bonding and hydrophobic interactions, with (S,S) conformers consistently demonstrating superior potency over (R,R) counterparts, attributed to strategic halogen substitutions and linker alterations.

• For phenolic derivative compounds, benzyl-containing derivatives uniquely achieved HO-2 selectivity by effectively targeting the HO-1 hydrophobic pocket due to their enhanced steric adaptability.

• For 3-hydroxy-β-lactam derivatives, studies revealed that (S,S) conformers exhibited enhanced stability and potency, with small halogen substituents being vital for boosting cytotoxic activity against the Colchicine Binding Site (CBS), underscoring the paramount importance of stereochemistry and halogen substitution.

• Sulfur-containing compounds featuring small electron-withdrawing groups, such as trifluoromethyl (-CF₃), displayed increased FOXM1 inhibition and anti-GAC activity.

• Polycyclic aromatic compounds showed anti-TNBC activity by inhibiting CDK2 and suppressing Wnt and SIRT1 pathways. Optimal effects were observed with methoxyl groups and bioisosteric substituents.

• Polypyridyl Ru(II) metal compounds required the presence of larger planar aromatic phenazines to achieve more potent tumor proliferation inhibition.

Graphical Abstract

1. Introduction

Triple-Negative Breast Cancer (TNBC) is a breast cancer subtype characterized by the absence of estrogen and progesterone receptor expression, as well as the lack of HER2 amplification. TNBC exhibits high chromosomal instability and an aggressive clinical phenotype [1]. TNBC occurs more frequently in women under 50 years of age and is more prevalent, among African-American and Black ethnic groups. It commonly as interval cancers and is associated with high chemosensitivity, a weak correlation between tumor size and lymph node metastasis elevated risks of brain metastasis and early recurrence (typically within 1–3 years post-diagnosis). TNBC also has shorter post-metastasis survival compared to other subtypes [2].

According to the Global Cancer Observatory (GLOBOCAN) 2020 data, breast cancer accounts for 16.6% of Indonesia’s 396,914 new cancer cases, with TNBC representing 10–15% of all breast cancer cases [3]. Notably, TNBC constitutes 24% of newly diagnosed invasive breast cancers and poses greater therapeutic challenges [4]. The prognosis for TNBC remains poor due to its aggressive nature, rapid growth, and frequent metastasis at diagnosis. Moreover, TNBC demonstrates higher recurrence rates post-treatment compared to other breast cancer subtypes [4]. In Indonesia, a 2015–2019 cohort study at Hasan Sadikin Hospital identified 628 TNBC cases among 4,050 breast cancer patients (15.5% prevalence), with 11 recurrence cases (1.7%) [4].

Currently, several chemotherapy options are employed to treat triple-negative breast cancer (TNBC), with the selection largely depending on the stage and progression of the disease. Platinum-based agents, such as cisplatin and carboplatin, are commonly administered during the early stages due to their DNA-damaging effects, which are particularly effective in TNBC cells that lack DNA repair mechanisms [5,6]. Microtubule-targeting agents like taxanes (paclitaxel and docetaxel) are also frequently used to disrupt cell division [7]. In addition, targeted therapies such as PARP inhibitors (e.g., olaparib and talazoparib) have shown efficacy, especially in patients with BRCA mutations [8], while angiogenesis inhibitors like bevacizumab (a VEGFR inhibitor) may be considered in specific cases [9]. Furthermore, immunotherapy has emerged as a promising approach, with the PD-L1 inhibitor pembrolizumab recently approved for use in combination with chemotherapy for PD-L1-positive TNBC [8]. Recently, the antibody-drug conjugate sacituzumab govitecan has been introduced as a treatment for patients with advanced or recurrent TNBC [10,11], offering a new therapeutic option for difficult-to-treat cases.

For decades, cancer management has relied on surgery, radiotherapy, and chemotherapy. However, developing effective treatments remains a major medical challenge. While chemotherapy is highly effective in elaminarting cancer cells, its systemic toxicity adversely affects healthy tissues. Beyond conventional modalities, there is a critical need to develop safer and more targeted anticancer agents [12]. For millennia, plants have served as sources of therapeutic compounds. Their vast chemical diversity offers immense potential for drug discovery. Given this vast natural abundance, high-throughput and rational approaches are now essential to uncover untapped anticancer compounds. Nevertheless, modern cancer therapies remain inaccessible to low-income populations [12]. As such, research has increasingly focused on discovering potent natural biomolecules with minimal side effects and developing cost-effective therapies. Medicinal plants, with their diverse chemical constituents, are pivotal for identifying novel antitumor agents. Over centuries, plant-derived secondary metabolites have played a central role in anticancer drug development. Notably, over 60% of FDA-approved anticancer drugs from 1983–1994 were derived from natural sources, and as of 2014, 771 new cytotoxic compounds were under development [13].

Most drugs act via specific interaction with macromolecular target, with their activity determined by chemical structure, dynamic conformational properties, and binding affinity [14]. Natural products and their derivatives have long served as key bioactive compounds, in treating human disease. To optimize their efficacy and pharmacological properties, modern strategies such as scaffold-based structural modifications, pharmacophore hybridization (combining critical functional moieties), and novel drug formulation approaches have been employed [15].

This review systematically evaluates the structure–activity relationships (SAR) of bioactive compounds reported from 2016 until February 2025 for TNBC treatment, identifying structural correlates of anticancer potency, target selectivity, and pharmacokinetic stability. Furthermore, it analyzes molecular modeling outcomes to advance structure-based drug design for TNBC.

2. Methods

2.1. Protocol and registration

This systematic review study was reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) for the guidelines. The study protocol has been prospectively registered in the Open Science Framework (OSF) under the identifier https://doi.org/10.17605/OSF.IO/XAVM4. Two authors independently performed steps of study selection, data extraction and reviews. As a referee in case of discrepancies dan disagreement, the third and fourth author was brought in for consultation. The main variables are TNBC cytotoxic activity of natural product derivatives, pharmacokinetic profiles that increased the anticancer activity, SAR, and molecular mechanism interactions over the compound and target receptor, including in silico approaches or in vivo and in vitro correlation.

2.2. Data sources and searches

Comprehensive literature searches were conducted across three major scientific databases: PubMed (National Library of Medicine), ScienceDirect, and Scopus, focusing exclusively on English-language peer review articles published between 2016 and February 2025. The restriction on the publication year range of selected research articles is intended to delineate and ensure the most current review of research advancements in compound modification for anti-TNBC activity. The search strategy utilized the Boolean keyword combination: ("TNBC") AND ("molecular modeling" OR "in silico" OR "computational") AND ("structure–activity relationship") to ensure comprehensive coverage of relevant publications.

2.3. Study selection

The study selection process followed a rigorous four-stage screening protocol without blinding. The first stage involved the initial screening of articles from the search sources, considering the publication year, article type, and accessibility. Results were uploaded to Rayyan web app as a systematic reviews tool manager. In the second stage, duplicate records were identified [16]. The third stage consisted of screening titles and abstracts to assess relevance based on the inclusion and exclusion criteria referred to in Table 1. In the fourth stage, full-text articles were reviewed, and studies that did not meet the criteria or did not align with the scope and objectives of this review were excluded. To minimize bias, the study selection carried out by two independent reviewers (RAF and DN) based on the predefined PICOS criteria and screener instruction. Discrepancies between the reviewers between screening process were resolved through a consensus meeting. Final binding decision was made by third and fourth reviewers and as a supervisor (NKKI and MM) in cases where consensus could not be reached.

Table 1.

Inclusion and exclusion criteria for article selection.

No.	Parameter	Inclusion	Exclusion
1	Language	English	Other languages
2	Study Type	In silico and/or in vitro, and/or in vivo studies	Articles focusing solely on compound synthesis without SAR methodology
3	Research Output	Explains structure–activity relationships for TNBC	Articles presenting only results without interpretation
4	Publication Type	Original research article	Reviews, book chapters, table of content, abstract, erratum, in press article, preprint article
5	Article Accessibility	Full text open access	Articles without full-text open access

2.4. Data extraction and synthesis

Two authors (RAF and DN) independently extracted data from selected studies, without blinding for the extraction and synthesis stage, as prior knowledge of the research questions is essential for accurate SAR interpretation. One author (DN) provided information and data extraction parameters contained in extractor instruction. The baseline characteristics that were remove include the first author’s last name, year of publication, synthesis protocols of compound, and biological assay specific methods. Our outcomes of interest for this systematic review were structure modification and correlations with cytotoxicity effect, either with biological assay or prediction activity, such as for pharmacokinetic profiles and interaction simulations between receptor and compound. Two reviewers (RAF and DN) as a extractor and synthesist were independently worked in extracted and synthesis process. The agreement was assessed with discussion. Discrepancies will be resolved by discussion, and in case anything unresolved discuss were adjudicated by third and fourth reviewer (NKKI and MM) as a supervisor. The chemical structures, tables and figures in this article were created using ChemDraw Proffesional 12.0, Biorender and Canva to ensure originality and avoid copyright issues.

2.5. Quality assessment and risk of bias analysis

The Quality Assessment Tool for In Vitro Studies (QUIN tool) was utilized to evaluate and quantify the risk of bias in individual in vitro studies as well as to enable comparison across multiple studies [17]. The evaluation was based on 12 criteria with scoring systems as listed in Table 2. Score 2 was assigned when the criterion was adequately specified and clearly stated in the articles, a score of 1 when it was inadequately defined, and a score 0 when it was not mentioned in the article. Studies are categorized as low risk of bias if scored over 70%, and those with value between 50–70% are categorized as medium risk, while studies scored less than 50% are considered as high risk of bias [17]. The critical appraisal was independently performed by two authors (RAF and DN) while the third and fourth authors (NKKI and MM) reviewed and resolved any discrepancies.

Table 2.

QUIN Tool assessment for in vitro study of the articles.

References	Clearly stated aims/objectives	Detailed explanation of sample size calculation	Detailed explanation of sampling technique	Details of comparison group	Detailed explanation of methodology	Operator details	Randomization	Method of measurement of outcome	Outcome assessor details	Blinding	Statistical analysis	Presentation of results	Total score	Final score %	Risk of bias
[18]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[19]	2	0	N/A	2	2	0	N/A	2	0	N/A	1	2	11	61.11%	Medium
[20]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[21]	2	0	N/A	2	2	0	N/A	2	0	N/A	1	2	11	61.11%	Medium
[22]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[23]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[24]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[25]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[26]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[27]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[28]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[29]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[30]	0	1	N/A	2	2	0	N/A	2	0	N/A	2	2	11	61.11%	Medium
[31]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[32]	2	0	N/A	2	2	0	N/A	2	0	N/A	1	2	11	61.11%	Medium
[33]	2	0	N/A	2	2	0	N/A	2	0	N/A	2	2	12	66.67%	Medium
[34]	2	0	N/A	1	2	0	N/A	2	0	N/A	1	2	10	55.56%	Medium

N/A = Not applicable.

3. Results

3.1. Study selection, extraction, and synthesis data

The comprehensive database search initially identified 53 potentially relevant articles. Following the PRISMA guidelines [35], the screening process eliminated 7 duplicate records, resulting in 46 articles proceeding to subsequent evaluation stages. Title and abstract screening excluded 21 non-conforming articles, with full-text assessment disqualifying an additional 8 publications that failed to meet the established criteria. Consequently, the final analysis incorporated 19 high-quality research articles that satisfied all methodological requirements for inclusion in this systematic review. This rigorous selection process (Figure 1) ensured the incorporation of only the most relevant and methodologically sound studies for SAR evaluation of TNBC therapeutics. There are not missing data from a study that marked as “Not Reported” due to clearly data contained in selected articles. Bias assessment for the in vitro studies revealed that the 17 studies contained in vitro assay had a medium risk of bias [17].

Figure 1.

Literature search strategy diagram based on PRISMA guidelines.

3.2. Structure–activity relationship collected

The articles reviewed provide detailed SAR insights for each compound modification, indicating changes that either enhanced or diminished (or even eliminated) activity. Modifications were diverse, encompassing the addition or alteration of substituents on pharmacophore groups or frameworks, changes in substituent position, conformational alterations, and variations in the length of linker compounds.

3.3. Cytotoxicity activity

The collected articles encompass the structural modification of derivative compounds and the evaluation of their cytotoxic activity against TNBC cells under varying treatment durations. Table 3 summarize the structural frameworks, the optimum compounds from the studies identified by the researchers in each article, their IC₅₀ values, and treatment durations. The IC₅₀ values for the optimum compounds across these articles indicate cytotoxic activity, ranging from 0.0007 to 17.2 μM within treatment periods of 24 to 72 h.

Table 3.

Biological activity values of the anti-TNBC derivatives.

Core structure	Derivative compound	Modification	IC50 (μM)	Treatment time	References
Benzothiazole hybrid thiazolidine-2,4-dione	5-[3-(Trifluoromethyl)benzylidene]thiazolidine2,4-dione (Cpd. KC21)	Trifluoromethyl substituted	10.77	96 h	[18]
Arylsulfonylhydrazone	N’-[(E)-(5-chloro-1H-indol-3-yl)methylidene]benzenesulfonohydrazide (Cpd. 1e)	Phenyl and 5-chloroindole substiuted	0.9	72 h	[19]
Penfluridol analog	1-(5,5-bis(4-fluorophenyl)pentyl)-3-(4-nitro-3-(trifluoromethyl)phenyl)urea (Cpd. 4q)	Fluoro substituted at meta- position linker	4.55	48 h	[20]
Aminothiazole	4-(2-((4-fluorophenyl)amino)thiazol-4-yl)benzonitrile (Cpd. C12.30)	4-F in para position substituted (ring A) and 4-CN in para position	17	72 h	[31]
Piperine based urea	1-((1E,3E)-4-(Benzo[d]1,3dioxol-5-yl)buta-1,3-dien-1-yl)-3-(4-chlorophenyl)urea (Cpd. 8q)	substituted 4-Cl	18.7	48 h	[22]
Tetrahydro-b-carboline-based	7-((6S-12aS)-6-(3-Bromophenyl)-1,4-dioxo-3,4,6,7,12,12a-hexahydropyrazino[1′.2′:1,6]pyrido[3,4-b]indol-2(1H)-yl)-N-hydroxyheptanamide (Cpd. 9c)	m-bromophenyl derivative	0.021	24 h	[23]
Amino-pyrazolo-pyrimidine core	[(1S,3S)-3-[[5-(1-Ethylpropyl)pyrazolo[1,5-a]pyrimidin-4-ium-7-yl]amino]cyclopentyl]ammonium Dichloride (Cpd. 28/KB-0742)	(S,S)-diaminocyclopentane with additional methyl	0.88	72 h	[24]
C3-aminopyridinyl riminophenazine	Riminophenazine 5-(4-(chlorophenyl)-3-((2-(piperazin- 1-yl)ethyl)imino)-N-(pyridin-3-yl)-3,5-dihydrophenazin-2-amine (Cpd. MU17)	ethylendiamine-spaced N-methylpiperazinyl derivative	1.1	24 h	[25]
Chiral trans 3-hydroxyl β-lactams	Fluoro-substituted (+)-(3S,4S)-4-(3-fluoro-4-methoxyphenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (Cpd. 27EN1)	B ring meta fluoro-substituted	0.0007	72 h	[26]
Isatin hybrid hydrazone based	(E)-1-Benzyl-3-((2-(4-nitrophenyl)-2-oxoethyl)imino) indolin-2-one (Cpd. 23)	Nitro substitution at the 4th position of the phenyl ring	15.8	48 h	[27]
3-substituted indole (bis-(3-indolyl) methanes)	3,3′-((4-chlorophenyl)methylene)bis(5-bromo-1H-indole) (Cpd. 5h)	Group I with 5-bromoindole and 4-chlorobenzaldehyde	8.73	72 h	[11]
Benzylbenzofurans and isoflavone derivatives	6-methoxy-4′,6′-dimethylisoflavone-2′,5′-quinone (Cpd. 39)	MeO moiety at isoflavone’s and methyl moiety at 4,6 benzene ring position	6.34	24 h	[28]
Quinoline sulfonate	5-Nitroquinolin-8-yl 5-chloro-2-fluorobenzenesulfonate (Cpd. 20)	Substituted NO2 at R1 and 5-Cl-2-Ph at R2	5.30	2 h	[32]
2-arylquinazolinechalcones	(E)-1-(3,4-dimethoxyphenyl)-3-(3-((2-(3,4-dimethoxyphenyl) quinazolin-4-yl)amino)phe nyl)prop-2-en-1-one (Cpd. 7n)	Methoxy groups at the phenyl ring of the quinazoline	0.45	72 h	[21]
Butylimidazolic pharmacophore with a hydrophobic moiety spaced	2-(4-(1H-imidazol-1-yl) butoxy)-N-benzyl-5-iodobenzamide (Cpd. VP 21-04)	Benzyl moiety substituted	0.9	48 h	[30]
TVS21	4-((3,4-Dichlorophenoxy)methyl)-1-(4-((1-methylpiperidin- 4-yl)methoxy)benzyl)piperidin-4-ol (Cpd. 104)	Methylen group (CH2) between piperidine ring B and phenyl ring C) and methyl moiety in piperidine ring D	4.4	72 h	[33]
Ruthenium polypiridine complex	Ru(bpy)2BEDPPZ	Benzo[e]dipyrido[3,2-b:2′,3′-h]phenazine substituted	17.2	72 h	[34]

3.4. Molecular interaction

Table 4 details the targeted receptors, revealing crucial strong interactions with specific amino acids that contribute to effective receptor inhibition. These receptors include EGFR, FOXM1, CDK9, and the Colchicine Binding Site. While each study utilized different receptor crystal structures, and some did not report binding energy values or docking scores, the visualized interaction results sufficiently illustrate the shared importance of hydrogen bonds, pi-pi stacking, and halogen bonds in the compounds’ inhibitory activity.

Table 4.

Type of anti-TNBC compound’s molecular interactions.

Compound	Receptor target	Binding affinity (kcal/mol)	Amino acid residue	Type of interaction	References
4-(4-chloropiperidin-1-yl)-N-[4-(3-piperidin-1-ylpropoxy)phenyl]pyrimidin-2-amine (Cpd. 15)	Thyroid Hormone Receptor (PDB ID: 1Y0X)	−7.4	GLU311 ARG429 VAL458 ILE303 LYS306 ARG383 PRO384	Conventional hydrogen bond interaction Carbon hydrogen bond interaction Alkyl interaction	[36]
5-[3-(Trifluoromethyl)benzylidene]thiazolidine2,4-dione (Cpd. KC21)	FOXM1-DNA Binding Domain (PDB ID: 3G73)	−6.8	ASN283 ARG286 TRP308 LEU291 LEU259 HIS287 ARG286 TRP308	Hydrogen bond interaction π-alkyl interaction π- π T-shaped interaction Van der Waals interaction	[18]
1-(5,5-bis(4-fluorophenyl)pentyl)-3-(4-nitro-3-(trifluoromethyl)phenyl)urea (Cpd. 4q)	ATP-binding cleft of FGFR1 (PDB ID: 4V05)	Not Available	LYS514 GLU531 ASP641 GLU562 ALA564	Hydrogen bond interaction	[20]
4-(2-((4-fluorophenyl)amino)thiazol-4-yl)benzonitrile (Cpd. C12.30)	Allosteric GAC binding site (PDB ID: 4JKT)	Not Available	LYS352C LEU328B PHE327B PHE327C TYR399B TYR399C LEU326B LEU326C	Polar interaction Hydrophobic interaction	[31]
1-((1E,3E)-4-(Benzo[d]1,3dioxol-5-yl)buta-1,3-dien-1-yl)-3-(4-chlorophenyl)urea (Cpd 8q)	VEGFR-2 (PDB ID: 4ASD)	−28.34	ASP1046 GLU885 LEU840 VAL848 PHE1047 CYS919 VAL899 PHE918 CYS1045 ILE592 ILE888 LEU889 ASP104	Essential hydrogen interaction Hydrophobic interaction	[22]
7-((6S-12aS)-6-(3-Bromophenyl)-1,4-dioxo-3,4,6,7,12,12a-hexahydropyrazino[1′.2′:1,6]pyrido[3,4-b]indol-2(1H)-yl)-N-hydroxyheptanamide (Cpd. 9c)	HDAC6 CD2 Domain (PDB ID: 5WGI)	Not Available	TYR745 HIS573 HIS574 PHE643 ASP460	Bidentat interaction Hydrogen interaction CH-pi interaction Halogen interaction	[23]
[(1S,3S)-3-[[5-(1-Ethylpropyl)pyrazolo[1,5-a]pyrimidin-4-ium-7-yl]amino]cyclopentyl]ammonium Dichloride (Cpd. 28/KB-0742)	ATP cognitive bindng pocket CDK9/siklin T1 (PDB ID: 3MY1)	Not Available	CYS106 ASP109 LEU156	Hydrophobic interaction	[24]
Fluoro-substituted (+)-(3S,4S)-4-(3-fluoro-4-methoxyphenyl)-3-hydroxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (Cpd. 27EN1)	Colchicine binding site (PDB ID: 1SA0)	Not Available	LYS B352 ASN B258 THR Α 179 THR B 353 LYS B 254 THR B 314 ASN B 350 CYS B 241 SER Α 178 GLN B 247 ILE B 378 ALA B 354 VAL B 318 LEU B 255 VAL B 315 VAL B 317 ALA B 316 LEU B 242 VAL B 238 ALA B 250 LEU B 248 ALA Α 180 VAL Α 181	Hydrophobic interqction Polar interaction	[26]
(E)-1-Benzyl-3-((2-(4-nitrophenyl)-2-oxoethyl)imino) indolin-2-one (Cpd. 23)	EGFR kinase (PDB ID : 1M17	−7.561	M769	Hydrogen bond interaction	[27]
3,3′-((4-chlorophenyl)methylene)bis(5-bromo-1H-indole) (Cpd. 5h)	PARP1 (PDB ID: 4ZZZ)	−11.3	HIS632 LYS587 ARG590 ARG488 THR 718	Van der Waals Interaction Conventional hydrogen bond interaction Halogen interaction π-Cation interaction Alkyl/π-Alkyl Interaction	[28]
6-methoxy-4′,6′-dimethylisoflavone-2′,5′-quinone (Cpd. 39)	SIRT1 (PDB ID: 4I5I)	−70.51	GLN345 ALA262 SER442 LYS444 ARG274 HIS363 PHE273	Hydrogen Interaction π- π Stacking Interaction	[29]
5-Nitroquinolin-8-yl 5-chloro-2-fluorobenzenesulfonate (Cpd. 20)	EGFR Kinase (PDB ID: 3W2S)	−10.73	THR790 LYS745	Hydrogen Bond Interaction	[32]
4-((3,4-Dichlorophenoxy)methyl)-1-(4-((1-methylpiperidin- 4-yl)methoxy)benzyl)piperidin-4-ol (Cpd. 104)	Hsp90β CTD binding site (PDB ID: 5FWK)	Not Available	ILE605B ALA608B ALA608A GLU489A ALA600A	Not Available	[33]

3.5. Other results

Some articles investigated the SAR in silico using Qualitative Structure–Activity Relationship (QSAR) methods. They employed software and biplot analysis to depict the structure–activity relationships of the studied derivative compounds, consequently not detailing the complete biological assay procedures. However, these studies still reported cytotoxicity activity values by converting IC₅₀ to pIC₅₀ values, illustrating the SAR for the developed derivative structural models.

4. Discussion

4.1. Structure–activity relationship of anti-TNBC compounds

4.1.1. Nitrogen heterocyclic compounds

Amino-pyrazolo-pyrimidine (Figure 2a, structure of amino-pyrazolo-pyrimidine scaffold) derivatives have been developed as selective CDK9 inhibitors for TNBC therapy, owing to their structural similarity to ATP and their ability to form hydrogen bonds with key hinge-region residues Asp109 and Glu107 [24,37,38]. Modifications at the R2 position (Figure 2a, blue circle), such as the replacing a cyclopentane ring with a cyclobutane ring, as explored by Freeman et al. [24], maintained potency due to preserved ionic and hydrogen bonding interactions and retained spatial flexibility [24,37]. In contrast, methyl substitution at R2 reduced activity, highlighting the importance of two hydrogen atoms in forming stable interactions with Asp109 and Glu107 [24]. Notably, hydroxyl substitution restored activity by enhancing polarity and hydrogen bonding potential [24,39].

Figure 2.

Core structure of bioactive anti-TNBC compounds, including (a–d) Nitrogen heterocyclic, (e) Phenolic derivative, (f) 3-hydroxy-β-lactam, (g, h) sulfur-based, (i–m) polycyclic aromatic, (n) polypyridyl Ru(II) metal, (o) Isatin-hydrazone derivative, and (p) others.

Chlorine substitution at position 3 of the pyrazolopyrimidine core improved CDK9 activity but reduced selectivity for CDK4, whereas a 3-pentyl group at R1 (Figure 2a, red circle) as contained in compound 28 (Figure 3a) yielded potent inhibition (IC₅₀ = 6 nM) and over 66-fold selectivity toward CDK9 via strong hydrophobic interactions with Leu156 and the glycine-rich loop [24,37]. R2 modifications involving acetamide, amide, or methyl sulfonamide groups had limited impact on potency, while methyl urea enhanced both activity and selectivity through dual N-H hydrogen bonds with Asp109 [24].

Figure 3.

Structure of derivative compounds of (a) amino-pyrazolo-pyrimidine, (b) tetrahydro-β-carboline, (c) phenolic, (d)3-hydroxy-β-lactams, (e) benzothiazole-thiazolidine-2,4-dione, (f) aminothiazole, (g) 3-arylquinazolinechalcone, (h) clofazimine, (i) benzoylbenzofuran, (j) indole analog, (k) piperine-based, (l) polypyridyl Ru(II)-DPPZ.

Stereochemistry play a crucial role with the (S,S)-isomer generally more potent than the (R,R)-isomer due to optimal orientation toward Cys106 [40,41]. However, for 3-aminopyrrolidine rings, the (R)-enantiomer showed superior potency, likely due to stronger hydrophobic interaction with Leu156 [24,42]. Overall, CDK9 inhibitor optimization is hinges on the nature, size, polarity, and orientation of functional groups that stabilize binding to Asp109 and Glu107, supported by ring conformational rigidity and charge distribution within the kinase binding pocket [24].

Optimization of the piperidine-based compound TVS21 (Figure 2b), a C-terminal domain (CTD) Hsp90 inhibitor for TNBC, highlighted the importance of spatial arrangement and hydrophobic interactions within the binding site [33]. The ether/amide linker between phenyl ring A (Figure 2b, blue grid) and piperidine acts structurally, rather than as a hydrogen bond donor/acceptor. The 3,4-dichloro substitution on ring A yielded the highest activity due to favorable hydrophobic interactions over halogen bonding [33]. Substitution of ring D (Figure 2b, red grid) with 4-methylpiperidine enhanced potency, while unsubstituted piperidine reduced it, indicating tight spatial constraints within the CTD [33]. A protonated amine on ring D was essential for activity, with bulky substituents negatively affecting affinity [33]. Replacing the carbonyl linker between ring B (Figure 2b, green side) and ring C (Figure 2b, yellow side), with a non-polar methylene improved antiproliferative activity (IC₅₀ in low micromolar range), while retention of the carbonyl resulted in inactivity [33]. The linker position is shown in Figure 2b (compound TVS21) with the grey spotlight. Compounds 89 and 104 (Figure 3a, compounds 89 and 104) demonstrated potent antiproliferative effects without triggering a heat shock response, suggesting an allosteric mechanism via binding to the post-ATP-bound closed Hsp90 dimer, selectively inhibiting PPID-CTD interaction (Kd = 490 μM vs novobiocin Kd = 1089 μM) [33]. Hydrophobic interactions with Ile605B, Ala608A/B, and halogen bonding with Glu489B were key to binding affinity [33,43], offering a strategic advantage in avoiding resistance commonly associated with N-terminal inhibitors [44]. Biologically, these compounds suppressed p-AKT, ERK, and HER2 oncoprotein expression and induced apoptosis in MDA-MB-231 cells. In vivo assays demonstrated comparable efficacy to AUY922 with a superior safety profile, underscoring their potential as promising TNBC therapeutics [33,45].

Fathy et al. [23] developed a novel series of diketopiperazine-fused tetrahydro-β-carboline (Figure 2c, structure of tetrahydro-β-carboline) derivatives bearing N-alkyl chains terminating in hydroxamic acids as selective HDAC6 inhibitors [23]. Derivatives with 4–6 carbon linkers and (R,R) configuration exhibited dual HDAC inhibition for compound PDE5 (Figure 2c, structure of PDE5), while substitution with carboxylates shifted selectivity to PDE5 alone [23]. In contrast, the (S,S) enantiomers lost PDE5 activity but retained selective HDAC6 inhibition, highlighting the stereochemical control of biological activity [23,46]. Consistent with previous studies, (S)-enantiomers displayed potent antiproliferative effects and HDAC6 selectivity in MV4-11 cells [47]. Comparative analysis of tadalafil-based PDE5 and its (S,S)-analogue showed that ortho/meta aromatic substitution improved HDAC6 selectivity and reduced off-target effects on HDAC1/8 [48]. This selectivity is attributed to halogen interactions (e.g., meta-Br, ortho-F/Cl) with Asp460 of HDAC6, absent in HDAC1/8 [23]. Compound 9c (Figure 3b) is one of these attributed halogen interactions compound due to the presence of meta-Br. Para substitution consistently diminished activity, whereas ortho-methyl substitution doubled potency compared to unsubstituted or para-substituted analogues [23]. At the C5 position, most mono- and di-substituted phenyl derivatives outperformed PDE5, with compound 9c being the most potent (IC₅₀ = 0.021 μM) [23]. Favorable patterns included electron-withdrawing ortho groups (F, Cl) and bulky meta substituents (Br, CF₃, NO₂, CN), while methoxy groups showed moderate but inferior activity relative to ortho-methyl [49–51].

As a cytoplasmic deacetylase targeting non-histone proteins such as α-tubulin, Hsp90, cortactin, and peroxiredoxin [49–51], HDAC6 modulates non-transcriptional processes in tumourigenesis. Its inhibition leads to Hsp90 hyperacetylation, client protein dissociation, and proteasomal degradation [50]. Compound 9c selectively increased α-tubulin acetylation and induced sub-G1 accumulation (apoptosis) in MDA-MB-231 cells at 10 μM without altering G1/S/G2-M distribution [23], in line with HDAC6’s roles in apoptosis and angiogenesis [52–56]. Dose-dependent apoptosis (3–50 μM) was confirmed via Annexin V/PI staining, with a twofold increase in activated caspase-3 and 25% reduction in Bcl-2 at 10 μM [23], consistent with mitochondrial-mediated caspase-9 activation [57,58]. Moreover, 9c suppressed PD-L1 expression by over 50% at 30 μM [23], implicating STAT3-mediated PD-1 or PD-L1 inhibition as a part of its mechanism [48,59]. Wound-healing assays showed up to 80% reduction in cell migration at 30 μM [23], likely due to α-tubulin hyperacetylation and ERK dephosphorylation, disrupting microtubule dynamics and receptor internalization [60–62]. Collectively, these findings underscore the importance of stereochemistry and substitution patterns in modulating potency and HDAC6 selectivity.

The limitations of conventional anticancer treatments have prompted the exploration of antipsychotic drugs, such as penfluridol analogs, as potential antitumor agents, owing to their ability to penetrate the blood-brain barrier, unlike most other anticancer agents [63]. Azhraf-Uz-Zaman et al. [64] modified compound 4a (Figure 2d) as a penfluridol analog compound focused on the monophenyl group, the urea linker, and the diphenyl group. This research revealed that mono-substitution with electron-withdrawing groups at the meta or para positions supported cytotoxic activity, whereas electron-donating groups or an increase in the size of para-substituents tended to abolish activity [64]. The meta-para di-substitution pattern generally enhanced cytotoxicity, showing a preference for non-hydrogen bond forming groups at the meta position and tolerance for H-bond acceptors at the para position (compound 4q) [64]. However, H-bond donors could abolish activity. Within the diphenyl "tail" group, strong electron-withdrawing groups were preferred, and the size of the para-substituent might be inversely proportional to activity [64]. Modifications to the urea linker indicated that linkers containing nitrogen exhibited lower activity, whereas an increase in linker length could slightly enhance cytotoxicity. Compound 4q (IC₅₀ 4.55 μM) was demonstrated to induce apoptosis in a dose-dependent manner in MDA-MB-231 cancer cells, activate caspase-3, and inhibit the FGFR1 signaling pathway, thereby elucidating the molecular mechanism of its cytotoxic effects [64].

4.1.2. Phenolic derivative compounds

The flexibility and size of substituents on phenolic derivatives (Figure 2e) play a crucial role in determining their inhibitory activity against HO-1. Rigid moieties such as benzhydryl, adamantyl, and 3-benzylphenyl exhibit poor activity (i.e., high IC₅₀ values) due to steric incompatibility within the hydrophobic pocket of HO-1 and obstruction at secondary binding sites involving residues Asn210, Ala31, Ile211, Ala28, and Glu32 [30,65]. In contrast, the benzyl derivative compound VP21-04 (Figure 3c) demonstrated a potent IC₅₀ of 0.9 ± 0.08 μM and a HO-2 selectivity index of 42, attributed to the freely rotating methylene group that facilitates steric adaptability [30]. The smaller hydrophobic cavity of HO-1 compared to HO-2 accounts for its lower affinity toward bulkier substituents [65]. Halogen substitution at the para-position of the aryl ring reduces affinity due to spatial constraints [30], whereas iodine at the 5-position is more favorable than phenyl or diphenyl substitutions, which decrease activity via steric hindrance although diphenylation may enhance halogen bonding and polarizability [30]. These findings underscore the importance of optimizing the balance between hydrophobicity, conformational flexibility, and electronic properties in the rational design of selective HO-1 inhibitors for TNBC therapy.

4.1.3. 3-Hydroxy-β-lactam compounds

Chirality and substituent positioning critically influence the antiproliferative potency of tubulin inhibitors, racemic 3-hydroxy-β-lactams (Figure 2f) target the colchicine binding site (CBS) [66]. However, stereochemical discrimination is essential due to enantiomer-specific differences in pharmacodynamics, pharmacokinetics, and toxicity profiles [67]. The 3S,4S enantiomer core compound is shown in Figure 2e as EN1, meanwhile, the 3 R,4R enantiomer is shown in Figure 2e as EN2. In Combretazet analogues as a base derivative of EN1 shown that the 3S,4S enantiomer (eutomer) exhibited superior antiproliferative activity compared to the 3 R,4R enantiomer (distomer) in MDA-MB-231 cells [26]. Meta-fluoro substitution on ring B (compound 27EN1, Figure 3d) achieved subnanomolar potency (IC₅₀ = 0.7 nM) in MDA-MB-231 cells, attributed to enhanced binding affinity through modulation of pKa, lipophilicity, and membrane permeability [26,68]. The meta-fluoro substituent outperformed 4-methylthio, meta-hydroxyl, and meta-methyl analogues [26], while halogen position was found to be critical for cytotoxicity [69]. Notably, meta-hydroxyl substitution enhanced the activity of the CBS-binding distomer 3 R,4R via polar interactions [26], and aromatic hydroxyl groups have been broadly associated with tubulin polymerization inhibition [70–73]. Compound 27EN1 induced microtubule depolymerization more effectively than both Combretazet and the distomer 27EN2 (Figure 3d), as evidenced by a significant reduction in AUC and Vmax values [26,74]. Overall, stereochemical optimization and the strategic incorporation of small halogen substituents are pivotal in enhancing tubulin inhibition for TNBC treatment [26,75,76].

4.1.4. Sulfur-containing compounds

4.1.4.1. Benzothiazole and thiazolidine-2,4-dione derivatives

The trifluoromethyl (-CF₃) substituent enhances FOXM1 inhibition in benzothiazole–thiazolidinedione (Figure 2g) derivatives developed for TNBC treatment [18]. Among these, KC12 (Figure 3e), a benzothiazole derivative bearing a para-positioned -CF₃ group on the phenyl ring, demonstrated the highest cytotoxicity against MDA-MB-231 cells, attributed to increased lipophilicity and binding affinity [18]. In contrast, compounds bearing 4-morpholinomethylphenyl or 4-methoxyphenyl substituents exhibited reduced potency, likely due to steric hindrance within the DNA-binding domain (DBD) of FOXM1 [18,77]. The positional flexibility of the -CF₃ group was evident in KC21 with meta-CF₃ (Figure 3e), which retained comparable activity to KC12 [18], supported by halogen interactions with Trp308, Ser30, and Arg297 [78,79]. Compound KC30 (Figure 3e) that incorporating 2-hydroxy-5-methoxy substituents, exhibited a two-fold increase in potency over KC12 via additional hydrogen bonding, whereas bulky or highly polar groups such as 4-dimethylamino and 3-nitro disrupted DBD interactions [18]. These findings reinforce the critical importance of small hydrophobic substituents in effectively occupying the FOXM1 binding pocket [79].

4.1.4.2. Aminothiazole compounds

Aminothiazole derivatives (Figure 2h) optimized by Costa et al. [31] were developed as selective glutaminase C (GAC) inhibitors for the treatment of TNBC. Among them, compounds C12 and C15.1 (Figure 3f) demonstrated MDA-MB-231-specific antiproliferative activity with a 5.9-fold greater effect compared to SKBR3 cells. Structure–activity relationship (SAR) analysis revealed that small substituents on ring A (Figure 2h) (e.g., meta/para-4-Me, 4-F, 3,5-bis-CF₃; IC₅₀ = 4–15 μM) enhanced anti-GAC activity while simultaneously reducing inhibition of glutamate dehydrogenase (GDH) [31].

On ring C (Figure 2h), small electron-withdrawing groups (e.g., 4-CN, 4-CF₃) improved potency and selectivity via hydrogen bond stabilization [80,81] and enhanced metabolic stability [31,82]. In bulky substituents (e.g., 4-NO₂, IC₅₀ = 99 μM; 4-COOH, IC₅₀ >200 μM) significantly reduced activity [31]. Incorporating a phenylacetate moiety further increased GAC affinity while reducing GDH inhibition [31]. Modifications on ring B (Figure 2h), particularly shifting the phenyl group from C4 to C5, improved GAC inhibition. Substitution with a 5-amino-1,3,4-thiadiazole moiety (as in compound C12.33) (Figure 3f) enhanced interactions with Lys325C and Phe327 via hydrogen bonding and hydrophobic contacts. Compound C12.30 (Figure 3f), which combines favorable substitution on rings A and C, exhibited an IC₅₀ of 17 μM with only 9% GDH inhibition. Meanwhile C12.33 demonstrated a superior selectivity index compared to the clinical inhibitor CB-839 [31].

4.1.5. Polycyclic aromatic compounds

3-arylquinazolinechalcone (Figure 2i) as a hybrid quinazoline–chalcone derivatives represent a polypharmacological strategy to overcome the limitations of combination therapy in TNBC [21,83]. Stringhetta et al. [21] reported that compounds 7b and compound 7n each featuring a methoxy substituent on the quinazoline ring (shown in Figure 3g), exhibited high selectivity toward MDA-MB-231 cells (selectivity index > 2) via dual inhibition of CDK2 [21], a critical cell-cycle regulator, and ATR, which modulates the DNA damage response [84,85]. The presence of both electron-donating and electron-withdrawing substituents (e.g., 7n, which bears methoxy-nitro groups) further optimized activity, consistent with the role of methoxy in enhancing CDK2 affinity [86]. Despite IC₅₀ values for MDA-MB-231 (2.93–2.95 μM) being higher than that of paclitaxel (0.014 μM), both compounds significantly outperformed 5-FU (74.255 μM) and cisplatin (56.7 μM) [21]. In a 3D spheroid model, 7n significantly reduced cell viability (Calcein fluorescence decreased to 0.45 ± 0.15, p = 0.0004) and increased cell death (Propidium Iodide increased to 1.70 ± 0.07, p = 0.0001) [21], maybe with low E-cadherin expression compromising spheroid integrity [87,88]. Mechanistically, apoptosis was induced via enhanced caspase-8 cleavage at 12 h, reduced ATR expression at 24–48 h, diminished CHK1 at 48 h, and DNA replication stress through inhibition of CDK2-mediated ATRIP phosphorylation [21], leading to replication-fork collapse [89,90]. These findings reinforce the potential of hybrid scaffolds targeting multiple pathways, consistent with the previously reported efficacy of quinazoline/chalcone analogues in breast cancer treatment [57,91,92].

Clofazimine (Figure 2j), a lipophilic riminophenazine derivative [93], was modified by Koval et al. [25] to yield more hydrophilic analogues with improved canonical Wnt-inhibitory activity, such as MU17 (Figure 3h) (IC₅₀ = 1.1 ± 0.1 mM; SI = 2.5), which incorporates an ethylenediamine linker and N-methylpiperazinyl group to enhance aqueous solubility without compromising Wnt suppression. This compound also reduced β-catenin expression after 24 h and did not precipitate in subcutaneous adipose tissue unlike clofazimine itself which suggests a lower risk of dermatological side-effects such as rash and ichthyosis [25,94]. Additional analogues bearing a 3-aminophenyl moiety at C-3 with dimethylamino substitution, displayed improved solubility. Whereas insertion of bulky aromatic or tertiary amines (e.g., quinolizidinyl or pyrrolizidinyl groups) largely abolished Wnt activity except for hexahydropyrrolizinyl-ethanaminyl and dimethylpropanadiaminyl derivatives, which retained micromolar activity but with nonspecific toxicity [25]. Compound SV12 (Figure 3h, structure of compound SV12) (IC₅₀ = 6.3 ± 0.9 mM; SI = 2.1), which loses two chlorine substituents but retains activity via compensatory basal group effects, contrasted with fluorine substitutions that abolished Wnt-inhibition [25]. Chlorine is known to enhance anticancer potency and modulate pharmacokinetics and lipophilicity, including in breast cancer proliferation assays [95,96]. The aminopyridinyl derivative GG08 (Figure 3h, structure of GG08) (IC₅₀ = 7.3 mM; SI = 2.2) demonstrated the importance of a C-2 base chain and optimal alkyl spacer length (butylene or propylene diamine) for effective Wnt inhibition and selectivity. However, increased toxicity was observed across most cell lines with the exception of MU17 [25].

Selepe et al. [29] demonstrated that halogenation at the 5′ position of the A-ring in benzoylbenzofuran (Figure 2k) derivatives, named with compounds 6 and 9 (Figure 3i), led to a reduction in SIRT1 activity by 42.2 % and 45.9 % respectively. In contrast, conversion to isoflavone scaffolds (Figure 2k, structure of isoflavone analogue) suppressed SIRT1 activity by up to 7.31 %. Isoflavones consisting of two aromatic rings (A and B) joined via a piran-4-one heterocycle (ring C) are known for broad antiproliferative activity [29]. Methoxy or hydroxy substituents at the 6-position of ring A (for example resorcinol and para-dimethoxy) conferred over 90 % inhibition of SIRT1, whereas excessive substituents such as piragolol or free hydroxy groups at the 2′ position reduced potency [97]. Methyl substitution on ring B decreased activity in benzoylbenzofuran derivatives but enhanced SIRT1 inhibition up to 42.7 % in isoflavone derivatives. Meanwhile, resorcinol on ring B delivered over 50 % inhibition [97,98]. The quinone-isoflavone derivative 6-methoxy-4′,6′-dimethylisoflavone-2′,5′-quinone (compound 39, the structure shown in Figure 3i) proved to be the most potent SIRT1 inhibitor (IC₅₀ = 1.62 μM), engaging Ser442 hydrogen bonding and π-stacking with Phe273, and inducing apoptosis in MDA-MB-231 cell lines [97]. Its derivative inhibited cell proliferation with IC₅₀ = 6.34 μM, outperforming benzo[b]furan derivatives 5-hydroxy-3-(2′-methoxy-4′,5′-methylenedioxybenzoyl)benzo[b]furan (3.86 μM), as well as the clinically-used inhibitor Suramin [97]. These findings align with previous studies on alkaloid-based SIRT1 inhibitors [99–102], and support the growing evidence that SIRT1 plays a critical role in promoting TNBC metastasis. As a class III NAD⁺-dependent HDAC, SIRT1 facilitates epithelial–mesenchymal transition (EMT) by suppressing E-cadherin expression and promoting tumor invasion in TNBC [103,104].

Indole scaffolds, particularly at the 3-position, have emerged as promising antineoplastic frameworks, as exemplified by bis-indolyl methane (BIMs) derivatives (Figure 2l) such as 3,3′-((4-chlorophenyl)methylene)bis(5-bromo-1H-indole that mentioned as compound 5h (Figure 3j) (IC₅₀ = 8.73 μM) reported by Ngo et al. [28], which outperformed 3-aminoalkyl indols (AAIs) (Figure 2l) in MDA-MB-231 cells through π- π interactions with PARP1 and topoisomerase I. Substituents such as 5-bromoindole, 4-chlorobenzaldehyde, and 4-methoxybenzaldehyde enhanced binding affinity via hydrophobic and halogen interactions. Conversely, derivatives containing indole-2-carboxylic acid or dimethoxybenzaldehyde displayed low membrane permeability and no cytotoxicity toward TNBC cells [28]. Molecular docking confirmed stable binding among the derivatives compounds to Lys587 in PARP1, supporting their relevance in DNA repair–deficient TNBC therapy [28].

Piperine exhibits multiple bioactivities, including enhanced bioavailability via modulation of P-glycoprotein and CYP450 systems [22], and demonstrates in vitro anti-TNBC activity through p21 activation, suppression of survival signaling pathways, and potentiation of γ-irradiation cytotoxicity [22]. Among its derivatives, piperine urea (Figure 2m) analogs such as compound 8q (Figure 3k) IC₅₀ = 18.7 μM) displayed superior antiproliferative potency compared with amide derivatives, due to hydrogen bonding with Asp1046 of VEGFR-2 and hydrophobic interactions via the para-chlorophenyl group [22]. In contrast, amides derivatives with para-ethoxy substituents such as compound 5c (Figure 3k) reduced MDA-MB-231 cell viability to 24 %, while urea derivatives reduced viability to 8–26 % at 50 μM [22].

Antiproliferative activity was found to correlate with ring size and lipophilicity, but not steric hindrance [105]. Urea moieties, functioning as hydrogen bond donors/acceptors, were essential for efficacy. As bioisosteres of amides, they displayed broader anticancer potential [106,107]. Both piperine urea and amide derivatives induced apoptosis via G₂/M phase arrest, mirroring the mechanism of natural piperine which inhibits STAT3 [22], transcription factor implicated in proliferation, angiogenesis, and EMT in hormone- and p53-independent TNBC cells such as MDA-MB-231 [108].

4.1.6. Polypyridyl Ru(II) metal compounds

Zhao et al. [34] reported that the polypyridyl Ru(II)–DPPZ (Figure 2n) complex, Ru(bpy)₂BEDPPZ (IC₅₀ = 17.2 µM) displays antiproliferative and antimetastatic activity against MDA-MB-231 cells via DNA intercalation, cytoskeletal disruption and enhanced ROS production, while maintaining high DNA/protein affinity, low toxicity, and the capacity to modulate key oncogenic signaling pathways [34,109]. Whereas earlier DPPZ complexes were hindered by poor nuclear uptake, introduction of a phenylethynyl group increased planarity and lipophilicity [34], thereby facilitating nuclear penetration and DNA damage induction [110]. The larger planar aromatic phenazine ligand also affords superior DNA binding relative to cisplatin through favorable electronic and steric effects [34,111]. Ru(bpy)₂BEDPPZ demonstrated greater potency than related analogues such as Ru(bpy)₂MDPPZ, Ru(bpy)₂BrDPPZ and Ru(bpy)₂BnDPPZ (each with IC₅₀ > 30 µM). In a zebrafish xenograft model, Ru(bpy)₂BEDPPZ effectively inhibited tumor proliferation and metastasis while exhibiting stronger anti-angiogenic activity than the reference inhibitor PTK787. These findings support its potential as a promising multitarget candidate for TNBC therapy [34]. The chemical structures of these derivatives are all presented in Figure 3l.

4.1.7. Isatin-hydrazone derivatives

Munir et al. [27] evaluated a series of N-benzyl isatin–hydrazone (Figure 2o) derivatives, which combine two pharmacologically active scaffolds, hydrazones known for their functional nitrogen lone pairs and bioactive isatin heterocycles. Among these, compound 23 (Figure 3m) bearing a para-nitro substituent on the phenyl ring, showed significant antiproliferative activity against MDA-MB-231 cells (IC₅₀ = 15.8 µM). Molecular dynamics simulations (100 ns) confirmed a stable interaction between compound 23 and Met769 of EGFR, underscoring the importance of electron-withdrawing groups such as NO₂ in enhancing inhibitory potency [27]. Derivatives bearing various rings on the hydrazide moiety including pyridyl, benzyl, phenyl, naphthyl, and furan demonstrated moderate to high activity (IC₅₀ = 16.8–29.2 µM), whereas para-methoxy substitution exhibited reduced efficacy (IC₅₀ = 25.8 µM). Halogen substituents such as fluoro and methyl at positions 2 and 4 of the benzyl ring improved potency (IC₅₀ = 22.2–25.4 µM), with disubstituted fluoro compounds (IC₅₀ = 22.2 µM; cell viability = 22.46%) outperforming dichloro analogues (IC₅₀ = 29.2 µM; viability = 34.56%) due to differences in electronegativity [27,112]. Substituent positioning was also critical, as ortho-halogenated compounds showed superior activity to para-substituted analogues, potentially due to decreased coplanarity and enhanced non-covalent interactions, such as π–π stacking and halogen bonding, with enzymatic targets including EGFR and tubulin [27,113,114]. Conversely, molecules bearing both electron-donating and withdrawing groups exhibited moderate to low activity, potentially due to steric repulsion or spatial incompatibility within the active site [115].

4.2. Structure–activity relationships based on QSAR prediction

Structure–activity relationship (SAR) exploration through QSAR modeling of on 2-anilinopyrimidine derivatives (Figure 2p), initially reported by Jo et al. [116], was further refined by Abdulrahman et al. [117], who identified key descriptors, such as SpMin1_Bhs (electron density) and C3SP3 (tertiary carbon atoms) that positively correlate with antiproliferative activity against MDA-MB-468 cells. These correlations are linked to optimal electrostatic and hydrophobic interactions [117], given that π-π stacking and halogen bonding between isatin–hydrazone derivatives and amino acid residues are critically dependent on local electron density and spatial positioning of substituents [36]. Compounds bearing halogen (Cl) and methylene (CH₂) substituents exhibited the highest activity, attributed to enhanced hydrophobic interactions and molecular flexibility [117]. Conversely, descriptors such as VR1_Dzv (large molecular size) and MOMI-R (rigidity) were associated with reduced activity, owing to steric hindrance that impairs optimal orientation of molecular groups within the enzyme binding pocket [117]. Molecular docking simulations further validated strong binding to TRβ1 through hydrogen bonding and hydrophobic interactions, resembling the binding mechanisms of known EGFR inhibitors such as erlotinib [117,118].

A related study of QSAR analysis pyrazole derivatives (Figure 2p) by Bennani et al. [119] showed that PEOE_VSA + 1 (positively charged van der Waals surface area) contributed positively to anticancer activity, while Q_PC– (net negative partial charge) reduced potency [119]. Compounds with moderate hydrophobicity (Q_VSA_HYD) demonstrated optimal membrane permeability, although excessive hydrophobic groups decreased aqueous solubility [119]. These findings support the design strategy involving electron-donating groups (e.g., dimethylamino) to enhance anti-TNBC activity [36,119]. The same trend is observed in N-benzyl isatin-hydrazone derivatives, where moderate hydrophobicity (Q_VSA_HYD) aligns with improved solubility and permeability. Conversely, compounds bearing large para-halogen or bulky aromatic substituents display reduced solubility and compromised membrane penetration [120].

The swift advancement of artificial-intelligence (AI) and machine-learning (ML) methodologies now complements conventional QSAR approaches by permitting high-throughput virtual screening and enhancing the efficiency of computational drug discovery outcomes [116]. 2D QSAR models constructed with principle component analysis (PCA) and partial-least-squares (PLS) have already demonstrated how statistical learning can extract the most predictive molecular descriptors and forecasts pIC₅₀ values for previously unsynthesized analogues [119]. Similar QSAR pipelines applied to arylsulfonylhydrazones yielded robust predictive equations that directed the design of novel breast-cancer agents, thereby confirming the capacity of descriptor-driven ML to prioritize compounds before synthesis [117]. Moreover, descriptor-effect investigations on 2-anilinopyrimidine derivatives identified specific positive and negative variables that modulate anti-proliferative activity, underscoring the interpretability of ML-derived models for rational scaffold optimization [118]. Integrating ML-based QSAR predictions with molecular interaction simulations can overcome the limitations of conventional consensus-docking protocols and improve success rates in identifying high-affinity ligand-target complexes [116].

4.3. Impact of structural modifications on pharmacokinetic profiles

Structural modifications profoundly influence the pharmacokinetic (ADME) properties and biological activity of arylsulfonylhydrazone derivatives in breast cancer models. Compound 1e (Figure 4), a derivative of arylsulfonylhydrazone, exhibited an IC₅₀ of 0.9 μM and a selectivity index (SI) of 7 in MDA-MB-231 cells, attributed to enhanced lipophilicity and hydrophobic affinity [117]. The presence of a chlorine substituent extended the plasma half-life to 7 h compared to 2 h for the methoxy analogue, likely due to increased resistance to CYP450-mediated oxidation. The indole scaffold proved essential for activity, as phenyl replacement resulted in a 70-fold reduction in potency.

Figure 4.

Compound structure and ADME properties of arylsulfonylhydrazone derivative [119].

Compound 1e displayed a favorable pharmacokinetic profile with logP ∼3.5, plasma protein binding over 90%, and low clearance, supporting its potential development as a TNBC therapeutic candidate, although in vivo validation remains necessary [117]. Supporting data from another study demonstrated that compounds bearing chloro substituents exhibited potent activity against both MDA-MB-468 and MCF-7 cell lines, with an IC₅₀ of 8.2 μM and high SI values (36.6–58.9) against HEK-293 normal cells [121]. Beyond selective in vitro activity, these chloro-containing compounds also displayed good biocompatibility and low toxicity [121]. Rational design integrating indole, morpholine, and halogen (e.g., Cl) moieties not only improved biological potency but also enhanced ADME characteristics by increasing membrane penetration and metabolic stability, particularly against microsomal oxidation pathways [121]. This indicates that chloro are involved in breast anticancer activity and their pharmacokinetic profiles.

4.4. Molecular interaction modeling

4.4.1. EGFR inhibition via hydrogen bonding and halogen bonding

Targeting the epidermal growth factor receptor (EGFR) is a central strategy in TNBC therapy, particularly in subtypes that overexpress EGFR. Recent findings revealed that compound 23, an N-benzyl isatin-hydrazone derivative, binds strongly to the EGFR kinase domain (PDB: 3W2S, with a docking score of −7.561 kcal/mol and a binding free energy of −55.35 kcal/mol, outperforming the control ligand staurosporine (−4.572 kcal/mol) [27]. Key interactions include (a) a hydrogen bond between the carbonyl oxygen of compound 23 and Met769 (1.92 Å), sustained for 96% of the MD simulation; (b) hydrophobic interactions with Leu694 and Phe856, which stabilize the ligand–receptor complex; and (c) halogen bonding between the chlorine atom and the carbonyl backbone of Glu762, which enhances affinity [19,122].

Other tested compounds exhibited substantial hydrophobic interactions yet lacked critical hydrogen bonds like those formed by compound 23. This suggests that only ligands with optimal spatial orientation and conformational flexibility, such as compound 23, can effectively and consistently access the EGFR binding pocket [27]. Compound 23 also showed low root-mean-square fluctuation (RMSF) and a highly stable binding energy (MMGBSA = −79.12 kcal/mol), significantly surpassing that of staurosporine (−51.31 kcal/mol), further supporting its candidacy as a potent EGFR inhibitor [27,123]. Hydrogen bonding between electron-withdrawing groups (e.g., –NO₂ and –SO₃) and active site residues like Lys745 and Thr854 also influences activity. Compounds 13 and 22 showed strong binding affinities (< −8.0 kcal/mol), with additional interactions involving Arg841 and Asn842 that reinforced the ligand–receptor complex through a combination of electrostatic and hydrophobic forces [123]. Compound 15 as a derivative of 2-anilinopyrimidine have also demonstrated favorable interactions with the thyroid hormone receptor β1 (TRβ1), and due to structural homology with EGFR kinase domains, such findings remain relevant. Conventional hydrogen bonds with Glu311 and Arg429, along with hydrophobic contacts with Val458 and Ile303 (Table 4), underscore how spatial and polar characteristics dictate ligand binding effectiveness within kinase-like domains [118].

Altogether, the success of EGFR inhibition depends on strategic substituent placement, ligand flexibility, and the ability to form specific hydrogen bonds with key residues such as Met769, Lys745, and Thr854 [118]. The addition of electron-withdrawing groups and molecular configurations that permit free bond rotation appear to be decisive in generating biologically active and stable ligand–protein complexes [118]. Molecular dynamics simulations confirmed the structural stability of these complexes, with RMSD values <2 Å and low RMSF within the binding site, corroborating minimal conformational disruption. These results align with previous studies where quinoline sulfonate derivatives formed strong interactions with Lys745 through their –NO₂ groups, with binding energies around −8.3 kcal/mol [123].

4.4.2. FOXM1 inhibition via π-stacking and halogen bonding

Benzothiazole derivatives such as KC21 and KC30 demonstrated binding energies around −6.8 kcal/mol toward the FOXM1 DNA-binding domain (DBD), comparable to the reference inhibitor FDI-6, and exhibited highly specific interaction patterns [18]. These interactions include hydrogen bonds with Asn283, Arg286, and Trp308, and π- π T-shaped interactions with His287, outlining the essential framework of the binding mechanism [18]. Docking and 100 ns molecular dynamics simulations validated the stability of the complexes through multiple π-alkyl, hydrophobic, and water bridge interactions, notably between the methoxy substituent of KC30 and Arg297, reinforcing its extended residence time and enhanced biological efficacy [18]. The focal interaction residues Arg286, His287, Asn283, and Trp308 suggest that substitution on aromatic rings may guide ligands toward specific polar–hydrophobic binding regions within FOXM1-DBD [24]. Simulations confirmed that the benzothiazole ring of KC21 consistently engaged in π-stacking with His287, while the methoxy group contributed to water bridge formation with Arg297 [24]. These data corroborate previous findings that aromatic modifications can significantly prolong ligand retention within FOXM1’s active site [24].

The orientation and position of substituents on the aromatic ring are therefore pivotal in optimizing FOXM1 inhibition. Moreover, the hybridization of aromatic and heterocyclic scaffolds appears to enhance binding specificity and pharmacodynamic potential. Collectively, these insights support a rational design approach targeting FOXM1 with structurally refined benzothiazole-based compounds for TNBC therapy.

4.4.3. PARP1 inhibition via NAD⁺ competition

Bis-indolyl (BIMs) compounds, especially compound 5h, demonstrated significant competitive inhibition at the NAD⁺-binding site of PARP1, with docking energies approximating −11.2 kcal/mol, outperforming analogous aminoalkylindole (AAI) derivatives (∼−9.6 kcal/mol) [18]. Bidentate hydrogen bonds with His862 and Gly863 and π–cation interactions involving Lys587 and Arg488 validated the stability and specificity of these inhibitors. Molecular dynamics simulations over 100 ns (pose 890 with 1T8I) further confirmed the persistence of dominant hydrophobic and hydrogen bond interactions that maintained ligand occupancy within the NAD⁺-binding pocket [18].

RMSF values remained between 2–3.5 Å, and water bridge interactions exceeded 60–70% of simulation time, reinforcing the predicted bioactivity [18]. The architecture of the NAD⁺-binding pocket, especially its ART triad (His-Tyr-Glu), is consistent with prior literature highlighting the necessity for stable ligand engagement to disrupt PARP1 catalysis effectively [99]. BIMs not only satisfy this mechanism but also exhibit hydrophobic and polar interaction dominance aligned with PARP regulation and DNA repair pathway inhibition.

The dual combination of polar bulk and π-alkyl interaction capabilities in BIM ligands supports their development as next-generation PARP1 inhibitors with lower toxicity and enhanced selectivity against homologous recombination-deficient TNBC subtypes [124]. These findings substantiate that NAD-competitive mechanisms provide a viable route to block PARP1 activity in aggressive breast cancers, and further optimization can exploit this framework [124].

4.4.4. Tubulin interaction via the colchicine binding site (CBS)

The colchicine-binding site (CBS) of tubulin represents a promising target for small-molecule intervention, due to its resilience to drug resistance mechanisms [56]. Effective ligand interaction with CBS is crucial for the disruption of microtubule polymerization, thereby halting mitotic progression in TNBC cells. Surflex-Dock and GROMACS-based studies indicated that the 3S,4S-27EN1 compound binds tightly to critical residues such as Cysβ241 and Valβ318 within the CBS, supporting potent inhibition and stable ligand–protein complex formation in dynamic simulations [26]. The molecule induces kinetic disruptions between αβ-tubulin heterodimers, leading to spindle formation defects that underlie its antimitotic action. Hydrogen bonds with Thr179 and Asn101, coupled with van der Waals interactions involving Leu248 and Ala250, further stabilize the docking conformation. Simulation results reported RMSD values <1.5 Å over 50 ns, indicating strong conformational stability of the tubulin–ligand complex [28].

These outcomes align with previous studies demonstrating that small halogen substituents (e.g., fluoro groups) enhance binding within the tubulin hydrophobic domain, effectively impeding microtubule polymerization [20,32,125–129]. Additional work by Boichuk et al. [130] reported that the β-tubulin T7 loop plays a vital role in ligand orientation and binding, where its conformational shifts facilitate deeper access to CBS, while steric clashes inhibit productive binding [131,132].

This suggests that ligand design for CBS targeting should accommodate the structural flexibility of tubulin helices to maximize binding efficiency. The design strategy should focus on conformational adaptability, halogenation for hydrophobic pocket fitting, and stereochemistry optimized for prolonged CBS occupancy. Collectively, these insights provide a solid foundation for developing CBS-directed antimitotic agents tailored for TNBC. The diverse classes of anti-TNBC compounds reviewed in this study exhibit a range of molecular interactions that underpin their therapeutic effects, including hydrogen bonding, hydrophobic contacts, π-π stacking, and halogen bonding with key protein targets. These interaction types are summarized in Table 4, which categorizes the compounds based on their primary molecular mechanism of action.

5. Strengths

The article search revealed diverse, though limited, discoveries. The articles that met the inclusion criteria provided a comprehensive discussion on the structure–activity correlation of the modified compounds. The systematic review conducted herein covered the initial structures, the modifications performed, and the resulting activity outcomes of compounds from several studies across different compound classes. This provided a broad and general overview yet remained specific regarding the roles of modifications in the SAR of anti-TNBC compounds.

6. Limitations

While this review generally outlines the necessary aspects for modifying TNBC-targeted compound structures, further focused analysis on specific compound derivatives are still warranted to enable a more detailed and exhaustive discussion. Additionally, statistical data analysis (e.g., meta-correlation analysis) remains essential for providing a concise and quantitative representation of the relationship between compound structure and biological activity. Such analyses would facilitate a clearer and more objective interpretation of the correlation between in vitro and in silico findings (IC₅₀ values and binding energy correlations). Owing to the heterogeneity observed among the included studies, the risk of bias assessment was selectively performed only for in vitro studies, thus providing only a partial overview of potential bias. Since the selected articles also included in silico methodologies, future systematic review should employ bias assessment tool specifically designed for evaluating predictive in silico studies.

7. Conclusion

This systematic review demonstrates that structure–activity relationship (SAR) approaches and molecular modeling play a crucial role in the discovery and development of anticancer compounds for Triple-Negative Breast Cancer (TNBC) therapy. Various compound classes, including nitrogen heterocycles, β-lactams, phenol derivatives, and halogenated compounds, have been evaluated through experimental and computational studies. Each class exhibits distinct contributions to cytotoxic potential, target selectivity, and molecular effectiveness. Structural modifications such as halogen substitution, optimization of hydroxyl and amino groups, and stereochemistry have been shown to enhance ligand affinity for targets like CDK9, EGFR, FOXM1, PARP1, and tubulin. Furthermore, pharmacophore structures of compounds were developed to create additional interactions, including hydrogen bonding, halogen bonding, and π − π stacking, thereby strengthening protein-ligand complex stability. Consequently, target-based compound design strategies have proven effective in improving the biological efficacy of TNBC anticancer candidates.

Molecular modeling techniques, such as molecular docking and molecular dynamics simulations, provide profound insights into the mechanisms of ligand-target interactions and the conformational stability of complexes over simulation time. Studies indicate that low binding energy parameters, the formation of key bonds, and stable Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) values during simulation are strong indicators of compound affinity and specificity for their targets. Additionally, pharmacokinetic predictions, including logP, half-life, plasma protein binding, and resistance to P450 metabolism, further support the potential development of these compounds as drug candidates. This reinforces the relevance of integrating experimental data and simulation approaches in efficient and rational modern drug development.

Overall, the findings of this review highlight the critical importance of structure-based design strategies to overcome the limitations of conventional TNBC therapies. Compounds exhibiting high activity, selectivity toward cancer cells, and favorable pharmacokinetic profiles offer significant opportunities for developing more effective, safe, and affordable therapies. Further validation through in vivo studies and clinical trials is imperative to ensure the long-term efficacy and safety of these prospective compounds. Future investigations should prioritize the validation of insufficiently characterized molecular targets such as specific kinases or epigenetic regulators uniquely dysregulated across receptor subtypes to diversify the therapeutic landscape. Additionally, systematic lead compounds optimization should aim to enhance compound stability and plasma half-life, thereby improving ADME properties through strategic incorporation of rational structural modifications. A concerted strategy that integrates the exploration of natural products, the synthesis of targeted analogues, and AI/ML for predictive modeling is expected to accelerate the discovery of personalized therapies for TNBC. The findings presented in this review aim to provide a robust scientific foundation for further research and interdisciplinary collaborations in the field of molecular oncology.

Author contribution statement

RAF, DN, and MM did the conceptualization. RAF and DN conducted the literature investigation and drafted the manuscript. RAF wrote the original draft. DN, NKKI, and MM reviewed the original draft, editing and supervision. All authors have read and agreed to the final version of the manuscript.

Disclosure statement

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.